On Recent Trends in Science and Technology Sunday, February 11, 2018

on Recent Trends in Science and Technology Sunday, February 11, 2018

Recent Trends in General Science Track A Physics, Chemistry, Computer Applications , Mathematics, Statistics, and Electronics

Recent Trends in Life Sciences Track B Biotechnology, Biochemistry, Bioinformatics, Microbiology, Botany, and Zoology

Organized by []Re -accredoted by[] ~~c~~ CHRIST COLLEGE, RAJKOT College Included under 2(f) & 12(8) by UGC

Sponsored by GUJCOST -Gandhinagar Investing in science Department of Science & Technology, G . C "I S . & 1i h I Investing in the future! GovernmenlofGujaral UJarat ounc1 on c1ence ec no ogy Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat.

ISBN: 978-81-929521-3-0

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www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. CONTENTS

CONTENTS

I. FOREWORD i II. ORGANIZING COMMITTEE ii III. ADVISORY COMMITTEE iii IV. MESSAGES iv

PAGE S.NO TITLE OF THE PAPER & NAME OF THE AUTHORS NO.

Section 1 CHEMISTRY

1 COMPARISON BETWEEN 1-[(Z)-PHENYLDIAZENYL]NAPHTHALENE-2-OL DYE 1-4 PREPARED FROM MINERAL ACID AND ORGANIC ACID Dr. Saurabh K. Patel, Bilal J. Mansuri, Rahul V. Gupta, Mehul B. Prajapati 2 RESOURCE OF WATER AND RELEATED POLLUTION 5-9 Digant R. Bhadja & Abhishek D. Bhadja 3. A SEASONAL STUDY OF PHYSICO –CHEMICAL ANALYSIS OF DRINKING 10-16 WATER OF VARIOUS AREA GUJARAT, INDIA. Piyushkumar J.Ribadiya, Divyaba B.Jadeja, Sapna H.Mahera, Dr.D.K.Bhoi 4. SYNTHESIS AND THERAPEUTIC EVALUATION OF SOME NEW QUINOLINE DERIVATIVES OF 4-CYCLOPROPYL-5,6,7,8 SUBSTITUTED-1, 2-DIHYDRO 17-21 QUINOLINE-2-ONES Vikram R. Dangar and Viral R. Shah

Section 2

PHYSICS AND ELECTRONICS

5. ACTIVATED CARBON FROM RENEWABLE BIOMASS FOR SUPERCAPACITOR – 22-27 A REVIEW Parth Joshi, Desai Riddhi and Chirag Patel 6. ELECTRON IMPACT SCATTERING STUDY WITH CF2 RADICAL 28-31 Divya Ahir, Rujuta Patel, Swati Shirsagar, Hardik Desai and Minaxi Vinodkumar 7. ELECTRON IMPACT IONIZATION CROSS SECTIONS OF HE AND NE ATOM 32-36 Malhar Bhatt, Ankit Tandel and Hardik Desai 8. STUDY OF GROWTH, STRUCTURAL, OPTICAL, THERMAL AND MECHANICAL 37-41 PROPERTIES OF Ba+2 DOPED KDP SINGLE CRYSTALS Haresh K. Patel, K.G.Raval, Paras H. Trivedi, J. P. Gandhi, B. J. Lad 9. DESIGNING A LOW-COST AMBULATORY ECG RECORDER FOR CARDIAC 45-45 PATIENTS Harikrishna Parikh, Jatin Savaliya, Gumjan Vyas, Ankit Sidpara, Dr. H. N. Pandya

10. E – COLOURING OF A GRAPH 46-50 D. K. Thakkar and V. R. Dave

Section 4 BIOINFORMATICS

11. COMPUTATIONAL APPROACHES FOR PROTEIN STABILITY STUDIES 51-56 Varun S. Nair, John J Georrge

12. TOOLS FOR LIGAND BASE DRUG DISCOVERY 57-64 Janvi Gajipara, John J Georrge

13. STATISTICAL ANALYSIS OF INDUSTRIALLY IMPORTANT THERMOPHILIC 65-72 ORGANISMS PRODUCING ALPHA-AMYLASE, DNA POLYMERASE AND PROTEASE Rija George, John J Georrge

14. IN SILICO PROTEIN ENGINEERING: METHODS AND TOOLS 73-80 Sneha Thomas, John J Georrge

15. NONCODING RNAs: DATABASE AND TOOLS 81-86 Rachel Jani, John J Georrge

16. TOOLS FOR ASSEMBLY AND ANNOTATION 87-96 Mishal John, John J Georrge

Section 5 BIOTECHNOLOGY

17. Aspergillus flavus - A MENACE TO FARMERS 97-109 Avani Ranipa, Anju Shrilal, Akash Nimavat, Jalpa Rank, Ramesh Kothari, John J. Georrge

Section 6 MICROBIOLOGY

18. THE FUTURE OF OUR ENVIRONMENT 110-114 Chetana Rajyaguru

19. BIOACTIVITY GUIDED FRACTIONATION OF PRICKLES OF BOMBAX CEIBA 115-116 LINN. FOR IN-VITRO ANTIMICROBIAL ACTIVITY WITH REFERENCE TO ACNE VULGARIS Darshan Vasani, Riddhi Gorasiya, Neha Chohala, Jigna Vadalia

20. EFFECT OF INCREASING CONCENTRATION OF SEAWATER IN SOIL ON 117-120 GROWTH OF PLANTS (RGR, NAR, LAR) OF PEARL MILLET CROP OF SAURASHTRA REGION Rasik C.Viradia

21. ANALYSIS OF PHYSICO CHEMICAL PARAMETERS OF DIFFERENT SOIL 121-122 SAMPLE OF SURENDRANAGAR Dr. Rutva H. Dave and Dr. Jignasa B. Joshi 22. FUNCTIONAL ABILITIES OF CULTIVABLE RHIZOSPHERIC BACTERIA IN 123-129 PHYLLANTHUS FRATERNUS AND THEIR POTENTIAL FOR PLANT GROWTH PROMOTION AND PHYTO-PATHOGEN CONTROL Kalpna D. Rakholiya, Krupali Sureja, Riddhi Chaniyara, Kana Parmar, Dhrupa Mayatra, Mital J. Kaneria, Satya P. Singh

23. CULTIVABLE PLANT GROWTH-PROMOTING ACTIVITIES OF RHIZOSPHERIC 130-135 BACTERIA ISOLATED FROM INDIGENOUS MEDICINAL PLANT CARDIOSPERMUM HALICACABUM Kalpna D. Rakholiya, Kana Parmar, Krupali Sureja, Riddhi Chaniyara, Dhrupa Mayatra, Mital J. Kaneria, Satya P. Singh

Section 8 ZOOLOGY

24. STUDIES OF BIOCHEMICAL PARAMETERS OF OREOCHROMIS NILOTICUS 136-140 EXPOSED TO CADMIUM CHLORIDE (CDCL2, 2H2O) Hetalben Parekh, Dr. Shantilal Tank 25. CAGE CULTURE TECHNIQUE OF PANGASIUS (Pangasius sutchi) FARMING IN 141-143 BANSWARA, RAJASTHAN Bhagchand Chhaba, Dr. Bhatt Nakul A., Vikash Chahar 26. COMMERCIAL IMPORTANT FISHING GEAR OPERATED AT GUJARAT COAST 144-151 R. A. Khileri, A. Y. Desai, S. I. Yusufzai, S. R. Lende 27. ANTIMICROBIAL PROPERTIES OF SKIN MUCUS FROM FRESHWATER FISHES 152-156 H.D. Kaher, S.S. Bhatt, A.V.R.L. Narasimhacharya, A.N. Upadhyaya 28. SALIVARY COMPONENTS OF THERAPEUTIC LEECH AND ITS USES 157-160 K.K.Koriya, A.N. Upadhyay, H.D.Kaher 29. DIVERSITY AND DENSITY OF MIGRATORY BIRDS AND ITS CORRELATION 161-168 WITH PHYSICO-CHEMICAL PARAMETERS OF LAKHOTA LAKE, JAMNAGAR, GUJARAT A.N. Upadhyaya, P. S. Oza and B. A. Jadeja, H.D. Kaher, K.K.Koria 30. A DIFFERENT CULTURE TECHNIQUES FOR MUD CRAB IN GUJARAT 169-172 Dr. Bhatt Nakul A., Dr. B. K. Sharma, Dr. D. T. Vaghela

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat.

FOREWORD

Science and Technology are fast developing and growing to gain more mileage in a multidisciplinary way. The importance of interdisciplinary research is increasing exponentially. The juncture of biology, chemistry, physics, mathematics, and social and behavioral sciences is where the excitement is more intense. The classroom teaching is inadequate to meet the demands of the time. The younger generation needs to be well-equipped to face the challenges of the morrow. They require a platform to come up to the level where they can be well-prepared to stand in this competitive world. In compliance with these objectives, Christ College, Rajkot is organizing the 10th National Science Symposium aiming at: • An exposure to the latest development in various fields of Science and Technology • A platform to interact with eminent scientists and subject experts • Knowledge enhancement in particular areas of interest • Career guidance for selecting a subject for higher studies • An opportunity to strengthen inter-institute collaboration and institute-industry networking.

Christ College Rajkot acknowledges the constant support and encouragement from the academic that sustains this initiative. We are thankful to GUJCOST for sponsoring the 10th National Science Symposium on the Recent Trends in Science and Technology.

Christ College appreciates the dedication and commitment of the staff and students responsible for this event.

Organizing Committee SS2018 Christ College, Rajkot

______www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130 Page No. i Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat.

Chief Patron

Most Rev. Bishop Jose Chittooparambil Bishop of Rajkot Diocese, Gujarat

Chief Guest

Prof. (Dr) Pratapsinh Chauhan Vice Chancellor, Saurashtra University, Rajkot, Gujarat

Keynote Speaker

Dr. Mahan MJ (Formerly, Mahan Mitra) Professor, School of Mathematics, Tata Institute of Fundamental Research. Mumbai

Guest of Honours

• Dr G. C. Bhimani Dean, Faculty of Science, Saurashtra University, Rajkot

• Dr. Rupani Mehul Pravinbhai Other than Dean, Faculty of Science, Saurashtra University, Rajkot

Organizing Committee

Patron : Rev.Fr. Benny Joseph Director, Christ Campus, Rajkot

Symposium Chair : Dr. Yvonne Fernandes Principal, Christ College, Rajkot

Symposium Convener : Dr. Sushmita Ganguly Head, Department of Chemistry, Christ College, Rajkot

Symposium Coordinators

• Dr. Aravindakshan, Head, Department of Physics • Dr. Charmy Kothari, Head, Department of Biotechnology • Dr. John J. Georrge, Head, Department of Bioinformatics • Dr. Shailendrasinh Jadeja, Head, Department of Computer Application • Mr. Usmangani Tabani, Head, Department of Microbiology • Dr. Padma Ambalam, Coordinator, MSc Microbiology • Mr. Jagrut Tripathi, Coordinator, Department of Mathematics

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NATIONAL ADVISORY BOARD

• Dr. A. K. Shah, Honorable Vice Chancellor, Gujarat Vidyapeeth, Ahmedabad • Prof.(Dr.) Bhasker Raval, Pro Vice Chancellor, Veer Narmada South Gujarat University, Surat • Prof. B. J. Rao, School of Biological Science, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai • Prof. Sunil Bhand, Dean (University wide), Sponsored Research and Consulting, BITS PILANI, GOA • Prof. Bengt Danielsson, CEO, Acromed Invest AB, Magistratsvagen 10, SE-226 43 Lund, Sweden • Prof. H.S. Misra, Head, Molecular Genetics Section, Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai • Dr. S. K. Khare, Professor and Senior Scientist, Department of Chemistry, IIT, Delhi • Dr. A. K. Goel, Scientist F, Biotechnology Division, DRDE, Ministry of Defence, Gwalior, MP • Dr. S. D. Bhatt, Rtd. Senior Principal Scientist, CSMCRI, Bhavnagar, Gujarat • Dr. C. G. Joshi, Professor, Dept. of Animal Biotechnology, Anand Agricultural University, Gujarat • Dr. S. R. Dave, Professor and Head (Rtd.), Dept. of Microbiology and Biotechnology, Gujarat University, Ahmedabad, Gujarat • Dr. S. P. Singh, Professor and Head, Dept. of Biosciences, Saurashtra University, Rajkot, Gujarat • Dr. C. K. Kumbharana, Professor & Head, Dept. of Computer Applications, Saurashtra University, Rajkot • Dr. P. H. Parsania, Professor & Head (Rtd.), Dept. of Chemistry, Saurashtra University, Rajkot • Dr. H. S. Joshi, Professor & Head, Dept. of Chemistry, Saurashtra University, Rajkot • Dr. H. H. Joshi, Professor & Head, Dept. of Physics Saurashtra University, Rajkot • Dr. D. K. Thakker, Professor & Head, Dept. of Mathematics, Saurashtra University, Rajkot • Dr. M. J. Joshi, Professor, Dept. of Physics, Saurashtra University, Rajkot • Dr. Shipra Baluja, Professor. Department of Chemistry, Saurashtra University, Rajkot • Dr. Ramesh Kothari, Professor, Dept. of Biosciences, Saurashtra University, Rajkot, Gujarat • Dr. Sabu M. C., Assistant Professor, St Albert College, Ernakulum, Kerala • Dr. Vijay Singh Rathore, Director at Shree Karni College & KISMAT, Jaipur & Professor, CSE, Rajasthan College of Engineering for Women, Jaipur • Mr. Suneel Dwivedi, Associate Professor & Head, Department of Computer Science, Maharaja Krishnakumarsinhji Bhavnagar Uni. Bhavnagar. Gujarat

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Message

As we are about to complete the second decade of 21st century, we are struck by a sense of wonder and bewilderment looking at breakthroughs in science and technology. We have cloned a pair of macaques; the frightening question is, what next? Science is a progressive medium for the development of mankind as it makes our life easy by harnessing technology for the collective good. Fundamentally, it shapes our lives through futuristic inventions, innovations and discoveries at both micro and macro level. However, the big challenge for scientists across the world is to ensure that the scientific as well as technological innovations are focused on the inclusive growth and sustainable development so that our today's solutions don't turn to be the problems of tomorrow.

Eventually, the task of making a further inquiry and propagation into the realm of science shifts to the erudite scholars, researchers and students. I’m delighted at the perennial efforts of Christ College, Rajkot, in organizing the National Science Symposium where the student fraternity from across the nation would be enlightened by the nexus of novel scientific ideas amidst the jubilant panel of speakers.

I congratulate and extend my best wishes to the budding scientists to keep up their sense of wonder and intrigue to contribute to our society with their scientific caliber and wisdom.

tJose Chittooparambil CMI Bishop of Rajkot

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SAURASHTRA UNIVERSITY Prof. Pratapsinh Chauhan University Campus, Rajkot - 360 005. Gujarat. India. Vice Chancellor Tel.: (0.) +91 {281 )-2577633, Fax: +91 {281 )-2576802 email: [email protected] em_ail : [email protected] Accredited With Grade "A" by NAAC

Message

I am pleased to know that Christ College Rajkot is organizing the 10th National Level Science Symposium on Recent Trends in Science and Technology. We are witnessing sweeping changes in the highly technologically advanced digital era driven by commercial activity. With this accelerating pace of the economy research in the field of science and technology becomes the call of the hour. In this context, the symposium will allow participating research scholars and students to deliberate on the new developments in the field. It's indeed a necessity to incorporate extracurricular and cocurricular activities along with regular teaching throughout the year for overall development of a students and teachers as well and the present symposium is a welcome step in this direction.

It is also extremely motivating to know that this is the 10th Science Symposium organized by Christ College. To start with a new endeavor is appreciative but to persist with the good work is worth high admiration.

I am sure that this symposium will be a source of motivation and encouragement for young, dynamic and enthusiastic students for their personal and career development. I wish the symposium grand success. Jay Hind.

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Dr. Girish Bhimani Department of Statistics Professor & Head Saurashtra University Member of Syndicate, Office : Dolarrai Mankad Marg, Rajkot - 360 005. Gujarat (India) Dean, Facultyof Science Tel: (0) 0281-2586756, Mo.94260 19046 Director, IQAC & Co-Ordinator, PGDHM Email : [email protected] (Acmdit

MESSAGE

It is indeed a pleasure to see the growth of Christ College, Rajkot, as an academic institution since its inception in 1998. It has been providing an excellent service to society by nurturing the overall personality of the student.

Science is a way of life rather than just being a body of knowledge and collection of information. Involving more and more students in science is a vision we all share as academician. The platform in the form of a symposium would provide an opportunity to students and faculty members to share and exchange knowledge and motivate the budding scientist to pursue career in science and research. Christ College has already set the benchmark by conducting quality national and an international seminar every year.

As the Dean of Science, Saurashtra University, I extend my warm wishes to the 10th National Science Symposium on “Recent Trends in Science and Technology” on 11th February 2018.

Prof. Girish Bhimani

Resi., : "SAMARTHYA", 1-ShreeRampark, University Road, Rajkot - 360 005. Tel : (R) 0281 - 2585899

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1ST CO LEGE (Affiliated to Saurashtra University, Rajkot) 'NiAc7 Vidya Noketan, Saurashtra University P. 0 . ~ : B.,.(CGPA2.8t.J Rajkot-360 005, Gujarat, India. College Included under 2(f) & 12(8) by UGC Phone: 9427164732: 9427164733 email: [email protected] Undergraduate & Postgraduate Studies wy-,tW.christcollegerajkot.edu.in

From the Director

It is my great pleasure to welcome you to the 10th National Science Symposium on Recent Trends in Science and Technology, organized by Christ College, Rajkot, on February 11, 2017. It has been more than a decade that Christ College, Rajkot, organizes science symposium on recent trends in science and technology every year. The Symposium is considered one of the most awaited academic pursuits at the National level.

The theme of the Symposium suggests the demand of the modern scientific age and how one branch of Science should recognize the other branches of it to work together. A symposium of this kind, I am sure, would bring together the well-known and young budding scientists of far and wide to generate innovative ideas of Science and Technology.

Christ College, Rajkot, takes pride in organizing National Science Symposium for 10th year consecutively and creating a benchmark in the academia.

I heartily welcome the publication of the Abstract Books and appreciate the team members involved in bringing out such a beautiful form with all their contents minutely reviewed and arranged. I hope this publication will be an encouraging effort to help teachers, researchers and students of Science and Technology in furthering interest in the development of modern trends in Science and Technology.

I wish the symposium and the publication all the best.

Director

Christ Campus Rajkot www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130 Page No. vii

f;:RA dited bc7 (Affiliated to Saurashtra University, Rajkot) Vidya Noketan, Saurashtra University P. 0 . ~ ! e.,.(CGPA2.8 r-.J Rajkot-360 005, Gujarat, India. College Included under 2(f) & 12(8) by UGC Phone: 9427164732, 9427164733 email: [email protected] Undergraduate & Postgraduate Studies wy.w.christcollegerajkot.edu.in

From the Principal

I am pleased that Christ College, Rajkot is organizing the 10th National Science Symposium, on the Recent Trends in Science and Technology on February 11, 2018. Our country plays a significant role in the contribution to high-quality scientific research and is ranked high globally in terms of the number of scientific publications and the number of patents filed. The government of India's move towards a digital economy and the availability of sufficient of digital talent have motivated organisations to set up innovation centres in India. With reference to the recent developments in science and technology, the research opportunities in India have increased inspiring the Indian scientist to come back to India to pursue research in our country. However, we still have a long way ahead. We require a critical resource pool for strengthening further and expanding the Science &Technology system and Research & Development base. This science symposium is a platform to expose the young minds to the excitement of science, to attract young science students to research and to give them a wide exposure. It is an excellent forum for exchanging scientific information and presenting the latest developments and trends in Science and Technology. Christ College, Rajkot, has been organizing science symposia since 2004. The success of this event over the years has compelled us to sustain this event and make it more meaningful and appealing to the scholars. The scientific events are multi-disciplinary and target a wide range of students - undergraduates, post graduates, research scholars and even academia. The overwhelming response to our call for papers indicates the popularity of this symposium and confirms that this event has become the nation-wide forum for all branches of science and technology. The symposium this year is marked by the key note address by Prof. Mahan MJ, Professor, School of Mathematics, Tata Institute of Fundamental Research, Mumbai, and a recipient of Shanti SwarupBhatnagar Award in Mathematical Science, 2011. The symposium also includes oral and poster presentations by undergraduate and postgraduate students, research scholars and academia, in the fields of Physics,Chemistry, Mathematics, Statistics, Electronics, Computer Science, Biotechnology, Biochemistry, Microbiology, Zoology, Botany and Bioinformatics. Over the years this symposium on the Recent Trends in Science and Technology has evolved and become a much awaited event. It has helped establish a networking among the various educational and research institutions. I place on record my sincere gratitude to the symposium convener and the entire organizing team for successfully organizing the event. '1 k...... , ,--J ~ Dr. Yvonne Fernandes Principal & Symposium Chair www.christcollegerajkot.edu.in, ISBN: 9788192952130 Page No. viii Christ College, Rajkot © Christ College, Rajkot, India

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Chemistry

Section 1

CHEMISTRY

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Chemistry

COMPARISON BETWEEN 1-[(Z)- PHENYLDIAZENYL]NAPHTHALENE-2-OL DYE PREPARED FROM MINERAL ACID AND ORGANIC ACID Dr. Saurabh K. Patel*, Bilal J. Mansuri, Rahul V. Gupta, Mehul B. Prajapati Department of Chemistry, Veer Narmad South Gujarat University, Surat, Gujarat ,395007 *[email protected] ABSTRACT: The study focuses on the comparison between the 1-[(Z)- phenyldiazenyl]naphthalene-2-ol formation by mineral acid and organic acid respectively. A distinct difference in the color intensity and intermediate mobility has been observed. The different solvents also affect the product yield. Major difference is noticed in the UV and chromatography data of the dye compound. The priority of the solvent stands for the environmental defense. INTRODUCTION The term azo comes from “Azote” which means to donate nitrogen group. Azo dyes contains at least one nitrogen-nitrogen double bond -N=N-; however many different structures are possible. Monoazo dyes have only one -N=N- double bond, while diazo and triazo dyes contain two and three -N=N- double bonds, respectively. The azo groups are generally connected to benzene and naphthalene rings, but can also be attached to aromatic heterocycles or enolizable aliphatic groups. These side groups are necessary for imparting the color of the dye, with many different shades and intensities being possible. An example of an azo dye is shown in figure 1. When describing a dye molecule, nucleophiles are referred to as auxochromes, while the –N=N- groups are called chromophores. Together, the dye molecule is often described as a chromogen. Synthesis of most azo dyes involves diazotization of a primary aromatic amine, followed by coupling with one or more nucleophiles. Amino- and hydroxy- groups are commonly used coupling components. Because of the diversity of dye components available for synthesis, a large number of structurally different azo dyes exists and are used in industry. Azo dyes acquired wide interest in application to biological system and indicator in complexometric titrations of analytical chemistry. Azo dyes are the most important group of synthetic colorants for clothing, plastics, cosmetics and food beverages. Color changes are caused by change in extent of delocalization of electrons. More delocalization shifts the absorption max to longer wave lengths and makes the light absorbed redder, while less delocalization shifts the absorption max to shorter wavelengths.

Figure 1: Example of a common azo dye structure EXPERIMENT Preparation of red azo dye in mineral acid Preparation of diazonium salt Solution of 1 mL of concentrated hydrochloric acid in 10 mL of distilled water added in a round bottom flask. While stirring 1 g of p-nitroaniline was added with stirring in ice bath. Then solution of 0.5 g of sodium nitrate in 2 mL of distilled water was added slowly. Synthesis of para red Solution of 1 g of 2-naphthol in 10 mL of 2.5 M sodium hydroxide was added into the flask with the diazonium salt in an ice bath, with vigorously stirring for a few minutes. The product was dried at 50 ˚C for 2 hours. The product was characterized by UV spectrophotometer and column chromatography as shown in figure 2.

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Table 1

Batch HCl red azo dye, direct.sre EV300 172103 Data Mode A 100%T Baseline Correction HCl red azo dye 1 2 3 4 Cycle01 Nm 230 243 318 412 Peaks A 3.965543 3.888075 3.099271 4.775892

THERMO ELECTRON -VISIONpro SOFTWARE V4.10

Operator Name (None Entered) Date of Report 1/9/2018 Department (None Entered) Time of Report 1:56:49PM Organization (None Entered) Information (None Entered)

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200 250 300 350 400 450 500 550 600 650 700 750 800 Wavelength(nm) --HAC red azo dye.sre:HAC red azo dye:Cycle01

Figure 2: UV-Visible curves of compound Preparation of red azo dye in organic acid Preparation of diazonium salt Solution of 1 mL of concentrated acetic acid in 10 mL of distilled water added in a round bottom flask. While stirring 1 g of p-nitroaniline was added with stirring in ice bath. Then solution of 0.5 g of sodium nitrate in 2 mL of distilled water was added slowly. Synthesis of para red Solution of 1 g of 2-naphthol in 10 mL of 2.5 M sodium hydroxide was added into the flask with the diazonium salt in an ice bath, with vigorously stirring for a few minutes. The product was dried at 50 ˚C for 2* hours. The product was characterized by UV spectrophotometer and column chromatography as shown in figure 3. Table 2

Batch HAc red azo dye 02.sre EV300 172103 I I I I I I I Data A 100%T Baseline Correction Mode HAc- red azo dye 1 2 3 4 5 6 7 Cycle01 nm 230 243 289 319 326 337 380 Peaks A 3.805982 3.742857 3.974833 4.894375 4.898755 4.863479 5.003208

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••• •• •• 3.5 >O 2$ I20 •• • 0 •• OO L 200 250 >00 >SO 400 ... 000 000 100 150 W•.,...noch(nm)... HAC •Mt •zo c,,,. .,,. HAC '" •.to c1.,.. e.,.c&eOt - -

Figure 3: UV-Visible curves of compound

Table 3 : UV-Visible transition Compound Type of transition Wave lengths, nm Red azo dye π π * 285 sharp n π * 325 shoulder π- π * 490 tautomerism - RESULTS AND DISCUSSION The synthesis of an azo dye requires two organic compounds – a diazonium salt and a coupling component. The diazonium salt reacts as an electrophile with an electron-rich coupling-component, like a -naphthol and naphthalene derivative through an electrophilic aromatic substitution mechanism. The prepared products as azo dye were characterized by various available techniques. The infrared and UV- visible spectroscopy is one of the most efficient techniques used in the characterization of organic compounds. The first dye prepared in mineral acid gives benzene diazonium chloride whereas benzene ammonium quaternary acetate salt is obtained in second dye prepared in organic acid. Chlorine is good replacing group as compared to acetate hence acetate gives slow reaction and formation of salt is slow. Dye prepared in mineral acid is more active whereas in organic acid it is found to be less active which may be due to the presence of the traces of chloride ions The color intensity of the dyes varies as shining brick red for mineral acid and light red orange color for organic acid. The mobility of the salt is also distinctive. The acetate ions being bulky reduces the mobility of the salt and hence dye prepared in organic acid form little slowly as compared to that prepared in mineral acid. However the UV data confirms the comparable dye structures form both the methods. The column chromatography shows the equivalent bonds but the elution of the mineral acid assisted dye is faster than the organic acid assisted dye.

CONCLUSION The study emphasizes on the selectivity of the mineral and organic acid for the preparation of 1- [(Z)-phenyldiazenyl]naphthalene-2-ol stands for the environmental defense. The products were monitored by column chromatography and characterized by UV-Visible spectrophotometer. The

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REFERENCES 1. Heinrich Z, Color Chemistry, Synthesis, properties and application of organic dyes and pigments, 1991. 2. Chandrashekhar Patil, Review of The azo derivatives of salicylic acid. 3. Ewelina Weglarz Tomczak, Review of Azo dyes –biological activity and synthetic strategy. 4. J.Griffiths, Organic Dyes. 5. Venkataramam, Dyes Chemistry.

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RESOURCE OF WATER AND RELEATED POLLUTION Dr. Digant R. Bhadja1* & Abhishek D. Bhadja2 1M. M. Science College, Morbi-363641, Gujarat 2Shree M.P. Patel B.Ed. & Science College, Jodhapar-Morbi-363641, Gujarat *Corresponding author: [email protected]

ABSTRACT: Water, H2O hydrogen oxide, is an extraordinary chemical compound of fundamental environmental importance. The electronic structures of its molecules impart the very special chemical and physical properties which lead to water being described as ‘the universal solvent’ or ‘the liquid of life’. Water in the gaseous state is made up of single molecules. Water in the liquid state is made up of groups of molecules associated together by linkages of hydrogen bonding. In ice the water molecule is associated in tetrahedral structure. The earth’s water resources, the ‘hydrosphere’, consist of the oceans and seas. Rainfall, even when it was in its wholly natural state. It is the part of the natural scheme of things that man, a saprophytic animal, should cause environment pollution in almost all he does. Therefore, water pollution take place with diffuse pollution. Awareness and treatment of purification required to solve this matter. Key words: Water, Resources, pollution, Treatment

1. INTRODUCTION The Earth’s water resources, the ‘hydrosphere’, consists of the oceans and seas, the ice and snow of the polar regions and mountain glaciers, the water contained in surface soils and the water in lakes, rivers and streams. Less than 1% of these resources consist of freshwater, some 2% is freshwater ice located seawater and sea ice. The annual evaporation of water from the hydrosphere, and its return as rainfall amounts to about 260 x 1012 m3. The total water content of the atmosphere is about 7 x 1012 m3, indicating that atmospheric water is replaced on the average some 37 times a year. For all practical purposes it can be said that the water of the hydrosphere was of natural quality until the Industrial Revolution in Europe and North America initiated the development of technology has been reached where the entire hydrosphere, except the ice which was formed before the industrial revolution began, is contaminated by the polluting activities of man.1 The pollution of water is not the only cause of manmade degradation of that environment. Water abstraction for domestic, industrial and agricultural supply, and the lowering of near surface groundwater levels to increase agricultural output, can bring about major depreciations of the aquatic environment.

2. THE NATURE AND COMPOSITION OF WATER The quality of water is defined in physical, chemical and biological terms. Rainfall, even when it was in its wholly natural state, was quite impure water in the scientific sense. Its physical characteristics in the liquid phase are that it would normally be naturally clear, colourless, and odourless with a temperature varying from ambient to colder depending on the nature and height of its precipitation. Rainfall naturally contains materials dissolved from the atmosphere. On reaching the ground, rainfall picks up more impurities naturally from vegetation and the ground surface. If the ground is permeable, all or some of the rainfall will pass underground according to the circumstances. Some will be evaporated and the balance will run off to streams and rivers and via underground route to the sea. On the average some 70% of the rainfall evaporates directly, or indirectly via plant transpiration. Of the remaining 30% more travels through underground state than along rivers and streams and about 90% of the total run-off reaches the oceans. Natural run-off from bare, substantially impermeable hard rock gathers material from gradual weathering of the rock to yield low concentrations of dissolved Calcium, Magnesium, Sodium and Potassium. Traces of other materials will be present according to the chemistry of the rock.2

3. FRESHWATER FROM RIVERS, STREAMS AND LAKES By and large the natural quality of rivers and streams at any point reflects the quality of the upstream contributions of surface run-off and groundwater discharge. Similarly, but to a lesser degree www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 5

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Chemistry according to circumstances, the natural quality of a lake reflects the quality of the inflows of water that maintain the lake level. However, these waters, open to the energy of sunlight, the solution of oxygen from the air and containing the mineral nutrients sufficient to support plant growth, naturally become the media for the growth of aquatic biota. The nature, variety and abundance of these biota-the biology of the water is essentially determined by the chemistry of water. The natural shape of the channel of a river at any point is determined by the rate of flow of water and the topology and geology of the terrain through which the river passes. These factors determine the gradient of the river, its velocity of flow, its depth and width, its shoals and pools, and the nature of the river bed at various points. Even as the growth of river has a secondary effect on the physical and chemical characteristics of the water, so the growth of the biota can influence the effective shape of the river channel. A good example of this is the heavy growth of rooted water plants which occurs in the shallow, calcareous waters of chalk streams. Heavy growths of these plants, if not controlled by cutting and removal from the rivers, often so reduce the flow cross-sections to cause flooding during heavy summer storms. Another major cause of quality variability is the biochemical activity which proceeds within micro-organism in the water, particularly bacteria, as key factors in the operations of the carbon and nitrogen cycles and other biochemical transformations. The most important of these are summarized.3

4. WATER POLLUTION It is part of the natural scheme of things that man, a saprophytic animal, should cause environment pollution in almost all he does. Pollution of water divided into two kinds namely into point sources and non-point sources. The point sources are mainly discharging of wastewaters from sewage works, factories and farms, while the non-point sources arise mainly from specific categories of general land use. As regards the type of pollution which occur, these can be listed according to the effect exerted by the polluting matter. (a) Substances actually toxic to man and/or aquatic flora and fauna (e.g. Lead, Mercury, Cadmium, Cyanide, Pesticides). (b) Substances are hazardous to man in causing chronic or long dormant cumulative, harm (e.g. polynuclear aromatic hydrocarbons, chlorophenols, trihalomethanes) (c) Substances at very low concentrations which are not highly toxic but which can either be rendered actually toxic by biochemical transformation in the water. (d) Substances which add to the eutrophication (plant-nutrient content) of the water (e.g. sewage, farm wastes) (e) Substances which have a detrimental effect on the physical appearance of the water (e.g. oil, detergent foam, suspended matter) (f) Substances which add to the load of biochemical oxygen demand in the water or in benthal (bottom) deposits (e.g. sewage effluent, food-industry water waste, farm wastes). (g) Substances which only have a polluting effect at relatively high concentrations in water (e.g. mineral salts such as sodium chloride).

5. DIFFUSE POLLUTION Pollution from non-point sources, or diffuse pollution, in its very nature presents difficult control problems. If it is known that a river, lake is polluted by a particular substances or group of substances, but the point of access of this pollution to the water cannot be located and no particular person or corporate body can be proved to have caused of know permitted the pollution to occur, the normal processes of pollutants and making them pay for remedial and preventive measures are thwarted. There are three main problems before us. The first is acidification of waters caused by acid rain and the second is the high and still rising level of nitrate in many waters, particularly groundwaters.4

5.1 ACIDIFICATION OF WATERS The acidification of rivers and lakes by acid deposition from the atmosphere is essentially a local problem. Much of the acidity is a part of the consequence of the major problem of atmospheric pollution in industrial areas. The effect of acid decomposition varies greatly according to the type of soil on which it falls. Alkaline soils based on limestone can neutralize large amounts of acid whereas www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 6

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Chemistry soils based on peat or granite cannot do so. There are two practically ways in which the impact of acidification can be cased. One is reduced the emission of the atmospheric pollutants. The second is to add a neutralizing alkali (such as powdered limestone) to the acid-sensitive areas. The key link between the emissions and their ecological impact is the transfer of the acidity from deposition to run-off to rivers and lakes, and understanding of this link is essentially a matter of the chemistry of the interactions between the soil, thin and base poor, are the most sensitive to acid deposition and are the main generators of acid rivers and lakes from that depositions.

5.2 NITRATE IN WATERS The quality of water intended for human consumption sets the maximum acceptable limit for nitrate in water as 50 mgL-1 (11.3 mgL-1 nitrate nitrogen). Nitrate problems in groundwater’s arise mainly because of heavy loadings of nitrogenous matter on land through which percolating rainfall recharges the underlying aquifer. The only practically way of reducing the rate of increase the nitrate concentration, and eventually reducing the actual concentration in the ground water, is to reduce the intensity of agricultural activity which produce the heavy nitrogenous loading. There is plenty of evidence that nitrate levels in many ground waters have increased greatly over recent time, in some cases to the extent that water abstractions from boreholes have to be curtailed or even abandoned. Further, computer models indicate that this position will worsen unless remedial action is taken.

6. WASTEWATER TREATMENT The present system of wastewater disposal worldwide is based on the provision and operation of public sewerage reticulations and sewage treatment plants in urban areas, and the private provision of drains and wastewater treatment arrangements at industrial premises and stock rearing farms. Much of the industrial wastewater produced in urban areas is disposed of, with or without pre- treatment, into position applies in many countries but in others, particularly developing ones industrial wastewater disposal direct to rivers is the norm. Wastewaters produced on farms are usually too strong in the load of organic matter they carry for disposal to public sewerage systems. Instead, efforts are made to contain the farm wastes on the producing farm where the fertilizer value of the wastes can be realized.5

6.1 TREATMENT PROCESS USED IN PURIFICATION OF PUBLIC WATER SUPPLY Process Purpose

1. Raw water storage (short term) Sedimentation, balancing of intake water quality, raw water reserve. 2. Raw water storage (long term) Oxidation of organic matter, partial removal of NO3,HCO3,PO4,SiO2 by algal uptake 3. Chemical precipitation using Al2(SO4)3 or Coagulations, flocculation and settlement of SiO2 or Fe salts plus polyelectrolytes turbidity and colour 4. Microstraning Straining through very fine-mesh rotary screens 5. Rapid filtration Rapid up or down-flow filtration through sand 6. Chlorination and/or ozonation or UV Disinfection, Removal of Ca and Mg hardness softening by lime, lime-soda 7. Activated carbon treatment by powder Reduction in residual organic matter addition before filtration or passage through active carbon filters

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6.2 MAIN SEWAGE TREATMENT PROCESSES Process Type of treatment Basic purpose 1. Preliminary Screening and grit removal Removal of gross of abrasive solids 2. Primary settlement Settlement in tanks Removal of solid and grease 3. Secondary treatment Activated slug percolating Bio-oxidatio,carbonaceous filter other bio-reactor plus matter and ammonia and settlement Removal of solids 4. Polishing treatment Sand filtration Removal of very fine solids microstrainning 5. Tertiary treatment Chemical precipitation Removal of N, P and organic residuals 6. Sludge treatment Digestion thickening CH4 production, preparation dewatering drying for disposal

7. RESULT AND DISCUSSION The primary requirement regarding the quality of public water supplied is the public health requirement that the water should be ‘wholesome’ for drinking. Wholesome in this context is generally interpreted as ‘promoting or conductive to health’. Quality of water intended for Human Consumption came into effect in 1985. Given that a public water supply complies with requirements for drinking water, it can be taken as axiomatic that the water is suitable for agricultural purposes. However public water supplies are not sufficient pure for many industrial purposes. For example, in steam raising, in the chemical and pharmaceutical industries, and in the health care and electronics industries. Each industry is expected to take its own steps to produce, from the available public supply, the precise level of purity of water is requires for its particular purpose.

7.1 NORMAL CHEMICAL CHARACTERISTICS OF PUBLIC WATER SUPPLIES IN ppm FROM DIFFERENT SOURCES Chemical Chalk borehole Upland river Lowland river Lowland characteristics (softened) reservoir 1. pH value 7.3 7.3 7.9 7.6 2. Colour(units) 0 2 5 4 3. NH3(N) 0 0.10 0.02 0.03 4. NO3(N) 3.0 1.2 4.0 1.7 5.Total solids 220 80 410 430 6. Total 150 45 260 225 hardness 7. Non-CO3 10 25 135 70 hardness 8. Alkalinity 110 20 130 160 9. Chloride 16 15 35 55 10. Iron 0.01 0 0.02 0 11. Manganese < 0.01 < 0.01 < 0.01 < 0.01

The physical characteristics of public supply are measured in terms of its colour, turbidity, odour, taste and pH value. The chemical characteristics cover a wide and complex field, embracing low concentrations of the acutely toxic inorganic chemicals, very low concentrations of the hazardous organic micro-pollutants such as polycyclic aromatic hydrocarbons, halo forms as produced in the chlorination of organic residuals in water, pesticides. Mercury and cadmium. Also, specific inorganic chemicals such as fluoride, nitrate, magnesium and sulphur which at particular levels of concentrations are or may be harmful to health. Furthermore, the parameters of water quality which are specified as limitations of the acceptability of water for particular use purposes. Such

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Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Chemistry specifications usually appear as ‘quality standards’, ‘quality criteria’ or ‘quality guidelines’ promulgated by international bodies and individual nations.

8. REFERENCES

1. The greenhouse Effect, Climate Change and Ecosystems,’(SCOPE 29), ed. Bolin, B.R. Doos, J. Jager and R.A. Warwick, John Wilet and Sons, Chichester, 1986. 2. V. Ramanathan, R.J. Cicerone, H.B. Singh and J.T. Kiehl, j. Geophysics. Res. 1985, 90, D3, 5547. 3. Department of the Environment, ‘Digest of Environment Protection and Water Statistics’, No. 13, HMSO, London, 1990. 4. G.W. Brummer, ‘The Importance of Chemical Speciation in Environmental Processes’, Springer Verlag, Berlin, 1986. 5. F.A.M. de Hann, ‘Scientific Basis for Soil Protection in Europe’, ed. H. Barth and P.L. Hermite, Elsevier, Amsterdam, 1987, p.211

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A SEASONAL STUDY OF PHYSICO –CHEMICAL ANALYSIS OF DRINKING WATER OF VARIOUS AREA GUJARAT, INDIA. Piyushkumar J. Ribadiya1, Divyaba B. Jadeja2, Sapna H. Mahera3, Dr.D.K.Bhoi 4* 1,2,4*Department of Chemistry, J & J. College of Science, Nadiad-387 001, Gujarat, India. 3ARIBAS, New Vallabh Vidyanagar-388 120, Gujarat, India. *4Email: [email protected], 1Email: [email protected] ABSTRACT: A seasonal study of Physico-chemical analysis such as temperature, pH, dissolved oxygen, total dissolved solids, chloride, total alkalinity, calcium and magnesium hardness, sulphate, phosphate, nitrate of bore wells water. The origin of water on the earth is not clear. When rain falls on the earth, two forces action it. One is the earth’s gravity, which pulls the water downward through any open path. The second force is the electrical attraction between water and the other materials to which water tends to stick. The significance of chemical analysis depends to a large extent on the sampling programme an ideal sample should be one which is both valid and representative. These conditions are met by collection of samples thought a process of random selection. The other body some Which it is collected and the sample shares the sample physico -chemical characteristic with the sampled at the time and site of sampling. The recreant factors, for any sampling program are, frequency of sample collection, total number of samples, size of each sample, sites of sample connection, method of sample collection, data to be collected with each sample and transportation and care of samples prior to analysis. Water quality characteristics of aruatic environments arise form a multitude of physical, chemical and biological interactions. Variation in values of parameters with distance in water sample was also studied. Our present investigation shows that some of the above mention parameters are within the permissible limit of WHO standards. So, its good indicator for drinking water. Physico-chemical analysis was carried out form fifteen sampling satations of various area, Gujarat during February 2017-July2017in order to assess water quality index. Keywords: Study, physico-chemical analysis, bore wells drinking water, Various area, Gujarat. INTRODUCTION In continuation of our earlier analysis on bore wells drinking water, here we report the physico- chemical analysis of bore wells drinking water of various areas, Gujarat. (D.K.Bhoi, A.K.Rana). Bore wells water is generally used for drinking and other domestic purpose in this area. The use of fertilizers, pesticides, manure, lime, septic tank, refuse dump, etc are the main sources of bore wells water pollution. (D.K.Bhoi, F.J.Thakor). In the absence of fresh water supply, people residing in this area use bore wells water for their domestic and drinking consumption. In order to assess water quality index, we have carried out the physico –chemical analysis of bore wells drinking water.

EXPERIMENTAL In the present study bore wells water sample form fifteen areas located in Gujarat. were collected in brown glass bottles with necessary precautions. All the chemicals used were of AR grade. Double distilled water was used for the preparation of reagents and solutions. The major water was used for the preparation of reagents and solutions. The major water quality parameters considered for the examination in this study are temperature, pH, dissolved oxygen (DO), total dissolved solid (TDS), total alkalinity, calcium and magnesium hardness, sulphate, phosphate and nitrate contents. (A.I.Vogel, D.G. Miller) Temperature, pH, DO, TDS, phosphate, nitrate values were measured by water analysis kit and manual methods. Calcium and magnesium hardness of water was estimated by complex metric titration methods. Chloride contents were determined volumetrically by silver nitrate titrimetric method using potassium chromate as indicator and was calculated in terms of mg/L. sulphate contents were determined by volumetric method. (P.A. Hamilton, D.K. Helsel)

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RESULTS AND DISCUSSION The physico-chemical data of the bore wells water samples collected in February 2017 and July 2017 are presented in Tables 1,2,3 and 4 respectively.The results of the samples vary with different collecting places because of the different nature of soil contamination.(D.K. Bhoi, H.R.Dabhi, A.K. Rana) Temperature: In the present study temperature ranged from 26 to 33ºC. (Table-1) pH: In the present study pH ranged from 7.2 to8.52. The tolerance pH limit is 6.5 – 8.5. The sample station No.7 shows higher pH than the prescribed range. (Table-1) TDS: In the present study TDS ranged from 240to1653 mg/L. According to WHO and Indian standards, TDS values should be less than 500 mg/L for drinking water. The sample station nos.4,6,7,8,9,13 and 14 higher ranged as prescribed by WHO and Indian standards. (Table-1) D.O.: The D.O ranged from 5 to 9.8 mg/L in present samples. The minimum tolerance range is 4.0 mg/L for drinking water. (Table-2) Total Alkalinity: The total alkalinity content in the samples is in between 46 to 132 mg/L.(Table-2) EC: In the present study EC ranged from 0.1 to 1.2. (Table-2) Chloride: In the present study chloride ranged from 7.1 to 26.01 mg/L, while the tolerance range for chloride is 200-1000 mg/L. (Table-3) Calcium Hardness: The calcium hardness ranged from 16.98 to 50.60 mg//L. The tolerance range for Ca-hardness is 75-200mg/L. (Table-3) Magnesium Hardness: Magnesium ranged from 14.70 to 98.30 mg/L. The tolerance range for Mg hardness is 50-100mg/L. (Table-3) Sulphate: Sulphate ranged from 57.64 to 430.47 mg/L. The tolerance range for sulphate is 200-400 mg/L. (Table-4) Phosphate: Phosphate ranged from 20 to 128 mg/L. The evaluated values of phosphate in the present study are higher than the prescribed values. The higher values of phosphate are mainly due to the use of fertilizers and pesticides by the people residing in this area. If phosphate is consumed in excess, phosphine gas is produced in gastro – intestinal tract on reaction with gastric juice. (Table-4) Nitrate: Nitrate ranged from 90 to 451 mg/L. The tolerance rang for nitrate is 20-45 mg/L. Nitrate nitrogen is one of the major constituents of organism along with carbon and hydrogen as amino acids proteins and organic compounds in the bore wells water. If the nitrate reduces to nitrite, then it causes methaemoglobinaemin in infants and also diarrhea. (Table-4)

ACKNOWLEDGEMENT The authors are thankful to the UGC for financial assistance in the form of Minor Research Project of Dr. D.K. Bhoi [F NO. 47-259/2001 (WRO) Dt. 01/11/2001]. The authors are also thankful to the Nadiad Education Society, Nadiad and the Principal, J. College of Science, Nadiad for providing necessary facilities.

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TABLE-1 ANALYSIS OR RESULT OF THE SAMPLES COLLECTED IN FEBRUARY 2017 AND JULY 2017

Temp TDS pH (ºC) (mg/L) Sample No. Station Feb July Feb July Feb July 2017 2017 2017 2017 2017 2017

1 J.&J. Boy’s Hostel 26 29 8.17 8.19 400 405

Vadodara 2 26 30 8.01 8.05 240 251 (station)

3 Bhumel 28 31 7.56 7.60 480 495

4 Visnoli 27 32 8.08 8.20 520 522

5 Borsad 27 32 7.72 7.81 320 318

Chokhandi 6 27 31 8.08 8.12 760 782 (Vadodara)

7 Segva 29 32 8.44 8.52 880 878

8 Maninagar 29 30 8.14 8.30 1080 1091

9 Satisana 28 31 7.61 7.81 640 638

10 Alindra 28 32 7.88 7.92 280 292

11 Limbadiya 28 33 7.90 7.99 280 290

12 Kumbharvadi 27 32 7.96 8.00 240 235

13 Virpur 26 31 7.74 7.84 1640 1653

14 Bakor 27 32 7.24 7.32 550 570

15 Vadagam 29 33 7.43 7.55 330 341

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TABLE-2 ANALYSIS OR RESULT OF THE SAMPLES COLLECTED IN FEBRUARY 2017 AND JULY 2017

Total DO Alkalinity EC (mg/L) (mg/L) Sample No. Station Feb July Feb July Feb July 2017 2017 2017 2017 2017 2017

1 J.&J. Boy’s Hostel 8 9.6 45 36 0.4 0.5

Vadodara 2 7 7.2 45 42 0.5 0.8 (station)

3 Bhumel 8 8.4 90 85 0.9 0.7

4 Visnoli 6 6.5 90 86 1.1 1.0

5 Borsad 8 8.2 107 100 1.2 1.1

Chokhandi 6 5 5.4 143 132 0.4 0.3 (Vadodara)

7 Segva 6.7 7.1 35 30 0.5 0.7

8 Maninagar 8.2 8.4 35 29 0.3 0.4

9 Satisana 8 8.5 70 65 0.1 0.2

10 Alindra 8.7 9.0 130 124 0.9 1.0

11 Limbadiya 5 5.6 79 75 0.7 0.6

12 Kumbharvadi 5.6 7.0 87 89 0.6 0.5

13 Virpur 7 7.5 102 104 0.7 0.6

14 Bakor 7.6 8.1 70 72 0.4 0.5

15 Vadagam 9 9.2 105 110 0.2 0.4

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TABLE-3 ANALYSIS OR RESULT OF THE SAMPLES COLLECTED IN FEBRUARY 2017 AND JULY 2017

Chloride (Ca-Hardness) (Mg-Hardness) (mg/L) (mg/L) (mg/L) Sample No. Station Feb July Feb July Feb July 2017 2017 2017 2017 2017 2017

1 J.&J. Boy’s Hostel 8.52 8.60 20.84 18.20 29.16 31.01

Vadodara 2 8.52 8.69 30.46 28.60 31.10 32.05 (station)

3 Bhumel 8.52 8.70 30.46 29.50 43.75 44.12

4 Visnoli 14.2 13.90 20.04 19.32 44.71 45.80

5 Borsad 12.74 12.90 19.23 18.40 34.02 35.08

Chokhandi 6 8.51 8.60 27.25 26.26 31.10 32.10 (Vadodara)

7 Segva 9.94 9.90 27.25 27.30 37.90 38.18

8 Maninagar 7.1 7.32 25.65 25.00 23.32 25.50

9 Satisana 21.3 22.4 28.85 29.00 97.20 98.30

10 Alindra 9.95 10.01 24.04 23.05 24.30 25.52

11 Limbadiya 18.35 17.50 54.51 50.60 14.70 16.80

12 Kumbharvadi 12.90 13.00 36.87 32.50 21.70 20.71

13 Virpur 19.88 20.00 44.88 45.00 75.90 74.80

14 Bakor 25.56 26.01 17.63 16.98 33.04 34.10

15 Vadagam 25.56 25.70 22.44 21.05 83.59 85.61

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TABLE-4 ANALYSIS OR RESULT OF THE SAMPLES COLLECTED IN FEBRUARY 2017 AND JULY 2017

Sulphate Phosphate Nitrate Sample (mg/L) (mg/L) (mg/L) No. Station Feb July Feb July Feb July 2017 2017 2017 2017 2017 2017 J.&J. Boy’s 1 19.21 20.10 20 21 176 150 Hostel Vadodara 2 115.29 110.18 32 34 98 90 (station)

3 Bhumel 165.29 160.22 53 52 240 242

4 Visnoli 172.93 178.93 71 75 245 241

5 Borsad 176.77 177.80 62 65 230 228

Chokhandi 6 57.64 58.02 24 25 140 135 (Vadodara)

7 Segva 103.76 104.86 28 27 320 322

8 Maninagar 149.87 150.00 53 54 232 230

9 Satisana 65.33 64.30 120 128 345 342

10 Alindra 230.58 231.12 64 68 247 240

11 Limbadiya 126.81 127.82 46 43 325 320

12 Kumbharvadi 230.47 221.60 64 65 310 315

13 Virpur 268.62 270.11 85 86 420 418

14 Bakor 430.17 425.18 35 39 340 319

15 Vadagam 222.52 223.50 55 60 467 457

REFERENCES 1. A.K. Rana, M.J. Kharodawala, J.M. Patal, R.K. Rai, B.S. Patel and H.R. Dabhi, Asian J. Chem., 14, 1209 (2002). 2. D.K. Bhoi, A.K. Rana, H.R.Dabhi, Orient J Chem., 24(3), 807 (2008). 3. A.K. Rana, M.J. Kharodawala, H.R. Dabhi, D.M. Suthar, D.N. Dave, B.S Patel and R.K. Rai, Asian J. Chem., 14, 1178 (2002). www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 15 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Chemistry

4. D.K. Bhoi, A.K. Rana, H.R.Dabhi, Asian J Chem. 21, (2009) 5. P.A. Hamilton and D.K. Helsel, Ground water, 33, 2 (1995). 6. D.K. Bhoi, Y. M. Mehata, D.S. RajM.B. Chauhan, M.T. Machhar, Asian J Chem.17(1), 1275 (2003). 7. E. Brown, M.W. Skovgested and M.J. Fishman, Method for Collection and Analysis of Water Samples for Dissolved Minerals and Gases, Vol. 5 (1974). 8. D.K. Bhoi, D.S. Raj, Y. M. Mehata, M.T. Machhar, M.B. Chauhan, Orient J Chem.20(2), 361-364 (2003). 9. N. Manivasagam, Physico-Chemical Examination of Water, Sewage and Industrial Effluents, Pragati Prakashan, Meerut (1984). 10. D.K. Bhoi, F.J. Thakor, A.A. Chary. Aqua. Biology. 25(1) 41-45, (2010) 11. A.I. Vogel, Text Book of Quantitative. Inorganic Analysis, 4th Edn, ELBS, London (1978). 12. D.K. Bhoi, F.J. Thakor, H.K. Dabhi, Nakitarai B. Chauhan, S.N. Pandya Current World Environment Vol 6(2), 225-231 (2011). 13. APHA: American Public Health Association, Standard Methods for Examination of Water and Wastewater, 16th Edn, APHA-WPCF-AWWA, Washington (1985). 14. D.K. Bhoi, F.J. Thakor, Chirag A. Acharya, Janki Vaidya, J.R. PrajapatiJ. Aqua. Biology.vol 25(1) 41-42 (2010) 15. International Standard for Drinking Water, 3rd End, WHO, Geneva (1971). 16. The Gazette of India: Extraordinary, Part-II, 3, 11 (1991). 17. A.J. Dhembare, G.M. Pondhe and C.R. Singh, Pool. Res., 17, 87 (1998). 18. J.E. Mekee and H.W. Wolf, Water Quality Criteria. The Resources Agency of California State Water Quality Control Board (1978). 19. APSFSL, Andhra Pradesh State Forensic Science Laboratories, Annual Report (1988). 20. D.G. Miller, Nitrate in Drinking Water, Water Research Centre, Medmenham (1981). 21. NEERI: National Environment Engineering Research Institute, Disinfection of Small Community Water SuSpplies, Nagpur (1972). 22. J.W, White, J. Agri, Food Chem., 23, 886 (1975). 23. D.K. Bhoi, F.J. Thakor, Y.D. Parmar, A.B. Parmar, M.D. Chauhan Asian Journal of Environment Science Vol 6(1) 37-41 (2011). 24. D.K. Bhoi, A.K. Rana, F.J. Thakor, S.N. Pandya Current World Environment Vol 8(1), 153- 156 (2013).

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SYNTHESIS AND THERAPEUTIC EVALUATION OF SOME NEW QUINOLINE DERIVATIVES OF 4-CYCLOPROPYL-5,6,7,8 SUBSTITUTED-1, 2-DIHYDRO QUINOLINE-2-ONES. Vikram R. Dangar1* and Viral R. Shah. Department of Chemistry, Kamani Science college, Amreli-365601, Gujarat, India. Corresponding author Email: [email protected] ABSTRACT: The Quinoline nucleus found in several natural compounds (cinchona alkaloids) and pharmacologically active substances displaying a broad range of biological activity. Like other heterocyclic compounds, quinolines with different functional groups exhibit wide range of applications in the field of pharmaceutical and bioorganic and bioorganometallic processes. Quinoline derivatives like some new 4-cyclopropyl-5,6,7,8 substituted-1,2-dihydro quinoline-2-ones derivatives of type (2a-l) have been prepared by the cyclocondensation of N-Aryl-3-cyclopropyl-3- oxo-propanamide derivatives of type (1a-l) with the help of concentrated sulphuric acid. All the prepared compounds were characterized by their spectral (I.R., N.M.R., Mass) data and screened for their antimicrobial activities. Key words: Knorr synthesis, Quinoline derivatives, Antimicrobial activities.

INTRODUCTION Quinoline derivatives have attracted considerable attention as they appeared of interest to possess antihypertensive1 , bacteriocidal2-4, analgesic5-6 & antidepressant7, herbicidal8-9, anticonvulsant10, antitumor11-12, antidiabetic13, antiulcer14, and anti-inflammatory15, anticancer 16, antimalarial17 activities. With an intention of preparing agent with better therapeutic potency the quinoline derivatives have been prepared by the cyclocondensation of N-Aryl-3-cyclopropyl-3-oxo- propanamide derivatives with the help of concentrated sulphuric acid. The structure of synthesized compounds was assigned based on Elemental analysis, I.R. 1H- NMR and Mass spectral data. The antimicrobial activity was assayed by using the cup-plate agar diffusion method 18 by measuring the zone of inhibition in mm. All the compounds were screened in vitro for their antimicrobial activities19-20 against varieties of bacterial strains such Staphylococcus aureus, Bacillus subtilis, Escherichia coli, P. aeruginosa and fungi Aspergillus niger at 40 μg concentration. Standard drugs like Ampicillin, Benzyl penicillin, Ciprofloxacin, Erythromycin and Griseofulvin were used for comparison purpose (Table-1).

EXPERIMENTAL SECTION Melting points were taken in open capillary tubes are uncorrected. IR spectra (cm-1) were recorded on SHIMADZU FTIR 8400 Spectrophotometer; Frequency range: 4000-400 cm-1 (KBr disc) and, 1H-NMR spectra on GEMINI-2000 spectrometer (300MHz) using TMS as an internal standard, chemical shift in δ ppm. General procedure for the preparation of N-Aryl-3-cyclopropyl-3-oxo-propanamide (1a-l) : A mixture of Methyl-3- cyclopropyl-3-oxo-propanoate (1.42 gm, 0.01 mol) and p- chloroaniline (1.27 gm, 0.01 mol) in toluene was refluxed for 12 hr. Methanol was removed using Dean & Stark azeotropic assembly. The mixture was cooled to room temperature and washed the reaction mixture with dil. HCl and than washed with water. Distilled out toluene under vacuumed.

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The resulting residue was crystallized into the n-Hexane. Yield 80 %, m. p. 1100 C, TLC System: Ethyl acetate: Hexane (2 : 3). General procedure for the preparation of 4-Cyclopropyl-5,6,7,8 substituted-1,2-dihydro quinoline-2-ones (2a-l): A mixture of 3-Cyclopropyl-N-(p-methylphenyl)-3-oxopropanamide (2.33 gm, 0.01 mol) and 5ml concentrate sulphuric acid was stirred for 5.0 hrs. at 50O C. The reaction mass pours on to crushed ice. Adjust pH of mixture with liq. ammonia. The resulting solid mass separated was filtered and 0 crystallized from dioxane. Yield 39 %, m.p.165 C Anal. Calcd. for C13H13NO Calcd: C,78.36; H,6.58; N, 7.03%, Found: C,78.50; H,6.72; N,7.25%. TLC System: Ethyl acetate : Hexane (2 : 3).Similarly, other 4-cyclopropyl-5,6,7,8 substituted-1,2-dihydro quinoline-2-ones were prepared. The physical data are recorded in Table No.1. 4-Cyclopropyl-5,6,7,8 substituted-1,2-dihydro quinoline-2-ones (2a-l): 0 Yield 39 %, m.p.165 C; IR(KBr): Alkane C-H str. (asym.) 2960, -CH3 C-H str. (sym.)2889,

C-H def.(asym.) 1458, -C-CH2 def. (sym.) 1361, Aromatic =C-H str. 3062, C=C str. 1610, 1419, C- H i.p.(def) 1245,1022, C-H o.o.p.(def). 825, Quinoline C=N str. 1560 , N-H str. 3523, Carboxamide -1 1 C=O str. 1647, N=H str. 3191 cm ; H-NMR (CDCl3) : δ 1.80- 2.44, (m, 4H, HC-(CH2)2 , 2.79-2.84

(m, 1H, (H2C)2-CH, 3.78-3.82 ( s, 3H, Ar-CH3 ) , 6.36 -7.56 ( m, 4H, Ar-H), 11.61 (s, 1H, N-H).

Mass m/z 199. M.F.: C13H13NO. Scheme-1

Reaction Scheme

O O

O 1 O R CH Con. H2SO4 2 3 Toluene R O NH + 4 R NH2 -~ 3 4 R N O R 4 H 1 3 R R R 1 3 2 (2a-l) R R R 1 2 3 2 (1a-l) R ,R , R , R R4,=CH ,OCH ,Cl,Br, * 3 3 NO ,F 2

Table-1

- i I I I Characterization data of the compounds (2a-l) - I 1 2 3 4 I l I I com R R R R Molecular Mole.W M.P. Nitrogen % - pd Formula t. (0C) Calcd Found no. 2a H H H H 7.56 7.78 C12H11NO 185 150 ' 2b Cl H H H 6.38 6.56 C12H10NOCl 219.5 200 - 2c H Cl H H 6.38 6.56 C12H10NOCl 219.5 250 - 2d Cl Cl H H C H NOCl 254 180 5.51 5.69 12 9 2 - 2e H F H H 10.51 10.71 C21H19N3O2ClF 399.5 112 - www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 18 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Chemistry

2f CH3 H H H C13H13NO 119 145 7.03 7.26

2g H CH3 H H C13H13NO 119 165 7.03 7.25

2h H OCH3 H H C13H13NO2 215 185 6.51 6.72

2i NO2 H H H C12H10N2O3 230 210 12.17 12.40

2j H NO2 H H C12H10N2O3 230 225 12.17 12.40

Table-2 Compd. Antibacterial Activity Antifungal (zone of inhibition in mm.) Activity No. B. subtilis S. aureus Aero P.aeruginosa A.niger genes 2a 14 12 16 10 15 2b 14 17 14 15 17 2c 16 14 18 19 15 2d 18 16 15 13 20 2e 17 20 14 17 18 2f 13 17 19 16 14 2g 14 14 17 18 16 2h 17 16 12 12 15 2i 19 18 13 14 17 2j 12 16 17 14 13 Amoxicillin 25 25 20 22 0 Benzyl penicillin 18 19 21 21 0 Ciprofloxacin 20 15 22 16 0 Erythromycin 22 21 19 23 0 Griseofulvin 0 0 0 0 26

Table-2-Chart: Antimicrobial activity: (zone of inhibition in mm) :

30 ~ Compd. 25 +--~---~0:::------..:,::....-- No. -&- ~ 2a 20 2b :12~~1tr~;--,:rr~-- : 2c 15 ~ 2d 2e 10 2f ~ 2g 2h 5 +------+-- ...--- _._2i -~ 2j 0 +--~ --...,..--g,-....,.-....,;;1P=:::1~.--~Gt--""l"""--tJ-----, ~ Amoxicillin 1 2 3 4 5 Benzyl penicillin -5 ~------~ Ciprofloxacin - ANTIBACTERIAL ACTIVITY It has been observed from the microbiological data that all compounds (2a-l) were found to be mild to moderately active against Gram positive and Gram-negative bacterial strains. However, www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 19 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Chemistry the maximum activity was observed in compounds (2d), (2i) against B. subtilis. The significant activity was observed in compounds (2e), (2i) S.aureus against. The maximum activity was displayed by the compounds (2c), (2f), against Aerogenes. The compounds (2c), and (2g) were comparatively more effective against P. aeruginosa. ANTIFUNGAL ACTIVITY The antifungal data revealed that compounds were least toxic to the fungal strain. However mild activity was shown by the compounds (2d), (2e) against A.niger. The antibacterial activity was compared with standard drug viz. Ampicillin, Benzyl penicillin, Ciprofloxacin, Erythromycin and antifungal activity was compared with standard drug viz. Griseofulvin.

RESULTS AND DISCUSSION Quinoline derivatives like some new 4-cyclopropyl-5,6,7,8 substituted-1,2-dihydro quinoline-2-ones derivatives of type (2a-l) have been prepared by the cyclocondensation of N-Aryl- 3-cyclopropyl-3-oxo-propanamide derivatives of type (1a-l) with the help of concentrated sulphuric acid. The formulas of the selected compounds were confirmed by the elemental analysis and their structures were determined by IR ,1 H-NMR, and mass spectral data.

CONCLUSION The present study leads to a convenient synthetic method for the synthesis of new compounds. Which show significant antibacterial and antifungal activity. Further investigation with appropriate structural modification of the above compounds may result in therapeutically useful products.

ACKNOWLEDGMENT The authors are thankful to authorities of Kamani Science College, Amreli for providing research facilities and we are also thankful to Department of Chemistry Saurashtra University Rajkot for I.R., N.M.R., Mass spectral & elemental analysis.

REFERENCES 1. Archibald, Ward, John, Terence James, White Janet Christine; Chem. Abstr., 107, 236532d (1987). 2. Renezelti A. and Villani F.; Bely. BE 902, 586 (Cl. CO7D) Chem. Abstr., 104, 2248338h (1986). 3. Vurbanova S., Chervenkov S.; Chem. Abstr., 107, 77601g (1987). 4. Shirahata Akira; Nakamura Toshiyo; Chem. Abstr., 104, 84642n (1986). 5. Negl N., Montsh, Kidwani M.; Chem. Abstr., 126,343537y (1997). 6. Renault, Christian, Mestre Michael; Eur. Pat. Appl. EP 155, 888 (Cl. C07 D 401/06); Chem.Abstr., 104, 68759a (1986). 7. Gujarati Vibha R., Sathi Garima, Sharma Manju nath Krishna P.; Chem. Abstr., 99, 175562f (1984). 8. Plath Peter, Meyer Norbert, Eichenauer Ulrich, Helmut Hage; Ger. Offen., DE 3, 593, 811 (Cl. CO7D 215/56); Chem. Abstr., 108, 94413k (1988). 9. Henry Lee, Strong; Chem. Abstr., 126, 17876b (1997). 10. Simand, Jacaues, Boigergrain, Robert Keane, Peter, Eugene, Verniver, Jean Claude; Fr. Demande FR 2 593, 811 (Cl. CO7D 215/56): Chem. Abstr., 108, 94413k (1988). 11. Atwal Graham J., Baguley Bruce C., Denny William A.; J. Med. Chem., 31 (5), 1048-1052 (1988); Chem. Abstr., 108,167271z (1988). 12. Sanders, Robert G., Kiline, Kimberly, Hurley, Laurence; PCT.Int. Appl. WO 01 58, 889 (Cl. www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 20 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Chemistry

CO7D 311/20); Chem. Abstr., 135,166941p (2001). 13. Uragami, Sadaji Oda, Akinori.; Chem. Abstr., 58, 133161n (1995). 14. Uchida Minoru, Komatsu Makoto, Moritu Seiji, Nakagawa Kazuyuki; JPN. Kokai Tokyo Koho JP 60, 142, 959 (Cl. CO7D 251/22); Chem. Abstr.,104, 88454a (1986). 15. Jinbo, Susuma, Kohno, Shoichi Kashima, Koichi, Suzuki, Yasuo, Kazuo,Mochida E.; Can. CA 1 189, 078 (Cl. CO7D 215/42) Chem. Abstr., 104, 68761v (1986). 16. Kreft, Antony, Kees Kenneth Lewis, Musser John Henry, Bicksker James Jacob; Chem. Abstr., 107, 96605c (1987). 17. Ghorab M. M., Nassan A. Y.; Phosphorus & Sulfur Relat. Elem. 134/35, 57-76 (Eng) (1998); Chem. Abstr., 133, 58762t (2000). 18. A. L. Barry, The Antimicrobial Susceptibility Test: Principle and Practices, Edited by Llluslea & Febiger, (Philadelphia), USA, 180; Biol. Abstr., 64, 25183 (1977). 19. R. S. Lodhi and S. D. Shrivastava, Indian J. Chem., 36B, 997 (1997). 20. G. S. Gadaginamath, A. S. Shyadligeri and R. R. Kavali, Indian J. Chem., 38B, 156-159 (1999).

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Section 2

PHYSICS & ELECTRONICS

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ACTIVATED CARBON FROM RENEWABLE BIOMASS FOR SUPERCAPACITOR – A REVIEW Parth Joshi, Desai Riddhi and Chirag Patel Department of Physics, Uka Tarsadia University, Bardoli, Gujarat-394350 Corresponding author: [email protected] ABSTRACT: It is important to find the way for the ever-increasing demand of energy supply and also the maintain environmental stability. Supercapacitors have been emerged as an important energy storage device. To attain higher capacitances supercapacitor uses electrodes having high specific surface area. With these properties it makes them have power densities greater than those of batteries and energy density greater than those of conventional capacitors. Since supercapacitor is mainly a pulse current device it is best used with devices that require high current for short duration of time. The electrode is the main part of the supercapacitors, so the factor of electrode is most important. Activated carbon which is the common material for commercial Supercapacitors electrodes, it can derived from the sugarcane baggase, rise husk, neem leaf, wood, coconut shell etc. The textural property of activated carbon depends on the method of preparation and starting materials. From the articles activated carbon electrode which is derived from renewable biomass using chemical activation with KOH, K2CO3, ZnCl2, NaOH etc. The performance of supercapacitors is based on the surface area and the development of the mesopores. In this review the surface modification on activated carbon properties is also discussed. The paper provides an overview of supercapacitors, its importance and processes of making supercapacitors using activated carbon. Keywords: Supercapacitors, activated carbon, production of supercapacitors. 1. INTRODUCTION High energy cost, continuous depletion of fossil fuel reserves and climate change due to excessive greenhouse gases lead to the society turning towards renewable and sustainable energy sources. As a result, there has been an increase in the production of green and clean energy from sources such as solar and wind and also the development of low CO2 emission electric vehicles/hybrid electric vehicles. A major requirement in harnessing the intermittent electricity being generated from these renewable sources is an efficient energy storage system. Batteries and electrochemical capacitors are the leading energy storage devices, but both have their short comings. Though, electrochemical capacitors can be charged–discharged in a matter of seconds but they have lower energy densities than batteries, however, much higher power densities[1] can be achieved within the same period than batteries. Thus, supercapacitors lying between electrochemical batteries and conventional capacitors are seeing as a promising replacement for batteries especially in the areas of load leveling and electrical energy storage devices; and also for applications that required maximum power, long cycle life, operational stability, fast charge–discharge time, low level of heating, appropriate dimension/weight and low cost[1,2]. Generally the mechanisms of energy storage in supercapacitors involve two modes: the charge accumulation in the double-layer formed at the electrolyte interface and a pseudo-capacitive contribution related to faradic reaction[3]. Nowadays carbon are the most studies electrode materials for supercapacitors, due to their high specific surface area, good electrical conductivity and low cost[4,6]. The high accessible surface area of carbon is applicable for more charge being stored at the electrolyte interfaces. The supercapacitors capacitance of activated carbon depends on the relative sizes of pores. In addition, researchers are developing pseudocapacitors in which activated carbon serves as the support backbone for pseudocapacitive materials[7−11]. Currently petroleum-derived coke, pitch, and coal are the common precursors for production of commercial activated carbon[12−14]. The decreasing availability of fossil-based carbon compounds has driven the industry to search for sustainable resources to synthesize activated carbon. For example, activated carbon has been produced from renewable biomass precursors such as cellulose, corn grains, sugarcane bagasse, and others[15,16]. Activated carbon is a form of carbon process to have small, low volume pores that increase the surface area available for adsorption or chemical reactions. Activated carbon is a predominantly amorphous solid carbon material with highly developed internal surface area, porous structure and high degree of surface reactivity. Common forms of activated carbon are beads, granules, pellets, fibers, cloths and powder. All activated carbons contain micropores, mesopores and macropores within their structures but the relative proportions vary considerably according to the raw material. Macropores having average pore diameter more than 50 nm, Mesopores have pore diameter 2-50 nm, Micropores having average pore diameter less than 2nm. Activated carbons is very much dependent on the precursor, the oxidizing agent employed, the temperature of activation and the degree of activation. Depending on these factors, activated carbons with moderate to high porosity can be achieved, as well as with varying surface chemistry (i.e. amount and type of www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 22 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Physics & Electronics oxygen groups). As a general trend, the higher the activation temperature/activation time, the larger the porosity development. However, higher porosity developments are usually accompanied by a broadening of the pore size distribution (PSD). These characteristics have widened the usefulness of activated carbons to more demanding applications, such as catalysis/electrocatalysis, energy storage in supercapacitors and Li-ion batteries, CO2 capture or H2 storage.

2. EXPERIMENTAL CONDITION 2.1. Preparation of activated carbon The preparation and production of activated carbon usually consist of carbonization and activation that can be done either separately in two-stage process or combined in a single stage process. The carbonization of a precursor takes place at a low temperature usually between 400°C and 850°C and it is done in the absence of oxygen. Activation is a process of converting carbonaceous materials into activated carbon by thermal decomposition in a furnace (convectional heating). Physical activation, chemical activation, combined chemical and physical (Physiochemical) activation are the methods being employed for the preparation of activated carbon. Table 1 Activation methods for preparation of activated carbon.

Activation method Activating Lignocellulosic materials References agent

Chemical KOH Bamboo species, distillers dried grains with [2,17,18,20,2 soluble, waste tea-leaves, oil palm empty 3,24,26,27– fruit bunches, argan seed shells, sunﬂower 33] seed shell, coffee endocarp, cassava peel waste, soybean oil cake ZnCl2 waste coffee beans, sugarcane bagasse, waste camellia oleifera [19,22,25,32– 34] NaOH Apricot shell [21]

K2CO3 Cocoa pod husk, straw pulping [31,33] Physical Steam, Corncob, bagasse bottom ash, macadamia [35] CO2 nut-shell, rice husk ﬂy ash, sawdust ﬂy ash Combined chemical and KOH/CO2 Oil palm empty fruit bunches, cassava peel [2,20] physical waste sugarcane baggase (Physiochemical)

2.2 Physical activation Physical activation is a process by which the carbonized product develops porous structure of molecular dimensions and extended surface area on heat treatment in the temperature range of 800-1000°C in presence of suitable oxidizing gases such as steam, CO2 or air [38, 45-46]. The most common method is the steam activation process, which is accomplished in two stages. The material is first carbonized to an intermediate product. Cleaning the clogged pore structure to produce a more accessible internal surface area is then achieved by chemically reacting the carbonized product with steam at a temperature between 800°C and 1000°C. The use of steam for activation can be applied virtually to all raw materials. A variety of methods have been developed and all have the same basic principle of initial carbonization at 500-600°C followed by activation with steam at 800-1100°C. Since the overall reaction of converting carbon to carbon dioxide is exothermic, it is possible to utilize this energy and have a self-sustaining process. 2.3 Chemical activation Chemical activation is achieved by degradation or dehydration of cellulosic raw material. The popular activating agents are phosphoric acid [37,39], zinc chloride [36] and sulfuric acid [40]. Activating agents, e.g. potassium hydroxide [41-42], calcium chloride, manganese chloride and sodium hydroxide [43-44], all of which are dehydrating agents have also been used. The precursor and reagent are mixed into a paste, dried and carbonized in a rotary furnace at 600°C. When phosphoric acid is the activating agent the carbonized product is further heated at 800-1000°C during which stage the carbonaceous materials are oxidized by the acid. The acid is largely recovered after the activation stage and converted back into the correct strength for reuse. The activated product is washed with water and dried. Activity can be controlled by altering the proportion of raw

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 23 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Physics & Electronics material to activating agent. By increasing the concentration of the activating agent, the activity increases although control of furnace temperature and residence time is also very important parameters. Generally, chemical activation is carried out with wood as the starting material. In the beginning, wood is impregnated with a concentrated solution of activating agents. It results in degradation of cellulosic material. Chemical-impregnated material is then pyrolyzed between 400°C and 600°C in the absence of air. Pyrolyzed product is cooled and washed to remove activating agent, which is recycled. On calcination, impregnated and chemically dehydrated raw material results in charring and aromatization, and creation of porous structure. All activating agents are dehydrating agents which influence the pyrolytic decomposition and inhibit the formation of tar. They also decrease the formation of acetic acid, methanol etc. and enhance the yield of carbon. 2.4 Surface modification Texture and surface chemistry are twin factors that determine the electrochemical performance of carbon materials. The nature and concentration of surface functional group may be modified using appropriate thermal and chemical post-treatments [47]. Modification of surface functional groups of carbon materials usually leads to changes in the double-layer properties such as wettability, capacitance, electrical conductivity, point of zero charge and self-discharge characteristics of the carbon [48].

3. COMPARISON OF CHARACTERIZATION 3.1. Physiochemical characterization Physiochemical characterization of carbons is done to provide clues as to the commercial Estimation of the electrochemical properties of activated carbon is usually done using cyclic voltammetry (CV), galvanostatic charge/ discharge (GCD) and electrochemical impedance spectroscopy (EIS). Viability of the experimental carbons [52]. Characterization of activated carbons by N2 and CO2 sorption analyses are normally carried out to gain understanding of the inﬂuence of production conditions on the pore structure development in carbons [22]. The classiﬁcation of pores in porous materials into micropores (< 2 nm), mesopores (2–50 nm) and macropores (450 nm) [53]. Each of these pore structure has their adsorption purpose; while microporous structure of activated carbon is usually desired for adsorption, the presence of mesopores in activated carbons are helpful in the adsorption of large molecules and where faster adsorption rate is required [51,54]. In order to compare the coffee ground carbon (CGC) produced with Maxsorb—a reference material commonly use in supercapacitor research [55], Rufford et al. [19] have calculated the pore size distribution in CGC and Maxsorb from CO2 at 273 K and N2 at 77 K adsorption isotherms. Rufford and his team found out that CGC displays a Type I isotherm characteristic of a microporous material just like commercial Maxsorb but has a greater ratio of narrow micropores (< 1 nm) to total pore volume than Maxsorb despite having less total pore volume than Maxsorb. 3.2. Electrochemical characterization While cyclic voltammetry measurements are commonly used to test the EDLC performances of the activated carbons, the galvanostatic charge/discharge measurements are conducted to test the capacitors performance [23]. The equivalent series resistance (ESR) of the activated carbons are commonly obtained from the electrochemical impedance spectroscopy measurements. Specific capacitance of porous carbons reported in the surveyed literature is presented in Table 2.

Table 2 Specific capacitance of porous carbons reported in literature

Sr. Materials Specific capacitance (F g-1) References No 1 Sugarcane bagasse 138 49 2 Rice husk 194 50 3 Waste coffee beans 368 19 4 Waste tea leave 330 30 Because different experimental methods were used by different authors to determine the specific capacitance, the values reported are sometimes not consistent. Furthermore, the calculation of specific capacitance also differs depending on the cell used (3 or 2 electrodes). Cheng et al. [56] prepared hierarchical porous carbons from chestnut shell by ZnCl2 activation. On employing as electrodes for supercapacitors containing 6M KOH solutions, specific capacitance of 105 Fg-1 at a current density of 0.1 Ag-1 and 92.0 Fg-1 at current density up to 10Ag-1 was exhibited by the porous carbon prepared at 700˚C. The authors suggested that the electrocapacitive performance of the porous carbon-based supercapacitor could be attributed to the surface area, pore structure, surface functional groups and the graphitic degree of the carbons. Rufford et al. [31] conducted preparation of activated carbons by ZnCl2 activation of sugarcane bagasse. Specific capacitance www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 24 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Physics & Electronics of 300 Fg-1 and specific energy of 10Whkg-1 was observed. When the sugarcane bagasse carbon was used in a two-electrode, sandwich type supercapacitor it containing 1M H2SO4 electrolyte. The result obtained clearly demonstrates the benefit of mesopores to double layer capacitance at fast charge/discharge rate; between 77% and 83% of the initial capacitance were retained after 5000 cycles at current density above 2 Ag-1. A combined chemical (KOH) and physical (CO2) activation process was adopted by Farma et al. [2] in preparing highly porous binderless activated carbon electrode from oil palm empty fruit bunches. The authors noted that in addition to producing porous carbon with well developed pore structure and good electrochemical performance the combined approach also has economic advantage since a small quantity of chemical agent is required resulting in reduced activation time. At optimum activation time of 3h, the values of specific capacitance, specific energy density and specific power density obtained from the electrochemical measurements of the supercapacitor cells fabricated using these electrodes are 150 Fg-1, 4.297 Whkg-1 and 173 Wkg-1, respectively.

3.3. Effects of surface modification on capacitive performance of active carbons Jin et al. [28] utilized biochar generated from the pyrolysis of distillers dried grains with solubles (DDGS) to prepared hierarchical carbon having high specific surface area of 2959 m2 g-1 and high pore volume 3 -1 of 1.65 cm g by KOH catalytic activation. The hierarchical carbon was further treated with 4M HNO3 at 150 1°C. A pronounced increase in oxygen content in the hierarchical carbon was noticed by the authors. When the hierarchical carbon was used as supercapacitor electrode in 6M KOH electrolyte and evaluated after 2000 constant current charge/discharge cycles, it showed a stable reversible capacitance of 260 Fg-1 at a constant current of 0.6 Ag-1 compared to specific capacitance of 200Fg-1 obtained with non-treated hierarchical carbon at a higher current of 0.5 Ag-1 after 2000 cycles.

4. CONCLUSION The variations in the performance of renewable biomass-based activated carbons produced under the same conditions but from different precursors have brought into for the influences of the preparation procedures and conditions as well as the effects of the structure and composition of the renewable biomass precursors. Capacitive performances of the electrodes have been found to depend not only on surface area of the activated carbons but also on other factors such as the pore structure and distribution, electrical conductivity and surface functionalities. In addition, ultra small micropores present in the low surface area active carbons have been found to contribute immensely to the capacitance observed in those carbons. Overall, electrochemical performance of the energy cells was also found to be dependent on the electrolyte system used. Currently, enormous research efforts are being channeled towards the development of low cost active carbon electrodes that will give high capacitance at low resistance with the ultimate goal of enhancing the specific energy of the carbon-based supercapacitors without compromising its high level specific power.

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39) I. D. Harry, B. Saha and I. W. Cumming, Carbon, 45, 766, 2007. 40) Yoon S-H, Korai Y, Mochida I., Carbon fibers and active carbon fibers. In: Marsh H, Rodrı´guez- Reinoso F, editors. Science of carbon materials, Alicante, Spain: University of Alicante;. p. 287–325, [chapter 8], 2000. www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 26 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Physics & Electronics

41) C. L. Burket, R. Rajagopalan, A. P. marencic, K. Dronvajjala and H. C. Foley, Carbon, 44, 2957, 2006. 42) Centeno TA, Fuertes AB., J. Membr. Sci., 160, 201, 1999. 43) Viboon Sricharoenchaikul , Chiravoot Pechyen, Duangdao Aht-ong, and Duangduen Atong, Preparation and Characterization of Activated Carbon from the Pyrolysis of Physic Nut Waste,2007 44) Ekpete O.A. and Horsfall M., Preparation and Characterization of Activated Carbon derived from Fluted Pumpkin Stem Waste (Telfairia occidentalis Hook F),2011. 45) A. Babu rajendran, G. Manivannan, k. Jothivenkatachalam and S.karthikeyan, Characterization Studies of activated carbon from low cost agricultural waste,2015. 46) Gamzenur ozsgn, production and characterization of activated carbon from pishtachio-nut shell, 2011. 47) Figueiredo JL, Pereira MFR, Freitas MMA, Orfao JJM. Modiﬁcation of the surface chemistry of activated carbons. Carbon;37:1379–89, 1999. 48) Pandolfo AG, Hollenkamp AF. Carbon properties and their role in supercapacitors. J Power Sources;157:11–27, 2006. 49) Si WJ, Wu XZ, Xing W, Zhou J, Zhuo SP. Bagasse-based nanoporous carbon for supercapacitor application. J Inorg Mater;26:107–12, 2011. 50) He X, Ling P, Qiu J, Yu M, Zhang X, Yu C, et al. Efﬁcient preparation of biomassbased mesoporous carbons for supercapacitors with both high energy density and high power density. J Power Sources;240:109–13, 2013. 51) Liu Q-S, Zheng T, Wang P, Guo L. Preparation and characterization of activated carbon from bamboo by microwave-induced phosphoric acid activation. Ind Crops Prod;31:233–8, 2010. 52) Ng C, Losso JN, Marshall WE, Rao RM. Physical and chemical properties of selected agricultural byproduct-based activated carbons and their ability to adsorb geosmin. Bioresour Technol;84:177– 85, 2002. 53) Guo J, Lua AC. Preparation of activated carbons from oil-palm-stone chars by microwave-induced carbon dioxide activation, Carbon;38:1985–93, 2000. 54) Huang L, Sun Y, Wang W, Yue Q, Yang T. Comparative study on characterization of activated carbons prepared by microwave and conventional heating methods and application in removal of oxytetracycline (OTC). Chem Eng J,171:1446–53, 2011. 55) Raymundo-Piñero E, Leroux F, Béguin F. A high-performance carbon for supercapacitors obtained by carbonization of a seaweed biopolymer. Adv Mater,18:1877–82, 2006. 56) Cheng L, Guo P, Wang R, Ming L, Leng F, Li H, et al. Electrocapacitive properties of supercapacitors based on hierarchical porous carbons from chestnut shell. Colloids Surf A: Physicochem Eng Aspects,446:127–33, 2014.

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ELECTRON IMPACT SCATTERING STUDY WITH CF2 RADICAL 1Divya Ahir, 1Rujuta Patel, 1Swati Shirsagar, 1*Hardik Desai and 2Minaxi Vinodkumar 1Department of Physics, UkaTarasadia University, Bardoli, Surat,Gujarat, India – 394350. 2V.P. & R.P.T.P Science college,VallabhVidhyanagar, Gujarat -388120. *[email protected] ABSTRACT: The results of calculations of Total (elastic + inelastic) and elastic cross section for electron scattering from the CF2 radical are presented. The energy range of the present investigation was from threshold to 5000 eV. The calculations were carried out using the Spherical Complex Optical Potential. Total elastic cross sections calculated in present study are fairly in a good agreement with the other data available in the literature except at the intermediate energies where present data overestimates. Present Total Cross Sections (TCS) data are in fairly good agreement with the lone results available in the literature. Keywords: SCOP

INTRODUCTION Molecular radicals play an important role in electron-driven processes, including radiation damage in tissues, gas discharges, low-temperature plasma etch environments and deposition technologies. In particular radicals of fluorocarbons play significant role in the etching process and evolution of plasma. It is now well established that concentration of CFx radicals has significant effect on the behavior of fluorocarbon plasmas [1, 2]. Additionally, fluorocarbon feedstock gases are frequently used as etching gases in many applications which finally lead to electron impact dissociation to the productions of CFx (x = 1-3) radicals that are responsible for several important chemical and physical processes in substrates. The bombardment of electrons on CF2 will result into fragments, about which very little is known. The experimental determination of electron collision cross sections for CF2 is difficult and hence the measured data is reported by only one group [3]. Francis et al. [3] used crossed-beam electron scattering experiment to measure e - CF2 differential cross sections at specific angles (20° – 135°) within the scattered electron range for incident energies 2 - 20 eV. Theoretical investigations of total elastic cross sections for e - CF2 scattering are reported by three groups [4 - 6]. Lee et al.[4] used Iterative Schwinger variational method and calculated elastic differential, integral, and momentum transfer cross sections as well as total absorption cross sections in the energy range 1 – 500 eV. Rozumet al. [5] Francis et al. [3] also reported calculated data for absolute differential cross sections and integral cross sections using Schwinger multichannel method for impact energies 2 - 20 eV. Antony et al. [6] reported total elastic and total inelastic cross sections using Spherical Complex Optical Potential (SCOP) for impact energies 50 - 2000 eV. Owing to the difficulties involved in experiments with CF2 radical, total ionization cross sections is reported experimentally by a lone group of Deutsch et al [7] and theoretical results are presented by three groups [6- 8]. Reviewing the literature it is quite clear that the work on e -CF2 is fragmentary, as most of the authors have focused their results over specific range of impact energies as well as for specific cross sections. Hence the present work will be able to fill this void and provide the complete study of several cross sections which will form an important database that can be employed for plasma modeling. The motive behind such study is twofold, one to study the resonance process which are more prominent at low impact energies below 10 eV and to compare the present results with available fragmentary data that are consistent and widely used in the specific range of impact energies. The computation for total cross section (elastic + inelastic) is carried out using quantum mechanical approach through Spherical Complex Optical Potential (SCOP) [9-10]. A detailed description of the theoretical methodologies employed presently is provided in the next section.

THEORETICAL METHODOLOGY We briefly discuss the theoretical methodology employed here for the calculation of thevarious cross sections. The details of the methodology can be obtained from our earlier publications [9-12]. As discussed in the earlier section that the scattering phenomenon ischaracterized quantitatively into two important processes namely elastic and inelastic. Theycombine to give the total scattering phenomenon. QT(Ei) = Qel(Ei) + Qinel(Ei) (1) Where (Qel) represents the elastic cross section and (Qinel) represents the total inelastic crosssections. The Spherical Complex Optical Potential SCOP formalism [11-12] to describe theelectron molecule scattering is represented by the interaction potential,

Vopt(Ei,r) = VR(Ei,r) + iVI(Ei,r) (2)

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 28 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Physics & Electronics where the real part VR(Ei,r), consist of static potential (Vst), exchange potential (Vex ), and polarization potential Vp and the complex part VI of the optical potential takes care of all the inelastic processes via absorption potential (Vabs ) which accounts for the total loss of fluxscattered into the allowed electronic excitation or ionization channels. It is to be noted that the SCOP by equation (2) are charge-density dependent. In the SCOP formalism [11-12], the complex optical potential is used to solve the Schrödinger equation numerically using the partial waveanalysis. Using these partial waves, the complex phase shifts aregenerated which are keyingredients to find the relevant cross sections. The phase shifts contain all the informationregarding the scattering events.Total inelastic cross sections are not directly measurable quantities and hence do not providesa direct comparison with experiment. But it is obtained by subtracting the elastic crosssection from the total cross sections. Though it is difficult to measure, but it contains animportant quantity which is directly measurable such that, VR(r,Ei) = Vst(r) + Vex(r,Ei) + Vp(r,Ei) (3) where, Ei is the incident energy. Eq.3 corresponds to various real potentials to account for the electron target interactionnamely, static, exchange and the polarization potentials, respectively. These potentials are obtained using the targetgeometry, molecular charge density of the target, the ionization potential and polarizability as inputs. The molecular chargedensity is derived from the atomic charge density by expanding it at the center of mass of the system. The molecular chargedensity so obtained is normalized to account for the total number of electrons present in the target. The atomic chargedensities and static potentials (Vst) are formulated from the parameterized Hartree–Fock wave functions given by Cox and Bonham.[14]The parameter free Hara’s ‘free electron gas exchange model[ 15] is used for the inclusion of exchange potential (Vex).The exchange potential takes care of exchange of scatteringelectron with one of the target electrons. The polarization potential (Vp) is formulated from the parameter-free model ofcorrelation–polarization potential given by Zhang et al. [16] Here, various multipole non-adiabatic corrections are incorporated inthe intermediate region which will approach the correctasymptotic form at large ‘r’ smoothly. The target parameterssuch as ionization potential (I) and dipole polarizability (훼휊) ofthe target used here are the best available from literature.[13]The imaginary part in Vopt, called the absorption potential Vabs accounts for the total loss of flux from the incident channel,scattered into the allowed electronic excitation or ionizationchannels. The expression used here is vibrationally and rotationally elastic. This is due to the fact that the non-sphericalterms do not contribute much to the total potential at thepresent high energy range. The well-known quasi-free model of Staszewska et al.[17] isemployed for the absorption part, given by,

푻푰푶푪 ퟖ흅 2 2 Vabs(r,Ei) = - ρ (r)√ ( ) θ (P – KF - 2Δ ) (A1 + A 2+ A3) (4) ퟐ ퟏퟎ푲 푬 푭ퟑ 풊 where TIOC is the local kinetic energy of the incident electronwhich is given by, TIOC =E- Vst+ Vex + Vp (5) 2 2 ퟏ/ퟑ Here P = 2Ei and KF = [3흅 ρ(r)] is the Fermi wave vector and A1, A2 and A3 are dynamic functions that depend differently on휃(퓍), I, Δ and Ei. These parameters are explicitly given in our earlier publication and references therein [18] and hence not repeated here. Here, I is theionization threshold of the target, 휃(퓍) is the Heaviside unitstep-function and Δ is an energy parameter below which Vabs=0. Hence, Δ is the principal factor which decides the values oftotal inelastic cross section, since below this value ionization orexcitation is not allowed. This is one of the main characteristics of the Staszewska model [17].This has been modified by us byconsidering Δ as a slowly varying function of Ei around I. Suchan approximation is meaningful since Δfixed at I would notallow excitation at energies Ei≤ I. However, if Δ is much lessthan the ionization threshold, then Vabs becomes unexpectedly high near the peak position. The amendment introduced is togive a reasonable minimum value 0.8I to Δ [19] and also to express the parameter as a function of Ei around I, i.e.,

Δ(Ei) = 0.8I + β(Ei – I) (6) here the parameter b is obtained by requiring that Δ= I (eV) at Ei=Ep, the value of incident energy at which present Qinel reaches its peak. Ep can be found by calculating Qinel by keeping Δ= I. Bey ond Ep, Δ is kept constant and is equal to I.The complex potential thus formulated is used to solve the Schr¨odinger equation numerically through partial wave analysis.This calculation will produce unique complex phase shift for each partial wave which carries the signature of interaction of theincoming projectile with the target. The phase shifts Δl thusobtained are employed to find the relevant cross sections, total elastic (Qel) and the total inelastic cross sections (Qinel) using the scattering matrix Sl(k) =exp(2i훿l).[20-21] Then the TCS (QT) isobtained by adding these two cross sections.[20].

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RESULTS AND DISCUSSIONS Figure 1 shows electron interaction with CF2 radical and report data on total cross section (elastic+inelastic) and total elastic cross section with available comparison for impact energies from threshold of the target to 5000 eV. Below 50 eV there is no total cross section data available in the literature to compare with. Above 50 eV present data finds over all very good agreement with the lone data reported by Antony et. al. [6]. At low energies from ionization threshold to 20 eV our elastic cross section are falling within the error bar reported by Yoon et. al. [22]. However, sharp dip at 18 eV reported by Yoon et al and by Maddern et. al. [23] is not seen in our data. This must be due to additivity rule that we used to calculate the scattering cross section. Beyond 20 eV our data are little over estimating compare to the data presented by other authors [3,6,22-23]. Beyond 80 eV present elastic cross sections are in good agreement with theoretical data reported by Francis et. al. [3] The over estimations in elastic cross section data at lower energy can be overcome by taking the screening corrections in to consideration.

40 Present Q - T

- I - I ■ Present Qel • Qel Yoon et. al.

) 30

2 Qel maddern et. al. (Exp.)

Å * ( ---- Qel Maddern et. al. (SEP) Fransic staite et al .. ·:______Antony 20

10 Total cross section section cross Total

10 100 1000 Energy (eV)

Figure 1: Electron impact scattering cross section of CF2 radical Present QT, (Solid Line ), Present Qel (Solid Dash Dot ) ,QelYoon et. al. (Solid Circle - •) [22], Qel maddern et. al.(Exp) (Solid star – ★)[23], Qel Maddern et.al.(SEP) ( Short dash )[23], Fransic staite et.al.(Dash) [3] , Antony (Dot) [6].

ACKNOWLEDGEMENT Minaxi Vinodkumar acknowledges DST-SERB, New Delhi, for the major research project (EMR/2016/000470) for financial support under which part of this work is carried out.

REFERENCES 1) P. Chabert, H. Abada, J. -P. Booth, and M. A. Lieberman, J. Appl. Phys. 94, 76 (2003). 2) T. Nakano and H. Sugai, J. Phys. D Appl. Phys. 26, 1909 (1993). 3) J. R. Francis-Staite, T. M. Maddern, M. J. Brunger, S. J. Buckman, C. Winstead, V. McKoy, M. A. Bolorizadeh, and H. Cho, Phys. Rev. A 79, 052705 (2009). 4) M. -T. Lee, I. Iga, L. E. Machado, L. M. Brescansin, E. A. y Castro, and G. L. C. de Souza, Phys. Rev. A 74, 052716 (2006). 5) I. Rozum, P. Lima˜o-Vieira, S. Eden, J. Tennyson and N. J. Mason, J. Phys. Chem. Ref. Data, 35, 267, (2006). 6) B. K. Antony, K. N. Joshipura, and N. J. Mason, J. Phys. B: At. Mol. Opt. Phys. 38, 189 (2005). 7) H. Deutsch, T. D. Meark, V. Tarnovsky, K. Becker, C. Cornelissen, L. Cespiva and V. Bonacic- Koutecky, Int. J. Mass Spectrom. Ion Process.137, 77, (1994)

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8) Y.-K. Kim and K. K. Irikura, Proceedings of the 2nd International Conference on Atom Molecular Data and their Applications, edited by K. Barrington and K. L. Bell, AIP Conf. Proc. No. 543(AIP, New York, 2000) p. 220 9) M. Vinodkumar, H. Desai, and P. C. Vinodkumar, RSC Adv. 5, 24564 (2015). 10) M. Vinodkumar, C. Limbachiya, H. Desai, and P. C. Vinodkumar, J. Appl. Phys. 116, 124702 (2014). 11) H Bhutadia, AChaudhari, M Vinodkumar, Mol. Phys., 2015, 113, 3654. 12) M Swadia, Y Thakar, M Vinodkumar, C Limbachiya, Euro. Phys. J. D 2017, 71,85. 13) Handbook of Chemistry and Physics, ed. D. R. Lide, CRC, BocaRaton, FL, 87th edn, 2007. 14) H. L. Cox and R. A. Bonham, J. Chem. Phys., 1967, 47, 2599. 15) S. Hara, J. Phys. Soc. Jpn., 1967, 22, 710. 16) X. Zhang, J. Sun and Y. Liu, J. Phys. B: At., Mol. Opt. Phys.,1992, 25, 1893. 17) G. Staszewska, D. W. Schewenke, D. Thirumalai andD. G. Truhlar, Phys. Rev. A: At., Mol., Opt. Phys., 1983, 28,2740. 18) M. Vinodkumar, C. Limbachiya, H. Desai andP. C. Vinodkumar, Phys. Rev. A: At., Mol., Opt. Phys., 2014,89, 062715. 19) M. Vinodkumar, K. Korot and P. C. Vinodkumar, Int. J. MassSpectrom., 2011, 305, 26. 20) C. J. Joachain, Quantum Collision Theory, Amsterdam, North-Holland, 1983. 21) M. Vinodkumar and M. Barot, J. Chem. Phys., 2012, 137, 074311. 22) J.S. Yoon J. Phys. Chem. Ref. Data, Vol. 39, No. 3, 2010 23) T. M. Maddern PRL 100, 063202 (2008).

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ELECTRON IMPACT IONIZATION CROSS SECTIONS OF HE AND NE ATOM Malhar Bhatt, Ankit Tandel and Hardik Desai* Department of Physics, Uka Tarasadia University, Bardoli, Surat,Gujarat, India – 394350 *Email: [email protected] ABSTRACT: Electron impact Scattering cross section of He atom is important in application of atmospheric physics, astrophysics, plasma physics and quantum electronics and Ne atom is important in many areas such as the study of novae atmospheres, lighting technology, laser system and plasma processing. In the present study we have reported the calculations of the electron impact total ionization cross sections (Qion) of the He and Ne Atom. Calculations have been carried out for electron impact energies ranging from threshold of the target to 5000 eV. For the present study we have employed the well-known Spherical Complex Optical Potential (SCOP) formalism [1,2], which provides the total inelastic cross-section (Qinel). Qinel is a sum of total ionization cross sections (Qion) and total excitation cross section (Qexc) for any energy of the electron impact beyond the ionization threshold of the target. On those bases we have developed a semi-empirical method, called Complex Scattering Potential-ionization contribution (CSP-ic) method [3] to extract Qion from calculated Qinel. Present calculations also provide data on the Qexc of these targets. The calculated cross-sections are examined as a function of incident electron energy. Present results are compared with the theoretical and experimental data available in the literature and found fairly good agreement. Detailed results will be presented at the time of conference. Keywords: Spherical complex optical potential (SCOP), Complex scattering Potential Ionization Contribution (CSP-ic), Ionization Cross section

INTRODUCTION Electron-impact ionization is one of the most fundamental processes in atomic collisions and a very active field, both theoretically and experimentally. Electron-impact ionization of He is important processes in atomic physics that have been investigated intensively for many years. From a theoretical point of view, He is an ideal candidate for testing different theoretical models developed for complex many-electron atoms because it represents the simplest closed-shell structure. The ionization process of He is not affected by inner-shell effects. Contributions of double ionization and autoionization from doubly excited states are only small. Moreover, from an experimental point of view, He is of considerable interest due to the need for an accurate and reliable cross-section database of He ionization cross sections required in many fields of applied research to model plasmas, controlled thermonuclear fusion and atmospheres[4]. The Ionization of He atom is measured by different method, R G Montague et al (1984) have measured by crossed electron-fast atom beam technique[5], A fast-neutral-beam method using by Robert C.Wetzel et al (1987)[6] and Ionization of Ne atom is measured using different method by Nagy et al (1980)[7],Rapp et al (1965)[8],Rejoub et al (2002)[9],Schram et al (1965)[10]. We used SCOP method to measured ionization crosss section of He atom; however, present Complex Optical Potential-ionization contribution (CSP-ic) method uses a dynamic energy dependant ratio to extract Qion from Qinel, which gives a better representation of the target and thereby producing improved results.

THEORETICAL METHEDOLOGY Total ionization cross section (Qion) upon electron impact for the He and Ne Atom are calculated using SCOP formalism. The present theory is based on a quantum mechanical calculation solved by the method of partial waves, giving complex phase shifts as the output. The phase shift thus obtained is the key ingredients and they carry the signature of the interaction of the incoming projectile and the target. These phase shifts are further used to evaluate the total elastic cross sections (Qel) and its inelastic counterpart (Qinel) [11] such that,l

()()()+= EQEQEQ (1) iT el i iinel

However, our objective in this study is restricted to the calculation of the Qion for He and Ne atom, since this cross section is central to many applications related to plasma and other technologies mentioned in the introduction. Ionization is the predominant scattering process in the inelastic channel for incident electron energy ranging from ionization threshold to about 5keV. Hence, we have limited our calculation to this energy where channels that lead to discrete as well as continuum transitions in the target are open.

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We represent the electron-atom system by a complex potential comprising of real and imaginary potentials as,

V(r, Ei) = VR (r, Ei) + i VI (r, Ei) (2) such that

VrE(,)()(,)(,)= Vr + VrE + VrE (3) R i st ex i p i where, Ei is the incident energy. The three terms on the RHS of the eqn 3 are various real potentials arising from the electron target interaction namely, static, exchange and the polarization potentials respectively. The basic input for evaluating all these model potentials is the electronic charge density of the target. For atoms the charge density is derived from the parameterized Hartree-Fock wave functions given by Bunge and Barrientos [12]. The imaginary part in Vopt, also called the absorption potential Vabs, accounts for the total loss of scattered flux into all the allowed electronic channels of excitation and ionization. Here we have neglected the non spherical terms arising from the vibrational and rotational excitation in the full expansion of the optical potential. This is due to the fact that anisotropic contribution from vibrational excitation will be significant at very low impact energies while it will be negligible at the energies of present interest.

Finally, for the absorption potential we have employed a well-known quasi-free model form of Staszeweska et al [13] given by,

T 8 22 V(,)() r E= − r loc (p− k − 2  )  ( A + A + A ) (4) abs i 2 10kE3 F 1 2 3 F-F i

The local kinetic energy of the incident electron is

TEVV= −() + (5) loc i st ex

The parameters A1, A2 and A3 are defined as 풌ퟑ ퟐ 푨 = ퟓ 풇 , 푨 = 풌ퟑ(ퟓ풑ퟐ − ퟑ풌ퟐ)⁄(풑ퟐ − ퟑ풌ퟐ) 푎푛푑 ퟏ ퟐ∆ ퟐ 풇 풇 풇 ퟓ ퟐ ퟐ ퟐ ퟐ ퟐ ퟐ ퟐ 푨ퟑ = ퟐ휽(ퟐ풌풇 + ퟐ∆ − 풑 )(ퟐ풌풇 + ퟐ∆ − 풑 )ퟐ⁄(풑 − 풌풇) (6)

The absorption potential is not sensitive to long range potentials like Vpol. Hence it is not included in the 2 2 1/3 representation of Tloc as shown in Eq. (5). In Eq. (6), p = 2Ei, kF = [3 ( r ) ] is the Fermi wave vector and  is an energy parameter. Further ( x ) is the Heaviside unit step-function, such that = 1 for x

≥ 0, and is zero otherwise. The dynamic functions A1, A2 and A3 occurring in the equation 4 depend differently on ρ(r), I, and Ei as evident from eqn. 6. The energy parameter determines a threshold below which Vabs = 0, and the ionization or excitation is prevented energetically. In fact is the governing factor which decides the values of total inelastic cross section and that is one of the characteristics of Staszewska model [13]. The original model of Staszewska et al [13] has been modified by us by considering as a slowly varying function of Ei around I. Briefly, a preliminary calculation for Qinel is done with a fixed value =I . From this the value of incident energy at which our Qinel reaches its peak, named as Ep is obtained. This is meaningful since fixed at I would not allow excitation at incident energies EIi  . On the other hand, if the parameter is much less than the ionization threshold, then Vabs becomes unexpectedly high near the peak position. The modification introduced in our paper has been to assign a reasonable minimum value 0.8I to and express this parameter as a function of Ei around I as follows.

EIEI =0.8 + − (7) ()()ii

Here the value of the parameter  is obtained by requiring that = I(eV) at Ei = Ep, beyond which is held constant equal to I. The expression for (Ei) is meaningful since fixed at I would not allow excitation at incident energy Ei ≤ I. On the other hand, if parameter is much less than the ionization threshold, then www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 33 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Physics & Electronics

Vabs becomes substantially high near the peak position. After generating the full complex potential given in equation 2 for a given electron - molecule system, we solve the Schrödinger equation numerically using partial wave analysis. At low energies only few partial waves are significant, e.g. at ionization threshold of the target around 5-6 partial waves are sufficient but as the incident energy increases more partial waves are needed. Using these partial waves the complex phase shifts are obtained which are employed to find the relevant cross sections vide Equation 1 [11].

The two fundamental ingredients of the total cross sections as discussed earlier are the total elastic and total inelastic cross sections, which takes care of all the processes that result from the interaction of the incoming projectile with the target. Out of these two, the total inelastic cross section is solely responsible for the loss of incident flux in the out going channel. The loss can further be accounted mainly by two very important processes which are termed as ionization and sum of all electronic excitations. These processes are individually measurable and are reported in the literature as total ionization cross sections and partial or total excitation cross sections. But the sum of these two cross sections i.e. the total inelastic cross section, Qinel, cannot be measured directly in experiments, but can be estimated by subtracting total integral elastic cross section from the measured grand total cross sections. The measurable quantity of applied interest is the total ionization cross section, Qion, which is contained in the Qinel which is rotationally and vibrationally elastic. The Qinel can be partitioned into discrete and continuum contributions, viz,

QEQEQE=+ (8) inel()()() i  exc i ion i where, the first term is the sum over total excitation cross sections for all accessible electronic transitions. The second term is the total cross section of all allowed ionization processes resulting in excitation to the continuum state induced by the incident electrons. The first term arises mainly from the low-lying dipole allowed transitions for which the cross section decreases rapidly at higher energies. The first term in equation 8, therefore becomes progressively smaller than the second at energies well above the ionization threshold. By definition,

 EQEQ (9) ()()iinel iion

In the present method Qion cannot be rigorously derived from Qinel but may be estimated by defining the energy dependent ratio of cross sections,

QE() RE() = ion i (10) i QE inel() i such that, 0 < R 1.

We are required to impose three conditions on this dynamic ratio which has well justified physical footings. The conditions are summarized in the mathematical form through equation 11.

R() Eii=0 for E I

==RP at Ei E P (11)

1 for EiP E where ‘Ep’ stands for the incident energy at which the calculated Qinel attains its maximum value. Rp is the value of R at Ei = Ep.

According to the first condition we require R=0 when Ei  I. This is an exact condition as there is no ionization process possible if the incident energy is less than or equal to ionization threshold of the target. On the other hand the third condition is also physically justified that at very high incident energy, the only dominant inelastic channel is the ionization as the electronic excitation channels are almost over. So the ratio turns out to be nearly approaching one. Now the second condition is empirical in the sense that for a number of stable atoms like He,Ne for which the experimental cross sections Qion [14-16] are known accurately, the contribution of Qion is found to be about 70–80% of the total inelastic cross sections Qinel. Here the upper bound is found only in rare case like that of Ne atom having an ionization potential of 21.56eV. For calculating the Qion from Qinel we need R as a continuous function of energy for Ei > I; hence we represent the ratio R in the following manner www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 34 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Physics & Electronics

()()i 1−= UfER (12)

Presently the above ratio has been determined using the following analytical form

 C2 ln()U  ()i 1 −= CER 1 +  (13)  + aU U  E where, U is the dimensionless variable defined by, U = i . I

We have adopted this particular functional form for f(U) in equation 13 due to its behavior with respect to the incident energy. As Ei increases above I, the ratio R increases and approaches unity, since the ionization contribution rises and the discrete excitation term in equation 8 decreases. The discrete excitation cross sections, dominated by dipole transitions, fall off as (lnU)/U at high energies. Accordingly the decrease of the function f(U) must also be proportional to ln(U)/U in the high range of energy. However, the two-term representation of f(U) given in equation 13 is more appropriate since the first term in the brackets ensures a better energy dependence at low and intermediate Ei. The dimensionless parameters C1, C2, and a, involved in equation 13 reflect the properties of the target under investigation. The three conditions stated in equation 11 are used to determine these three parameters and hence the ratio R. This is called the CSP-ic method. Having obtained Qion through CSP-ic method [3], the summed excitations cross sections  Qexc can be easily calculated vide equation 8.

RESULT AND DISCUSSION The theoretical approach of SCOP along with our CSP-ic method discussed above offers the determination of the total ionization cross sections, Qion along with a useful estimate on electronic excitations in terms of the summed cross section ∑Qexc.The results of He And Ne atom are compared with theoretical and experimental data. Fig,. 1 and Fig. 2 show that Present results are in good agreement with the available calculations and measurements.

- Present

) e - He 2 0.4 Montague Å

( Nagy Scharm Wetzel Sorokin 0.3

0.2

0.1 Total Ionization Cross sections sections Cross Ionization Total

0.0 10 100 1000 Energy(eV)

Figure 1: Electron impact Total Ionization cross sections of He atom.(Solid Line)Present Data, (Open Circle)R.G.Montague et al(1984), (Open Square)P.Nagy et al(1980),(Solid star)Schram et al(1965), (Open Diamond)R.C.Wetze et al(1984), (Open Triangle)A.A.Sorokin(2004).

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Present - Joshipura ) 0.8 e - Ne Krishnakumar 2

Å Nagy ( Rapp Rejoub Schram 0.6

0.4

0.2 Total Ionization Cross sections sections Cross Ionization Total 0.0 10 100 1000 Energy (eV)

Figure 2: Electron impact Total Ionization Cross Sections of Ne atom.(Solid line)Present Data,(Dash )K.N.Joshipura et al(2001), (Dot)E.Krishnakumar et al(1988), (Open Triangle) P.Nagy et al(1980),(Open Diamond)Rapp et al(1965),(Open Square)R.Rejoub et al(2002),(Solid Star) Schram et al(1965).

CONCLUSIONS • A calculation to obtain total ionization cross sections for He and Ne atom were carried out. We have employed the well-known SCOP and CSP-ic formalisms to perform these calculations. The results obtained are presented in the article and are compared with other available measurements and theories. • Comparing the two plots depicted in Fig.1 and Fig.2 revealed that with increase in target electron scattering probability increases leading to the increase in cross sections Unavailability of required data set, especially reliable measurements makes this study very imperative, since most of the previous studies are fragmented. • The results obtained for the atoms are in good agreement with the available data for low and intermediate energies, however at high energies present data overestimates the measurements. It’s worth notice that despite the underestimations it follows the same trend as followed by the measurements giving the hint of parametric improvement in Eq. (13)

REFRENCES [1] A. Jain and K. L. Baluja, Phys. Rev. A: At., Mol., Opt. Phys., 45 (1992) 202. [2] M. Vinodkumar, C. Limbachiya, H. Desai and P.C. Vinodkumar, RSC Advances, 5 (2015) 69466. [3] M. Vinodkumar, K. Korot, P.C. Vinodkumar, Int. J. Mass Spect. 305 (2011) 26. [4] A A Sorokin, I L Beigman, S V Bobashev, M Richter and L A Vainshtein, J. Phys. B: At. Mol. Opt. Phys. 37 (2004) 3215–3226. [5] R G Montague, M F A Harrison and A C H Smith, J. Phys. B: At. Mol. Phys. 17 (1984) 3295-3310. [6] Robert C. Wetzel, Frank A. Baiocchi, Todd R. Hayes, and Robert S. Freund,Phys. Rev. A 35,no.2(1987) [7] P Nagy, A Skutlartz and V Schmidt, J. Phys. B: Atom. Molec. Phys. 13 (1980) 1249-1267. [8] Rapp D and Englander-Golden P 1965 J. Chem. Phys. 43 1464-79 [9] R. Rejoub, B. G. Lindsay, and R. F. Stebbings, Phys. Rev. A, 65, 042713 [10] B. I. Schram, F. J. DE Heer, M. J. Van Der Wiel and J. Kistemaker(1965), Physica 31 94-112. [11] C.J. Joachain, Quantum Collision Theory, North-Holland, 1983 [12] C. F. Bunge, J. A. Barrientos, At. Data Nucl. Data Tables 53 (1993) 113. [13] G. Staszewska, D.W. Schwenke, D. Thirumalai, D.G. Truhlar, Phys. Rev. A 28 (1983) 2740. [14] R. Basner, M. Schmidt, V. Tarnovsky, K. Becker, Int. J. Mass Spectrom. Ion Proc. 171 (1997) 83. [15] Krishnakumar E and Srivastava S K 1988 J. Phys. B: At. Mol.Opt. Phys. 21 1055–82 [16]G.P. Karwasz, R.S. Brusa, A. Zecca, Riv. Nuovo Cimento 24(1) (2001) 1. [17] K.N. Joshipura, B.K. Antony,Phys. Lett. A, 289 (2001) 323–328.

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STUDY OF GROWTH, STRUCTURAL, OPTICAL, THERMAL AND MECHANICAL PROPERTIES OF Ba+2 DOPED KDP SINGLE CRYSTALS Haresh K. Patel1*, K.G.Raval1, Paras H. Trivedi2, J. P. Gandhi1, B. J. Lad1 1*Narmada College of Science & Commerce, Zadeshwar, Bharuch, Gujarat, India, 2 Shree Jayendrapuri Arts & Science College, Bharuch, Gujarat, India *Email: [email protected], Mobile: 09227177138 ABSTRACT: Single crystals of potassium dihydrogen phosphate (KDP) with doping of 0.1% and 0.2% concentration of Ba+2, were grown by slow evaporation solution growth technique at room temperature. The objective of the study is to find out the influence of Barium metal on the growth morphology, optical and thermal characteristics of the KDP single crystals. The unit cell dimensions and crystalline nature of the grown crystals were verified by X-ray diffraction technique. The presence of Barium was confirmed by EDAX. The optical nature of the grown crystals was analyzed using the UV-Vis spectra. The hardness of the crystals was determined by Vicker’s Micro hardness test. Thermo gravimetric analysis (TGA) shows the thermal stability of the grown crystals. The addition of Metallic impurity of Baruim enhances the quality, yield and growth rate of the KDP crystals. The optical quality, thermal and mechanical stability showed the suitability of the Barium doped KDP crystals for device applications. Key words: KDP, UV-Vis, Barium, XRD, TGA, EDAX

1. INTRODUCTION Potassium Dihydrogen Phosphate is a technologically important inorganic crystals and studies on KDP crystals attract interest because of their unique piezoelectric, anti ferro electric, electro optic, dielectric, acousto optical, phase matching, optical mixing and non linear optical (NLO) properties. Numerous applications of the NLO property have been discussed in the field of science and technology such as second, third and fourth harmonic generators for Nd:YAG, Nd:YLF lasers and for electro optical applications such as Q-switches, for Ti:Sapphire, Alexandrite lasers, as well as for acousto optical applications1-7. As a representative of the hydrogen bonded material, KDP matrixes readily accept both organic and inorganic dopants. Recent work has been done on the growth and characterization of pure and doped KDP crystals with an aim to improve and engineer the performance of various device based on the KDP crystals with series of organic dopants like ammonium malate, thiourea, L-lysine mono hydrochloride dyhydrate, L-alanine, L- arginine, ammonium acetate and bi-metallic impurities (Ni3+, Mg2+, Ba2+, Fe3+, Co2+ etc) 8-21 and their effect on various property of KDP crystals were studied and reported that the addition of impurities in KDP crystal has turned various properties of the material. It is also reported that the second and third harmonic generation efficiency of KDP crystals has been found to be better with doping. 22-23. The attempt is made to introduce Ba2+ as an impurity in the KDP crystal matrix with different concentrations. Pure and doped KDP crystals were grown and characterized structurally, optically, thermally and mechanically by using the suitable standard methods and reported results are discussed.

2. EXPERIMENTAL SECTION 2.1 CRYSTAL GROWTH Good quality and reasonable size of single crystal is pivotal for practical applications. In the existent work analytical reagent grade (AR) samples of KDP and BaCl2 along with deionised water as a solvent were used for the growth of single crystals24-25. The supersaturated solution of pure KDP and 0.1 mol% and 0.2 mol% BaCl2 as a doping agent with KDP was blended well to attain the homogenous solution. All three supersaturated solutions of 500 ml filtered and seed crystal of KDP was allowed to grow with slow evaporation technique at room temperature. After a period of three weeks, transparent colorless crystals of size 30 x 10 x 10 mm3 of pure KDP, 28 x 10 x 10 mm3 of 0.1 mol% Ba doped KDP and 48 x 10 x 12 mm3 of 0.2 mol% Ba doped KDP crystals were harvested. The photograph of all three grown crystals of pure and Ba doped KDP is shown in figure 1 below which depicts good transparency, well defined faces and the morphology of the grown crystals and also the growth rate is more along the crystallographic ‘a’ axis.

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PureKDP

Figure 1: Photographs of the Pure and Ba doped KDP crystals 2.2 CHARACTERIZATION XRD patterns were recorded by Philips Xpert MPD system. The data was analyzed using the powder X software. EDAX analysis of the grown crystals was done using Nova NanoSEM 450 to verify the functional groups present in the sample. Transparency of the crystals in the wavelength range 400-1100 nm was studied by using Perkin Elmer Lambda 19. The thermal analysis was carried out using the LINSEIC STA-PT 1600 TGA, DTA set up. Micro hardness of the grown crystals was measured using Vickers Micro Hardness tester with constant indentation period.

3. RESULTS AND DISCUSSION 3.1 EDAX ANALYSIS In order to confirm the presence of functional group, the samples of grown crystals were subjected to EDAX analysis using the Nova NanoSEM 450. The EDAX spectra for pure and 0.1mol% and 0.2mol% of Barium doped KDP crystals were recorded and analyzed. The figure 2 below confirms the presence of C, O, P and Ba in 0.1 mol% and 0.2 mol% Barium doped KDP crystalline lattice. The observed weight percentage of elements in the 0.1 mol% and 0.2 mol% Barium doped KDP crystals are given in the table 1 below, where the presence of Barium is quite low compared to oxygen, carbon, phosphorous and potassium in both the samples.

-ll, - Ill lO

!U Ill

m IU

Ul Ill

Ill Ill

!l 0 !l I! I! 0

u •1 ~o ( ~o ( ll I 111 , 0 lllt 0 ~It 0 co lD •o V ?~) 1l.O 1:1 11.0 l!.O ~ lD 10 lO lO ~o 1l.O IU 110 IU " Figure 2: 0.1 mol% and 0.2 mol% Ba doped KDP crystals

Table 1: “EDAX data” Sr. Element 0.1 mol% Co doped ADP 0.2 mol% Co doped ADP No. Weight % Atomic % Weight % Atomic % 1 C K 1.07 2.05 2.36 4.64 2 O K 42.58 61.16 37.20 54.90 3 P K 23.90 17.73 25.04 19.09 4 Cl K 0.47 0.30 0.35 0.23 5 K K 31.61 18.58 34.78 21.00 6 Ba L 0.20 0.03 0.14 0.02

3.2 X RAY DIFFRACTION ANALYSIS X- ray diffraction analysis of 0.1 mol% and 0.2 mol% Ba doped was carried out by Philips Xpert MPD system which confirms the single-phase nature of the samples and Bragg’s peaks were obtained at 2 θ angles indicating that crystals are in order.

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0.2mol% Ba doped KDP

O.lmol¾ Ba doped KDP

Pure KDP

10 20 30 -40 50 60 70 BO :28(deg rree)

Figure 3: XRD patterns for 0.1 mol% and 0.2 mol% Ba doped and pure KDP crystals

The ‘d’ spacing hkl values for prominent peaks in the spectrum were identified and compared with ICDD as shown in Figure 3 above. The XRD patterns of doped KDP had six prominent peaks at (101), (200), (112), (220), (301), (312). The cell parameters were found as shown in table 2 below which shows good agreement with the reported value. There is slight increase in cell parameter values due to doping and both samples belong to tetragonal system with I-42d space group.

Table 2: “Unit cell parameters” Sample Lattice Parameter Cell Volume α = β = γ (Å) (Å3) a = b c Pure KDP 7.454 6.973 387.0 90° 0.1 mol% doped KDP 7.413 6.954 380.0 90° 0.2 mol% doped KDP 7.394 6.936 377 90°

3.3 UV-VIS-NIR SPECTROSCOPY The UV-Vis-NIR spectra observed using Perkin Elmer Lambda 19 for all samples grown is shown in figure 4 below. 5 mm thick crystal wafers are used for the transmission study. The observed optical transmission intensity and cut-off wavelengths indicate that both samples exhibit good transmittance towards the visible and infra-red region and low cut-off wavelengths. This transparent nature of the grown samples is the desirable property to have NLO applications.

Uv-Vis-NIs Spectra

500 400 Pure KDP 300 Intensity 200 0.1 mol% Ba 100 doped 0 0.2 mol% Ba

Transmission 400 500 600 700 800 900100011001200 doped Wavelength (nm)

Figure 4: UV-Vis-NIS spectra for 0.1 mol% and 0.2 mol% Ba doped KDP crystals www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 39 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Physics & Electronics

3.4 THERMAL ANALYSIS Thermal analysis of the 0.1 % and 0.2 % Ba doped KDP crystals were recorded using the LINSEIC STA-PT 1600 TGA, DTA set up at a heating rate of 20 °C/min to determine the thermal stability of the crystals. The recorded curves shown in figure 5 below shows that there is no weight loss ~ 170 °C in both the samples, indicating no inclusion of water in the crystal lattice which is used as a solvent for crystallization. The decomposition of KDP and Ba starts at 170 °C and terminates at 300 °C and 210 °C and 300 °C respectively. It is observed that 0.2 % Ba doped crystals show slow weight loss percentage. The results confirm the increase in thermal stability of Ba doped KDP crystals as compared to pure KDP crystals.

" ::_-:_:i WP•9'(t..l ,~ 171t1V•

IS » a » Ttmper,u u~ (OfirttCtlsiuJ) Ttmptnturt (Dtj!r ec- Ct lsiu~) Figure 5: TGA curves of 0.1 % and 0.2 % Ba doped KDP crystals 3.5 MICROHARDNESS STUDY The good quality crystals are needed for various applications not only with good optical performance but with also good mechanical behavior. Hardness test is useful to find the mechanical hardness of the crystals and to estimate the threshold mechanical stress. Hardness of crystal is due to the resistance offered by a solid to the movement of dislocation, practically which is caused by scratching or indentation. Vicker’s hardness measurement of pure KDP and 0.1 % and 0.2 % Ba doped KDP crystals were noted by applying various load for an indentation time of 10 sec for each trial. The collected data is presented in table 3 below. Table 3: Microhardness value of doped KDP Load P 0.1 mol% Ba doped KDP 0.2 mol% Co doped ADP (gf) X (microns) Y (microns) Hardness HV X (microns) Y (microns) Hardness HV 10 12.8 11.2 129 11.9 11.2 139 25 19.8 17.8 131 19.2 17.1 141 50 24.3 23.8 160 23.1 22.5 178 100 33.2 32.9 170 32.1 31.9 181 200 51.2 50.8 143 51.2 49.2 154

190 180 170 160 150 140 0.1 mol% 130 0.2 mol% 120

Hardness (Vicker's) 110 100 0 100 200 300 Load (gf) Figure 6: Microhardness Results

Results show that between 50 to 100 gm load hardness value increases in both the samples. It also shows that the hardness value of 0.2 mol% Ba doped KDP crystals are higher than that of 0.1 mol% Ba doped KDP crystals for constant indentation time which can been seen from Figure 6 above. This is because of the addition of Barium ions into superficial crystal lattice and removing defect centers reduce the weak lattice stresses on the 2 surface. Vicker’s micro hardness number was determined using relation Hv = 1.854P/d .

4. CONCLUSION By employing slow evaporation solution growth technique good quality colorless and transparent single crystals of 0.1 mol% Ba doped and 0.2 mol% Ba doped Potassium Dihydrogen Phosphate were grown. EDAX confirms the presence of barium in the lattice of both the crystals. Single crystal XRD analysis reveals the www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 40 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Physics & Electronics tetragonal structure of KDP is slightly changed due to the addition of barium. The observed optical transmission intensity and cut-off wavelengths in UV-Vis-NIR analysis indicate that both samples exhibit good transmittance towards the visible and infra-red region and low cut-off wavelengths. The TGA analysis confirms the increase in thermal stability of Ba doped KDP crystals as compared to pure KDP crystals. The Vicker’s micro hardness study shows that addition of Barium ions into superficial crystal lattice and removing defect centers reduces the weak lattice stresses on the surface and increases the mechanical stability. This study may prove to be helpful to obtain high quality single crystals for various device applications.

5. REFRENCES 1. Marder SR, Tiemann BG, Perry JW et al. Materials for Non-linear optical chemical perspectives. American Chemical Society: Washington; 1991. 2. Muncheryan HM. Lasers and opto-electronics devices. Hemisphere Pub. Co: New York; 1991. 3. Shen YR. Principles of non-linear optics. Wiley: New York; 1984. 4. Meystrey P, Seargent M. Elements of Quantum Optics. Springe-Verlag: Berlin; 1991. 5. Sakai JI. Phase Conjucate optics. Mc Graw Hill Inc: New York; 1992. 6. Ramaswamy P, Raghavan PS. Crystal Growth Process and Methods. KRV Pub: Kumbakonam; 1999. 7. Ramaswamy P, Raghavan PS. Recent Trends in Crystal Growth. Pinsa 68: New Delhi; 2002. 8. Essentia AA, Mahadevan C. Bull of Material Science. 28.2005: 415. 9. Xu DL, Xue DF. J. of Cryst. Growth. 2006: 286; 108. 10. Rajesh NP, Kannan V, Raghavan PS, Ramaswamy P, Lan CW. Mater. Chem. and Phy. 2002: 76; 181. 11. Mullin JW, Amata AV. App. Chem. 1967: 17; 151. 12. Rajesh P, Ramaswamy P. Mater. Lett. 2009: 63; 2260. 13. Dhanraj PV, Bhagavanarayana G, Rajesh NP. Mat. Chem. Phys. 2008: 112; 490. 14. Rajesh P, Ramaswamy P, Bhagavanarayana G. J. Crystal Growth. 2009: 311; 4069. 15. Jairama A, Dharmaprakash SM. Indian J. of Pure and App. Phys. 2005: 43; 859. 16. Rajesh P, Ramaswamy P, Mahadevan CK. J. Cryst. Growth. 2009: 311; 1156. 17. Ferdousi A, Poddar J. J. Cryst. Process and Tech. 2011: 1; 18. 18. Muley GG, Rode MN, Waghuley SA, Pawar BH. Optoelectronics and Advan. Mater. 2010: 4; 11-14. 19. Rajesh P, Ramaswamy P, Boopathi K. J. of Crystal Growth. 2011: 318; 751. 20. Claude A, Vaithianathan V, Ganesh RB et al. J. App. Sci. 2006: 6; 85. 21. Hudson JA, Mahadevan CK, Padma CM. Intl. J. of Engg. Res. and appl. 2014: 4; 257-266. 22. Feng X, Zhu L, Li F et al. RSC Adv.2016: 6; 33983. 23. Kochuparampil AP, Joshi JH, Joshi MJ. Mod. Phy. Letter B.[online]. 2017 [cited 2017 August] Available from: URL: https://www.researchgate.net/publication/318906282/volumes-and- issues/2017/vol-31-issue-27. 24. Delci Z, Shyamla D, Karuna S et al. Intl. J. of ChemTech Research. 2012: 4; 816-826. 25. Raja R, Saravanan S, Santhanam V, Kurinjinathan P. Intl. J. of Tech. Research and Appl. 2016: 38; 5-8.

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DESIGNING A LOW-COST AMBULATORY ECG RECORDER FOR CARDIAC PATIENTS Harikrishna Parikh, Jatin Savaliya, Gumjan Vyas, Ankit Sidpara, Dr. H. N. Pandya Department of Electronics, Saurashtra University, Rajkot E-mail: [email protected] ABSTRACT: Ambulatory monitors play an important role in diagnosis of difficult to identify symptoms like dizziness, syncope and palpitation. Also, it is very useful in monitoring efficacy of drug over prolonged period before or after a specific treatment. Here is presented a small work to record single channel- single lead ECG signal in ambulatory fashion with our design. We designed it with AD8232 based amplifier and atmega328 (Arduino mini pro) based microcontroller system. It was tested and found to be useful for the limited objectives fixed for. Further enhancement in its functionality is expected and we are working over it. Keywords: Ambulatory monitors, single-channel single-lead ECG, AD8232, Atmega328

INTRODUCTION Bio-Electrical signals monitored have great sense; as they are actually responsible for the normal mechanical function of heart [1]. ECG provides valuable information about a wide range of cardiac disorders such as the presence of an inactive part (infarction) or an enlargement (cardiac hypertrophy) of the heart muscle. ECGs are primarily used to detect disturbances in the cardiac rhythm [2]. The traditional medical examination involves a number of chemicals, physical and electrophysiological measurements. These measurements are of very short duration and hence represent the patient’s condition for the time during which tests are carried out or its proximal timing. But if it’s needed to perform functional tests on a patient, which are in some or other way related to or dependent on his activities in normal life, the test measurements have to be carried out allowing the patient to lead his normal life and get involved in daily activities in his native environment. Ambulatory monitoring ensures such tests/measurements in real time on patients during everyday stress and activity as well as during periods of sleep. Extending the time of monitoring enhances the diagnostic yield by monitoring infrequent rhythms of signal under monitoring [3][4]. Therefore, it can be stated that ambulatory monitoring records one or more physiological parameters either continuously or at a predefined sampling period, allowing the patient (rather person being monitored) be free from the jargon of traditional laboratory instruments and without influencing the variable being measured. Ambulatory monitoring is a valuable aid in the diagnosis of many difficult to identify symptoms like dizziness, syncope and palpitation [1][5] and also very precise tool that provides accurate data for the evaluation of drug therapy, stress testing, artificial pacemakers, status of myocardial infarction [1][6] and several other problems in research programmes. The technique was introduced by Dr Norman Holter in 1962, and since then there has been lot many advancements have taken place, including size and weight of the recorder, types of sensors, type of recording systems etc. Now ambulatory monitoring is accepted worldwide by physians just like other traditional diagnosis tools. Block diagram of a typical ambulatory monitoring system is shown here in fig.1. Depending upon functionality, the ambulatory monitors can be classified in two categories: 1. Continuous Recorders: Such recorders record signals or parameters continuously, leading to pile of data from which important/meaningful information is to be searched while analysis. This is a cumbersome task unless is supported by tools like automatic analysers.[1][7][8] 2. Event based/ Intermittent Recorders: such devices record parameter of interest only in case of relevant events. Such events are supposed to generate trigger to start recording. These triggers may be generated by person under monitoring (manual) or by some threshold circuits that determine trigger points (self-triggered) for starting recording.[1][7][8]

Operation of ambulatory recorder is comparatively simple. Designs of the systems are also such that it creates minimum trouble in leading normal day to day life. The person under monitoring is instructed to wear the system and connect the electrodes of ECG, as this require frequent replacement of electrodes (at least once/twice a day). Also, the person is supposed to keep time record of all activities and specifically time when he/she feels some symptoms of abnormality and what they are. [7]

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Interface

ECG Amplifier Microcontroller

BP Measurement

Skin Response Recording Real Time Multiplexer Media Clock EMG Amplifier

Fig. 2: Block diagram of the typical ambulatory patient monitoring system

MATERIALS AND METHOD We started with the only objective to design an extremely low-cost working ECG recorder that can record at least one lead of ECG signal on real-time basis. Block diagram of the system is as shown below in fig.2:

AD8232 Arduino mini pro based ECG based System Amplifier

Micro Real Time SDcard Clock

Fig. 2: Block diagram of the ambulatory ECG recorder

0.33µF

+Vs 0.33µF ri REFOUT

1.4MCl HPDRIVE HPSENSE +IN IAOUT • OUTPUT -IN REFIN 0.1µF RLDFB +Vs 10MO

RLD GND AD8232 SW FR

OPAMP+ AC/DC LO+ GND REFOUT SON ECG OPAMP- LO+ } TO DIGITAL Output INTERFACE OUT LO-

SIGNAL OUTPUT

Fig.3: Typical circuit diagram of ECG amplifier using AD8232 Fig.4: Microcontroller based system for recording (courtesy: Analog Devices Inc.) ECG

In order to provide with maximum uninterrupted recording and enhance battery life, it was decided to use as minimum hardware as possible. We selected extremely low power ECG amplifier available based on AD8232 from Analog Devices. This amplifier works on a single power supply of 2.0 to 3.5V and consumes <200µA current.[9] With this specification it can be operated on small CR2032 (3.0V, 240mAh) coin cell for 3-4 days www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 43 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Physics & Electronics without any problem. The amplifier offers reasonable CMRR of 80dB in range of DC to 60Hz, [9] which is sufficient to reject line noise which is most evil factor in ECG amplification. And additionally, has lead-off detection feature. This feature is very much useful, as the person under monitoring is moving and possibly one or more electrodes may get disconnected from the ECG amplifier. Such events can be notified to the person by audio beep to make corrective action. Typical circuit of an ECG amplifier based on AD8232 is shown here in fig.3.

Another block, where we worked, is on microcontroller-based system for recording the processed ECG signals from ECG amplifier. The microcontroller-based system has to perform following tasks. 1. To sample the signals available from ECG amplifier at predefined sampling rate (250 Samples/Second in our case) and convert it to 8-bit digital equivalent.[10][11][12] 2. To record the sampled data of ECG signals to recording device (here microSD card) in prescribed file format (here CSV file) with timing details from real time clock. 3. To check the status of lead-off detector and generate audible signal for such case. This will ensure that, if electrode connection is broken, the patient can reconnect the same and ensure continued recording. 4. To keep track of time for normal and lead-off conditions. Here we adopted Arduino platform for designing microcontroller-based system. We used Arduino mini pro (atmega328 operated at 16MHz & 5V) [13] because it does not have on board USB chip for direct USB communication, and provides minimum power consumption amongst atmega328 processor boards. It was programmed to keep track of real time clock DS1307, sampling ECG signals at 250 Samples/Second with 8- bit resolution and store the sampled data on microSD card. The data was stored on microSD card in csv form (each sample separated by ‘comma’). In case of lead-off condition detection samples were stored as ‘LO’ instead of numeric values. Microcontroller program was such that it could generate separate files on hourly basis with filename “ARhhddmm.csv”. The system was powered with 2000mAh power bank. The complete system is depicted here in form of connection diagram in fig. 4. RESULT AND DISCUSSION Implementation of the above-mentioned system was carried out at our laboratory and was tested upon author for a period of a week. The system exhibited satisfactory results over the period except failures recorded twice, when the signals could not be recorded in spite of the healthy lead connections. Overall the system was found to be compact and cost effective. Results show reasonable agreement with parallel connected commercial ECG machine. Recorded signals were verified to be in order with normal ECG signal that could be recorded with commercially available ECG machines. Plot of such sample ECGs recorded with recorder is shown in fig. 5.

0 2 4 6 8 10 12

Fig. 5: Sample ECGs recorded with our ambulatory ECG recorder

FURTHER WORK From the recorded sample plot, it is evident that, there is some noise present in the recorded signal along with ECG signal. Some work is needed to be carried out in this direction. Also, if we can manage for lead selector circuit with help of analog multiplexers, then the recorder will be able to record all 12 leads in 3-channels at a time format. This is requiring additional hardware and upgradation of microcontroller, as the current hardware is only for single channel-single lead recording as well as the processor cannot go for more than 400 Samples/Second as per our observation. Also it makes additional power requirements leading to replacing battery used already. CONCLUSION As a whole, the system was found to be compact and cost effective. Results exhibited reasonable agreement with parallel connected commercial ECG machine. Therefore, once it achieves industry standards it can be used commercially.

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REFERENCES 1. R.S.Khandpur: Handbook of Bio-Medical Instrumentation, Tata McGraw-Hill Pub., 2nd Ed. 2003, p-156 2. S Stern and D Tzivoni: Early detection of silent ischaemic heart disease by 24-hour electrocardiographic monitoring of active subjects, British Heart Journal. 1974 May; 36(5): 481–486. 3. Zimetbaum P, Goldman A. Ambulatory arrhythmia monitoring: choosing the right device. Circulation. 2010;122:1629. DOI: 10.1161/CIRCULATION AHA.109.925610. 4. Brignole M, Vardas P, Hoffmann E, et al. Indications for the use of diagnostic implantable and external ECG loop recorders. Europace. 2009;11:671–87. DOI: 10.1093/europace/eup097 5. Olson JA, Fouts AM, Padanilam BJ, et al; Utility of mobile cardiac outpatient telemetry for the diagnosis of palpitations, presyncope, syncope, and the assessment of therapy efficacy. Journal of Cardiovascular Electrophysiology 2007 May 18(5):473-7. Epub 2007 Mar 6. 6. H. Mickley Ambulatory ST segment monitoring after myocardial infarction, British Heart Journal. 1994 Feb; 71(2): 113–114. 7. Robert W. Rho, Richard L. Page, Ch-69 Long-Term Monitors and Event Recorders, Ambulatory Electrocardiography e-pub 21/06/2015 8. Ritsuko Kohno, Haruhiko Abe, David G. Benditt, Ambulatory electrocardiogram monitoring devices for evaluating transient loss of consciousness or other related symptoms, Journal of Arrhythmia 33(2017) 583-589 9. Data-sheet: www.analog.com/media/en/technical-documentation/data-sheets/ AD8232.pdf 10. Peter R. Rijnbeek, Jan A. Kors, Maarten Witsenburg, Minimum Bandwidth Requirements for Recording of Pediatric Electrocardiograms, Circulation, AHA (2001)104:3087-3090 11. Laszlo Hejjel, Elizabeth Roth; What is the adequate sampling interval of the ECG signal for heart rate variability analysis in the time domain?, Physiol. Meas. 25 (2004) 1405-1411 12. G. D. Pinna, R Maestri, A. Di Cesare, R. Colombo and G. Minuco, ”The accuracy of power spectrum analysis of heart rate variability from annotated RR lists generated by Holter systems,” Physiol. Meas. , 15-2(1994), 163-179 13. https://store.arduino.cc/usa/arduino-mini-05

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r Section 3

MATHEMATICS & STATISTICS

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E – COLOURING OF A GRAPH D. K. Thakkar1 and V. R. Dave2 1Department of Mathematics, Saurashtra University, Rajkot – 360 005, India Email id: [email protected] 2Shree M. & N. Virani Science College, Kalavad Road, Rajkot – 360 005, India Email id: [email protected] ABSTRACT: In this paper we introduce a new colouring of graphs called E – Colouring. This is not a proper colouring in general. We also define E – Chromatic Number and E - Homomorphism of a graph. We prove several related results. In particular we prove that for a given E – chromatic colouring there is a vertex in every colour class which has an equitable neighbour in every other colour class. Keywords: E – Colouring, E – Chromatic Colouring, E – Chromatic Number, E – Homomorphism, Equitable Independent Set, Maximal Equitable Independent Set, Transversal, Colour Class. AMS Subject Classification (2010): 05C15, 05C69. INTRODUCTION The concept of an equitable edge was introduced in [2]. An edge uv of a graph is said to be an equitable edge if | d (u) – d (v) | ≤ 1. Two vertices u and v are said to be equitable adjacent if there is an equitable edge containing u and v. The concept of an equitable independent set was introduced in [6]. A set S of vertices of a graph G is said to be equitable independent if no two vertices of S are equitable adjacent. An equitable independent set S is said to be a maximal independent set [6]if for every vertex v in V(G) – S , S  {v} is not an equitable independent set. We assume that our graphs are finite, simple and undirected.

E – COLOURING OF A GRAPH We introduced here a new concept of colouring. Definition: 1 ( E – Colouring of a Graph) Let G be a graph. An E – Colouring of the vertices of the graph G is an assignment f : V(G) → {1, 2, …….. , k} such that if u and v are equitable adjacent then the colours f (u) and f (v) are different. An E – Colouring f : V(G) → {1, 2, …….. , k} is also called a k-E-Colouring. Note that an E – Colouring of a graph G need not be a proper colouring of G.

Example: 1

Let G = (V, E) be a graph. V (G) = {a, b, c, d}. The function f: V (G) → {1, 2, 3} defined as f (a) = 1, f (b) = 2, f (c) = 3, f (d) = 3. a The above colouring is an E – Colouring of the graph and observe that it is not a proper colouring of the graph.

b C d

Figure 1 Definition: 2 (Equitable Independent Set) [6] Let G be a graph and S  V (G), then S is said to be an equitable independent set if no two vertices of S are equitable adjacent. Obviously, every independent set is an equitable independent set but an equitable independent set need not be an independent set. Remark: 1 Let G be a graph and f: V (G) → {1, 2, …….., k} be a k-E-Colouring of G. We assume that this assignment f of colours is onto.  i let Ci = {v  V (G) / f (v) = i} ( i = 1, 2, ….. , k) Then obviously each Ci is non - empty equitable independent subset of G and Ci  Cj =  if i ≠ j

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In fact the sets C1, C2, …….., Ck is a partition of V(G) into equitable independent subset of G. These sets Ci are called colour classes corresponding to this E – Colouring. Conversely if we have a partition of V(G) say C1, C2, …….., Ck consisting of equitable independent sets then we have an E – Colouring arising naturally as follows. Define f: V (G) → {1, 2, …….., k} as f(v) = i if v  Ci for some i, for this E – Colouring obviously the sets C1, C2, …….., Ck are the colour classes. For example, the colour classes corresponding to the E – Colouring in the above graph are C1 = {a}, C2 = {b}, C3 = {c, d} Definition: 3 ( E – Chromatic Number & E – Chromatic Colouring) Let G be a graph. The smallest positive integer k such that G admits a k – E - Colouring is called the E – Chromatic Number of the graph. Such a k – E – Colouring is called the E – Chromatic Colouring of a graph. Notation: The E – Chromatic number of a graph is denoted by E (G) or E – ch (G). Example: 2 Consider the graph

If we assign colour – i to vi (i = 1, 2, 3, 4) & colour – 4 to v5 then this colouring is an E – Chromatic Colouring of G and note that E – Chromatic number of this graph = 4. Note that this colouring is not a proper colouring of the graph. Also it is observed that there is a proper colouring of G which is an E – Chromatic Colouring. This colouring is as follows. Assign colour – i to v (i = 1, 2, 3, 4) & assign colour – 1 i Figure 2 to v5. Definition: 4 (Homomorphism of Graphs) [5]: Let G and H be two graphs. A function f : V (G) → V (H) is said to be a homomorphism from G to H if whenever u and v are adjacent vertices of G , f(u) and f(v) are adjacent vertices of H. Definition: 5 (E - Homomorphism of Graphs): Let G and H be two graphs. A function f : V (G) → V (H) is said to be anE- homomorphism from G to H if whenever u and v are equitable adjacent vertices of G , f(u) and f(v) are adjacent vertices of H. Example: 3 Consider the following two graphs G and H. Graph G has the vertex set {v1, v2, v3, v4} which is as shown below.

Figure 3

The vertex set of the graph H is {v1, v 2, v3, v4} and it is as shown below.

• Figure 4

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Define the function f : V (G) → V (H) as follows. f ( vi ) = vi ( i = 1, 2, 3, 4) It is obvious that f is an E – Homomorphism from G to H. Let g = f -1 then it is obvious that g : V (H) → V (G) is also an E – Homomorphism from H to G. Remark: 2 Suppose f is an E- homomorphism from G to H which is onto. -1 Let y  V(H) and consider S = f (y). Suppose x1, x2  S and suppose they are equitable adjacent. Since f is an E- homomorphism, f(x1) and f(x2) are adjacent and therefore they are distinct. However f(x1) = f(x2) = y. This is a contradiction.  S must be an equitable independent set. Now, we will see that an n – E – Colouring of a graph G gives rise to an E- homomorphism from G → Kn. Remark: 3 Suppose f is an E- homomorphism from G to H and G is regular or bi – regular. Therefore any two vertices of G are adjacent iff they are equitable adjacent. Therefore f is a homomorphism from G to H. Theorem: 1 Let G be a graph and f is an n – E – Colouring of a graph G, then f can be regarded as an E- homomorphism from G → Kn Proof: Let f: V (G) → {1, 2, …….., n} be an n – E – Colouring of a graph G. Consider the complete graph Kn whose vertices are {1, 2, …….., n} If we regard f as a function from V(G) → V(Kn) then if u and v are equitable adjacent vertices of G then f(u) and f(v) are distinct members of the set {1, 2, …….., n}. This means that f(u) and f(v) are adjacent vertices of Kn. Thus, f is an E- homomorphism from G → Kn. ∎ Remark: 4 Considering the above theorem we can say that the E – Chromatic number of a graph G is the smallest positive integer n such that there is an E- homomorphism from G → Kn. Remark: 5 Let G be a graph and suppose f: V (G) → {1, 2, …….., n} is a chromatic colouring of the graph. ( i.e. (G) = n ) then obviously f is an E – Colouring of the graph.  The E – Chromatic number of G ≤ n = Chromatic number of G Notation: Suppose G and H are graphs. If there is an E- homomorphism from G to H then we will write 퐸 G→ H. E If there is no E- homomorphism from G to H then we denote it as G H If there is an E- homomorphism from G to H and If there is an E- homomorphism from H to G, we denote this 퐸 by G ⇄ H . Proposition: 1 퐸 Let G and H be two graphs  G → H then E (G) ≤  (H). Proof: Suppose  (H) = n and let f: V (H) → {1, 2, …….., n} be a chromatic colouring of H. f can be regarded as a homomorphism from H → Kn. Let g be an E – homomorphism from G to H. Now, f∘g : V(G) → V(Kn) is an E – homomorphism from G to Kn. E (G) ≤ n =  (H). ∎

Let G be a graph and f: V (G) → {1, 2, …….., n} be an E - Chromatic Colouring of G. Let C1, C2, …….., Cn be the colour classes of G corresponding to this E – Colouring. We have the following theorem Theorem: 2 In every colour class C there is a vertex v such that v has an equitable neighbour in every other colour class. Proof: Suppose there is a colour class in which no such vertex exists. For simplicity we may assume that this colour class is C1.  v  C1 there is a colour class say Cj in which v has no equitable neighbour. (This j depends on vertex v) Now, we obtain a new E – Colouring of G as follows. www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 48 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Mathematics & Statistics

Assign colour j to v if v  C1 and v has no equitable neighbour in Cj . Repeat this  v in C1. Thus, now we have an E – Colouring of G consisting of colours { 2, 3, ……, n} This is a contradiction because the given colouring f: V (G) → {1, 2, …….., n} is an E - Chromatic Colouring.  The statement of the theorem holds. ∎

Now, we prove a necessary condition under which the chromatic number of a graph is same as the E – Chromatic number of a graph. Theorem: 3 Let G be a graph. If Chromatic Number of G = E – Chromatic Number of G then for every chromatic colouring of G and for every colour class C of this chromatic colouring there is a vertex v in C such that v has an equitable neighbour in every other colour class of this colouring. Proof: Suppose there is a chromatic colouring f of G which fails to satisfy the condition. Then there is a colour class C of f such that  v  C there is a colour class C of f such that C ≠ C and v has no equitable neighbour in C. We obtain a new E – Colouring f  by assigning the colour of C to v,  v  C. Then the E – Colouring f  uses one less colour than f. Which is a contradiction.  The statement of the theorem holds. ∎ Definition: 6 (Maximal Equitable Independent Set ) [6] Let G be a graph and S be an equitable independent subset of G. Then S is said to be maximal equitable independent set if for every vertex v in V(G) – S , S  {v} is not an equitable independent set. Example: 4 Consider the graph G which has the vertex set {v1, v2, v3, v4} which is as shown below.

v2 v3 v4 Figure 5

Obviously, S = {v3, v4} is a maximal equitable independent set. There are other maximal equitable independent set also. For example, T = {v2, v4} is also a maximal equitable independent set. Theorem: 4 Let G be a graph and S be an equitable independent set which is not a maximal equitable independent set then there is an E – Chromatic Colouring of G such that S is not a colour class in this E – Chromatic Colouring. Proof: If S is not a colour class in every E – Chromatic Colouring then the theorem is proved. Suppose there is an E – Chromatic Colouring f of G such that S is a colour class in this E – Chromatic Colouring. Since S is not a maximal equitable independent set, therefore  a vertex x which is not in S and x is not equitable adjacent with every vertex of S. Now, x lies in some colour class S such that S ≠ S. Suppose S = {x} then by theorem 2 x is equitable adjacent with some vertex of every colour class. In particular, x is equitable adjacent with some vertex of S also. Which is not true. Thus, S  contains some other element y also. i.e. S  has at least two vertices. Now, we obtain a new E – Colouring as follows. Assign the colour of S to the vertex x and do not change colours of any other vertex. Obviously, this colouring is an E – Colouring and uses the same number of colours as f. We call this colouring as f . Obviously, S is not a colour class in this colouring f . Thus, the theorem is proved. ∎

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Definition: 7 ( Transversal ) Let S be a non-empty set and { S1, S2, ……. Sk } be a family of nonempty subsets of S. A subset T of S is said to be a transversal of this family if T  Si ≠ ,  i  { 1, 2, ……. , k}. Theorem: 5 Let G be a graph and S be an equitable independent subset of G. Suppose f is an E – Chromatic Colouring of G. Then either V(G) – S is a transversal of the colour classes of f or there is an E – Chromatic Colouring f  of G such that S is a colour class in this E – Colouring. Proof: Let f be an E – Chromatic Colouring of G. Suppose V (G) – S = T is not a transversal for this E – Colouring f of G. Therefore, T does not contain any vertex of a particular colour of this colouring. This means that for a particular colour of this colouring the corresponding colour class C does not intersect T.  C  S If C = S then S is a colour class for this colouring f itself. Suppose C  S Also y  S – C , then the colour of y is different from the colour of C and also {y} cannot be a colour class for this colouring because otherwise y would be equitable adjacent to some vertex of C ( by theorem 2). Which is a contradiction as S is an equitable independent set. Therefore, there is a vertex y  outside S which has the same colour as y. Thus we have proved that for every y in S – C there is a vertex y  such that y  is not in S and y  has the same colour as y. Now we define a new colouring f  as follows. Assign the same colour as that of C to all vertices of S and do not change the colours of vertices which are outside of S. Then this is an E – Chromatic Colouring and S is a colour class in this colouring. ∎

REFERENCES [1]Berge C., Theory of Graphs and its Applications, Methuen, London, (1962) [2] Dharmalingam K., A Note on The Equitable Covering and Equitable Packing of A Graph, Bulletin of International Mathematical Virtual Institute, 3 (2013), 21-27 [3] Haynes T. ,Hedetniemi S. and Slater P., Domination In Graphs Advanced Topics,, Marcel Dekker, Inc,, New York, 1998 [4] Haynes T. ,Hedetniemi S. and Slater P. , Fundamentals of Domination in Graphs, Marcel Dekker, Inc,, New York, 1998 [5] Pavol Hell and Jaroslav Nesetril, Graphs and Homomorphisms,Oxford University Press(2004) [6] Swaminathan V. and Dharmalingam K., Degree Equitable Domination on Graphs, Kragujevac Journal of Mathematics, 35(1) (2011), 191 – 197 [7] West D., Introduction to Graph Theory, 2nd Edition, Pearson Education, India, (2001)

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Section 4

BIOINFORMATICS

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COMPUTATIONAL APPROACHES FOR PROTEIN STABILITY STUDIES Varun S. Nair 1, John J Georrge 2* 1, 2 Department of Bioinformatics, Christ College, Rajkot-360 005, Gujarat, India Email ID: [email protected], [email protected] * ABSTRACT: Protein stability is the net balance of forces, which determine whether a protein will be in its native folded conformation or a denatured state. Protein stability usually depends on thermodynamic stability. Protein stability is an important feature in protein engineering. Stability of protein has been important as it decides the fate of food and drug processing. There are many factors affecting protein stability, such as pH, ligand binding and disulphide bonds. Protein stability is depending on amino acid composition and the structure. The alteration in the amino acid residues may change the structural stability. Many protein stability tools have been focused: STRUM, CUPSAT, mCSM, SDM (Site Directed Mutator), MAESTROweb, INP-SMD and iStability. Studying the effects of mutation on protein stability and function is important in understanding its role in disease. Thermostable proteins have many industrial applications in detergent industry, cosmetics, food industry, etc. Studies on the stability of these thermostable proteins are of great interest among researchers. Keywords: Protein Stability, Factors, Stability Tools, Industrial Applications 1. INTRODUCTION Proteins are probably the most important class of biomolecules. Proteins are the basis of major structural components of human and animal tissue. For the optimal function, these proteins require stability. Proteins must fold to a globular conformation to carry out the most important tasks in living organisms. Protein stability can be defined as the net balance of forces, which determine whether a protein will be its native folded conformation or a denatured (unfolded or extended) state (Niesen, Berglund, & Vedadi, 2007). The net stability of proteins is quite small and is the difference between two large opposing forces. The folded native state of protein structures is stabilized by various atomic/group interactions, such as hydrophobic, electrostatic, hydrogen bonding, van der Waals, and disulphide, and the unfolded state is dominated by entropic and nonentropic free energies. Protein stability normally refers to physical stability rather than chemical stability (Fágáin, 1995) (Niesen et al., 2007). 1.1 Chemical Stability Chemical stability involves formation or destruction of covalent bonds within a peptide or protein molecule, such as deamidation of asparagine and/or glutamine residues, oxidation of methionine and other amino acids, reduction of disulphide bonds and disulphide interchange and hydrolysis of peptide bonds. Chemical stability involves loss of integrity due to bond cleavage (Volkin). ➢ Determination of asparagine or glutamine residue ➢ Hydrolysis of peptide bond ➢ Oxidation of methionine at high temperature ➢ Elimination of disulphide bonds ➢ Disulphide interchange at neutral pH

2. FACTORS AFFECTING PROTEIN STABILITY 2.1 pH Proteins are more stable in the vicinity of their isoelectric point pI. In general, electrostatic interactions are believed to contribute to a small amount of stability of the native state. 2.2 Temperature Increase in temperature can disrupt the hydrogen bonds and other non-polar hydrophobic interactions. The reason behind this is increased temperature increases the kinetic energy and causes the molecules to vibrate so rapidly and violently that the bonds are disrupted. 2.3 Oxidation The stability of protein s denatured during oxidation. To prevent from oxidation, strong reducing agents such as DDT are usually added. www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 51

2.4 Ligand binding It has been known for a long time that binding ligands increases the stability of proteins.

2.5 Disulphide bonds If disulphide bond is denatured, many proteins are denatured or unfolded (Arakawa, Prestrelski, Kenney, & Carpenter, 2001).

3. DETERMINATION OF PROTEIN STABILITY Protein stability is based on the thermodynamic properties such change in enthalpy (ΔH), change in entropy (ΔS) and heat-capacity change (ΔCP) to gain a deeper insight to the forces that stabilizes a unique three- dimensional structure. The Gibbs Free energy change for the equilibrium reaction is referred as the stability of protein and is defined by: Folded (N)  Unfolded (D) (Eidhammer et al., 2004). A simple method to check the effect of compound / drug in protein stability is Differential Scanning Fluorimetry (DSF) (Subramani, 2013). nanoDSF is a modified version of DSF to determine the protein stability by employing tyrosine. Differential Scanning Calorimetry (DSC) is also a widely used for studying the thermodynamics of protein unfolding. DSC enables precise determination of thermodynamic values Tm, ΔHunf and ΔCPunf and these values can be determined with sufficient accuracy (Niesen et al., 2007). 3.1 Differential scanning fluorometry For studying the protein unfolding, differential scanning fluorometry is the most widely used tool as it does not have any models of prior assumptions. DSC enables precise determination of thermodynamic values Tm,_Hunf, and_Cpunf, and these values can determined sufficiently within the deviations of ± 0.1 K, ± 2% in _Hunf and ± 5% in _Cpunf. In DSC, the heat capacity of protein in solution is directly measured with temperature as shown in Fig 1.

Tm 60 ;:, t 50 i B 40 jf' t:Ji : /1!,CpdT .c: :.:: 30 ig"i. 8~ 20 !,j ;i c- 10 !!! _j__ I1!,Cp & 0 ~ -10 30 40 50 60 TO 80 90

Temperature ('C) Fig 1: Heat capacity versus temperature obtained by differential scanning fluorometry (Eidhammer, Jonassen, & Taylor, 2004).

4. STABILITY OF THERMOPHILIC PROTEINS (Adaptation Mechanisms) In order to function at elevated temperature, thermophilic proteins must preserve their tertiary folds in order to maintain their biological function. Certain thermophilic life-forms exist which can withstand temperatures above this and have corresponding adaptations to preserve protein function at these temperatures. These can include altered bulk properties of the cell to stabilize all proteins, and specific changes to individual protein (Neves, da Costa, & Santos, 2005). The presence of certain types of salt has been observed to alter thermostability in the proteins, indicating that salt bridges likely also play a role in thermostability. Other factors of protein thermostability are compactness of protein structure, oligomerization, and strength interaction between subunits (Das & Gerstein, 2000). Thermal resistance enzymes determine a free-energy consumption necessary for the transformation from the folded to the unfolded state (Shiraki, Fujiwara, Imanaka, & Takagi, 2001). Thermostability of some enzymes can be assured by the environmental factors such as: protein concentrations, increased intracellular salts, synthesis of different stabilizers and chemolithoautotrophic mode of nutrition. www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 52

Enzyme Papain Bacillus Bacillus Thermolysin Caldolysin Thermus properties neutral alkaline strain Tok3 protease protease protease

Activity Low Low High Medium High High

Stability Low Low High High High High

Effect of None Inhibited None Inhibited Reduced None chelation stability

Cost Low Medium Medium High Very high Very high

Table 1: Stability of thermophilic proteases (Cowan, Daniel, & Morgan, 1985)

Slight changes in amino acid and sequences increase the number of stabilizing interactions in the folded protein, such as: additional ion-pairs, disulphide bridges, hydrogen bonds and hydrophobic Interactions (Fujinaga, Cherney, Oyama, Oda, & James, 2004). Other stabilizing mechanisms include filing cavities in molecular structure of proteins, shortening of loops reduction of accessible hydrophilic surface area. Other adapted strategies to environmental factors include the composition of minerals and gases, pH, redox potential, salinity and temperature (Fukuchi & Nishikawa, 2001). Chemolithotrophic mode of nutrition is famous among thermophilic proteins. Inorganic redox reactions serve as energy sources (chemolithotrophic) and CO2 is the only carbon source required to build up organic cell material (autotrophic). Therefore, these organisms fix CO2 by chemosynthesis and are designated chemolithoautotrophs. Chemosynthesis is the biological conversion of one or more carbon containing molecule and nutrients into organic matter using the oxidation of inorganic compounds rather than sunlight (Chela-Flores, 2000). Other aerobic and anaerobic types of respirations are important for energy-yielding reactions. Anaerobic respiration types are the nitrate-, ferric iron-, sulphate-, sulphur- and carbon dioxide respirations. H2 serves as an important electron donor. Within the natural environment, it may be a component of volcanic gasses. Other electron donors are sulphide, sulphur and ferrous iron (Bengtson, 1994). Thermal resistance of the DNA double helix appears to be improved in hyperthermophiles by reverse gyrase, a unique type I DNA topoisomerase that causes positive supertwists for stabilization. In proteases, specific binding of metal ions (particularly of calcium) further enhances molecular stability (Roche, Voordouw, & Matthews, 1978). For example, the increased stability of caldolysin over thermolysin can be attributed almost entirely to the binding of six calcium ions to the former ~9, as opposed to four calcium ions to the latter2. The abnormally high frequency of tyrosine in thermolysin 23has also been implicated in its thermostability, although this proposed mechanism seems to be unique. The stability of thermophilic proteases is not restricted to temperature but also includes resistance to denaturing agents, detergents and organic solvents. The stability of thermophilic protease is much greater than that of their mesophilic counterparts (Roche et al., 1978); however, this is yet to be proved. Other methods for thermophilic proteases through covalent cross linking have been well established. Deeper insights into the stabilizing principles of thermophilic proteases will most likely be obtain. Although, adaptation of thermozymes to act at elevated temperatures is mainly achieved by exchange of few amino acid residues and/or their different localization in molecule, the homologous thermostable and thermolabile enzymes are similar and have the same catalytic mechanisms after comparison of three-dimensional structures with homologous mesophilic proteases. In short, the main proposed mechanisms/indicators of increased thermostability include a more highly hydrophobic core, tighter packing or compactness, deleted or shortened loops, greater rigidity, (for example through increased Proline content in loops), higher secondary structure content, greater polar surface area, fewer and smaller voids, smaller surface area to volume ratio, fewer thermolabile residues, increased hydrogen bonding, higher isoelectric point, more salt bridges and network of salt bridges. More ion pairs have been strongly and consistently linked with thermostability in the literature. Water has a dielectric constant of about 80 at 0°C, which drops to 55 at 100°C and is lower still at the extreme pressures near hydrothermal vents in the deep sea where some hyperthermophilic organisms live. A lower dielectric constant makes electrostatic interactions stronger and therefore ion pairs should have a greater stabilizing effect at high temperatures and pressures. Also, better packing has always been a main reason for the higher stability of thermophilic proteins www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 53

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Bioinformatics and hence, smaller and less numerous cavities (Zehfus & Rose, 1986). One can study the packing of a protein by computing its compactness. Ca2+ is a known activator of protease activity and also offers protection against thermal inactivation. All proteases are stabilized in the presence of a certain level of free Ca+2. Therefore, 100- 1000 ppm of Ca+2 is normally added to liquid detergents that contain protease. Other agents such as glycine will partly inhibit the activity of proteases (e.g., by binding on the active side). However, the protease will recover its full activity when the detergent is diluted in the washing process.

4.1 Amino Acid Properties and Protein Stability Investigations have been carried out using the differences in amino acid composition between Mesophilic and Thermophilic proteins for understanding the stability. (Argos et al., 1979) observed that Gly, Ser, Asp and lys in mesophiles are generally substituted by Ala, Thr, Arg and Glu in thermophiles to enhance the stability. In addition, it has been reported that the substitution of Trp by Tyr increased the stability. (Vijayakumar, Ponnusamy, Balakrishnan, & Kothandaraman, 1982) analysed the relationship between amino acid composition and thermal stability and reported that the set of amino acid residues Asp, Cys, Glu, Lys, Leu, Arg, Trp, and Tyr enhances the stability and the set of residues Ala, Asp, Gly, Gln, Ser, Thr, Val, and Tyr decreases the stability. They found a correlation between the melting temperature and the amino acid composition of a stabilizing and destabilizing group of amino acids.

5. COMPUTATIONAL TOOLS FOR PROTEIN STABILITY PREDICTION 5.1 mCSM (URL: http://biosig.unimelb.edu.au/mcsm/stability) mCSM relies on graph-based signatures. These encode distance patterns between atoms and are used to represent the protein residue environment and to train predictive models. In order to understand the roles of mutations in disease, we have evaluated their impacts not only on protein stability but also on protein-protein and protein-nucleic acid interactions (Pires, Ascher, & Blundell, 2013). Features • Protein Stability Change Upon Mutation • Protein-Protein Affinity Change Upon Mutation • Protein-DNA Affinity Change Upon Mutation 5.2 SDM (Site Directed Mutator) (URL: http://131.111.43.103/) Studying the effects of mutation on protein stability and function is important in understanding its role in disease. Site Directed Mutator (SDM) is a computational method that analyses the variation of amino acid replacements occurring at specific structural environment that are tolerated within the family of homologous proteins of known 3-D structures and convert them into substitution probability tables (Worth, Preissner, & Blundell, 2011). 5.3 CUPSAT (CUPSAT: Cologne University Protein Stability Analysis Tool) (URL: http://cupsat.tu-bs.de/) CUPSAT is a tool to predict changes in protein stability upon point mutations. The prediction model uses amino acid-atom potentials and torsion angle distribution to assess the amino acid environment of the mutation site. Additionally, the prediction model can distinguish the amino acid environment using its solvent accessibility and secondary structure specificity. Predict Mutant Stability from Existing PDB Structures and Custom Protein Structures

5.4 iStability iRDP (in-silico Rational Design of Proteins) web server, a one-stop tool for protein design which implements an in-silico “Analyze and Build” approach to the rational protein design problem. The tool is divided into four modules, namely iCAPS, iMotifs, iMutants and iStability. Protein Stability forms an important feature of Protein Engineering. The module aims to implement specific pre-defined protein engineering strategies like disulphide bond insertion, stabilization by insertion of proline residues, and release of conformational strains, which are reported to alter the stability of the protein structures. On implementation of these strategies, the user is informed about the predicted stability of the protein due to the mutations carried out. Apart from the above strategies, the user can also gain knowledge about the predicted stability of customized mutants. When selected, the user is also informed of the residue conservation of the wild-type residue (Panigrahi, Sule, Ghanate, Ramasamy, & Suresh, 2015).

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6. INDUSTRIAL APPLICATIONS OF THERMOSTABLE PROTEINS Thermostable enzymes have been an important and indispensable part of industrial applications such as Starch Degradation, Laundry detergents, Paper and Pulp industry, Molecular biology industry, Meat tenderizing, Cosmetics etc; Starch is a ubiquitous polysaccharide composed of two high-molecular weight components: amylose and amylopectin. The most important industrial processes in which starch is used as a natural source of sugars are the production of glucose/ fructose syrup, the synthesis of non-fermentable carbohydrates, and the synthesis of anti-staling agents in baking. In addition, the glucose resulting from these processes can be used to produce ethanol, amino acids or organic acids (Banerjee, Sani, Azmi, & Soni, 1999).

The first thermostable a-amylases were isolated from Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis.

Extremophile Enzymes Applications

Thermophile Amylases Glucose, fructose for sweeteners

Xylanases Paper Bleaching

Proteases Baking, Brewing, Laundry

Psychrophile Proteases Cheese maturation

Amylases Polymer degradation in detergent

Halophile Ion exchange resin regenerate disposal

Piezophile Whole Microorganisms Formation of Gels and starch granules

Metalophile Whole microorganisms Ore-bioleaching, bioremediation

Radophile Whole Microorganisms Bioremediation

Table 2: An overview of industrial applications of extremophiles (Alquéres et al., 2007)

Alcalase have found many applications today, including food industry, skin care ointments, cosmetics, contact lens cleaners. For example, this enzyme is important in the processing of soy meal which results in soluble, non-bitter hydrolysate, used as component of protein-fortified soft drinks and dietetic food. Thermostable DNA polymerases are responsible for the elongation of the primer strand of a growing DNA molecule during DNA amplification by PCR. The isolation of Taq DNA polymerase from the thermophilic bacterium Thermus aquaticus, and in particular its cloning and expression in E. coli, was truly a revolutionary event in the field of Biotechnology (Gupta, Beg, & Lorenz, 2002).

7. CONCLUSION Various biological experiments have been done for genomic and proteomic information, stability studies are needed to be explore more to understand deeply. To study mutational stability and to improve the stability and activity through protein engineering are biologically time consuming which can be streamlined through bioinformatics analysis as currently, many bioinformatics tools are available and lots of data analysis can be performed.

8. REFERENCES Alquéres, S. M. C., Almeida, R. V., Clementino, M., Vieira, R. P., Almeida, W. I. d., Cardoso, A. M., & Martins, O. B. (2007). Exploring the biotechnologial applications in the archaeal domain. Brazilian Journal of Microbiology, 38(3), 398-405. www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 55

Arakawa, T., Prestrelski, S. J., Kenney, W. C., & Carpenter, J. F. (2001). Factors affecting short-term and long- term stabilities of proteins. Advanced drug delivery reviews, 46(1-3), 307-326. Argos, P., Rossmann, M. G., Grau, U. M., Zuber, H., Frank, G., & Tratschin, J. D. (1979). Thermal stability and protein structure. Biochemistry, 18(25), 5698-5703. Banerjee, U. C., Sani, R. K., Azmi, W., & Soni, R. (1999). Thermostable alkaline protease from Bacillus brevis and its characterization as a laundry detergent additive. Process biochemistry, 35(1-2), 213-219. Bengtson, S. (1994). Early life on Earth: Columbia University Press. Chela-Flores, J. (2000). Terrestrial Microbes as Candidates for Survival on Mars and Europa Journey to Diverse Microbial Worlds (pp. 387-398): Springer. Cowan, D., Daniel, R., & Morgan, H. (1985). Thermophilic proteases: properties and potential applications. Trends in Biotechnology, 3(3), 68-72. Das, R., & Gerstein, M. (2000). The stability of thermophilic proteins: a study based on comprehensive genome comparison. Functional & integrative genomics, 1(1), 76-88. Eidhammer, I., Jonassen, I., & Taylor, W. R. (2004). Protein Bioinformatics: An algorithmic approach to sequence and structure analysis (Vol. 1): Wiley Online Library. Fágáin, C. Ó. (1995). Understanding and increasing protein stability. Biochimica et Biophysica Acta (BBA)- Protein Structure and Molecular Enzymology, 1252(1), 1-14. Fujinaga, M., Cherney, M. M., Oyama, H., Oda, K., & James, M. N. (2004). The molecular structure and catalytic mechanism of a novel carboxyl peptidase from Scytalidium lignicolum. Proceedings of the National Academy of Sciences of the United States of America, 101(10), 3364-3369. Fukuchi, S., & Nishikawa, K. (2001). Protein surface amino acid compositions distinctively differ between thermophilic and mesophilic bacteria1. Journal of molecular biology, 309(4), 835-843. Gupta, R., Beg, Q., & Lorenz, P. (2002). Bacterial alkaline proteases: molecular approaches and industrial applications. Applied microbiology and biotechnology, 59(1), 15-32. Neves, C., da Costa, M. S., & Santos, H. (2005). Compatible solutes of the hyperthermophile Palaeococcus ferrophilus: osmoadaptation and thermoadaptation in the order thermococcales. Applied and environmental microbiology, 71(12), 8091-8098. Niesen, F. H., Berglund, H., & Vedadi, M. (2007). The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nature protocols, 2(9), 2212. Panigrahi, P., Sule, M., Ghanate, A., Ramasamy, S., & Suresh, C. (2015). Engineering proteins for thermostability with iRDP web server. PloS one, 10(10), e0139486. Pires, D. E., Ascher, D. B., & Blundell, T. L. (2013). mCSM: predicting the effects of mutations in proteins using graph-based signatures. Bioinformatics, 30(3), 335-342. Roche, R. S., Voordouw, G., & Matthews, B. W. (1978). The structural and functional roles of metal ions in thermolysin. CRC critical reviews in biochemistry, 5(1), 1-23. Shiraki, K., Fujiwara, S., Imanaka, T., & Takagi, M. (2001). Conformational stability of a hyperthermophilic protein in various conditions for denaturation. Subramani, B. (2013). How to determine protein stability? Retrieved from https://www.researchgate.net/post/How_to_determine_protein_stability Vijayakumar, C., Ponnusamy, E., Balakrishnan, T., & Kothandaraman, H. (1982). Thermal and pyrolysis studies of copolyesters. Journal of Polymer Science Part A: Polymer Chemistry, 20(9), 2715-2725. Volkin, D. B. Chemical Stability. Retrieved from https://mvsc.ku.edu/content/chemical-stability Worth, C. L., Preissner, R., & Blundell, T. L. (2011). SDM—a server for predicting effects of mutations on protein stability and malfunction. Nucleic acids research, 39(suppl_2), W215-W222. Zehfus, M. H., & Rose, G. D. (1986). Compact units in proteins. Biochemistry, 25(19), 5759-5765.

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TOOLS FOR LIGAND BASE DRUG DISCOVERY Janvi Gajipara1, John J Georrge1* 1 Department of Bioinformatics, Christ College, Rajkot-360 005, Gujarat *Corresponding author: [email protected] & [email protected]

ABSTRACT: The ligand Base drug design also called indirect drug design which relies on knowledge of other molecules that bind to the biological target of interest. These other molecules may be used to derive a Pharmacophore model that defines the minimum necessary structural characteristics a molecule must possess in order to bind to the target. Alternatively, a quantitative structure-activity relationship (QSAR), in which a correlation between calculated properties of molecules and their experimentally determined biological activity may be derived. Target fishing, or target identification, is an important start step in modern drug development, which investigates the mechanism of action of bioactive small molecules by identifying their interacting proteins. Reverse or inverse docking is proving to be a powerful tool for drug repositioning and drug rescue. It involves docking a small-molecule drug/ligand in the potential binding cavities of a set of clinically relevant macromolecular targets. This chapter covers all available tools for the ligand-based drug discovery, which will be beneficial to the researchers who are working on medicinal and/or natural product chemistry.

Keywords: Drug Design, Pharmacophore, QSAR, Reverse Docking

1. INTRODUCTION Ligand-based drug design or relies on knowledge of other molecules that bind to the biological target of interest. These other molecules may be used to derive a pharmacophore model that defines the minimum necessary structural characteristics a molecule must possess in order to bind to the target. In other words, a model of the biological target may be built based on the knowledge of what binds to it, and this model in turn may be used to design new molecular entities that interact with the target. Alternatively, a quantitative structure-activity relationship (QSAR), in which a correlation between calculated properties of molecules and their experimentally determined biological activity, may be derived. These QSAR relationships in turn may be used to predict the activity of new analogs. Ligand- based drug design uses Compound Drug Discovery Cycle ligands of the drug target— CollKtKIRS that is, molecules that bind to the drug target. S('cond..try A~1.ir, Design focuses on t-fficacy b,o.i.-a,labil,ty the structure of the ligands, tO>,;J(lt~ 1t"i ..,l\,O for example, by the use of pharmacophore models or by QSAR models. The former model seeks to lud Compounds determine what ligand •nd $AJt structures are necessary for target binding. QSAR models, on the other hand, Structural Chatac1er1z•oon Canchdate suggest that molecular of Prot ein- Ug.1.nd Compfo 0Ng similarity, through Direct combination molecular descriptors, predicts biological activity of the drug (Cereto-Massagué et al., 2015). The method available for Ligand based Drug Discovery are QSAR, Pharmacophore, Target Fishing and Reverse Docking. Based on these methods various research works were successfully completed (Chirag N. Patel et al., 2017; Gauravi Trivedi, 2016; John J Georrge, 2015; John J. Georrge, 2016; John J Georrge et al., 2011; John J Georrge et al., 2012; John L, 2012; Joseph et al., 2015; Kotadiya et al., 2015; Nishita NV, 2016; Patel et al., 2017; Rutvi Chovatiya, 2016; Soni et al., 2016; Vyas et al., 2015).

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2. QSAR Quantitative structure–activity relationship models are regression or classification models used in the chemical and biological sciences and engineering. Like other regression models, QSAR regression models relate a set of "predictor" variables (Wolber et al.) to the potency of the response variable (Y), QSAR and Drug Design while classification QSAR models relate the predictor variables to a categorical value of the response variable. The number of compounds required for synthesis in order to place 10 different groups in 4 positions of benzene ring is 104. Solution: synthesize a small number of compounds and from their data derive rules to predict the biological New compounds with activity of other compounds improved biological activity (Buratto, 2015).

2.1 VEGA platform [https://www.vegahub.eu/portfolio-item/vega-qsar/] Using the VEGA platform, you can access a series of QSAR models for regulatory purposes, or develop your own model for research purposes. QSAR models can be used to predict the property of a chemical compound, using information obtained from its structure.

2.2 DEMETRA [http://www.demetra-tox.net/] DEMETRA is an EU-funded project. This project aim has been to develop predictive models and software which give a quantitative prediction of the toxicity of a molecule, in particular molecules of pesticides, candidate pesticides, and their derivatives. The input is the chemical structure of the compound, and the software algorithms use “Quantitative Structure-Activity Relationships” (QSARs). The DEMETRA software tool can be used for toxicity prediction of molecules of pesticides and related compounds. The DEMETRA models are freely available. Five models have been developed to predict toxicity against trout, daphnia, quail and bee. The software is based on the integration of the knowledge acquired in the DEMETRA EU project in a homogeneous manner using the best algorithms obtained as the basis for hybrid combinative models to be used for predictive purposes (Kharkar et al., 2014).

2.3 T.E.S.T [https://www.epa.gov/chemical-research/toxicity-estimation-software-tool-test] Toxicity Estimation Software Tool (T.E.S.T.) will enable users to easily estimate acute toxicity using the above QSAR methodologies.

2.4 OCHEM [https://ochem.eu/home/show.do] The OCHEM is an online database of experimental measurements integrated with the modeling environment. Submit your experimental data or use the data uploaded by other users to build predictive QSAR models for physical-chemical or biological properties (Lavecchia et al., 2016).

2.5 E-DRAGON [http://www.vcclab.org/lab/edragon/] E-DRAGON is the electronic remote version of the well-known software DRAGON, which is an application for the calculation of molecular descriptors developed by the Milano Chemometrics and QSAR Research Group of Prof. R. Todeschini. These descriptors can be used to evaluate molecular structure-activity or structure-property relationships, as well as for similarity analysis and high throughput screening of molecule databases.

2.6 SeeSAR [https://www.biosolveit.de/SeeSAR/] SeeSAR is a software tool for interactive, visual compound prioritization as well as compound evolution. Structure-based design work ideally supports a multi-parameter optimization to maximize the likelihood of success, rather than affinity alone. Having the relevant parameters at hand in combination with real-time visual computer assistance in 3D is one of the strengths of SeeSAR (Lee et al., 2016).

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2.7 Dragon [https://chm.kode-solutions.net/products_dragon.php] Dragon calculates 5,270 molecular descriptors, covering most of the various theoretical approaches. The list of descriptors includes the simplest atom types, functional groups and fragment counts, topological and geometrical descriptors, three-dimensional descriptors, but also several properties estimation (such as logP) and drug-like and lead-like alerts (such as the Lipinski's alert).

2.8 PaDEL-Descriptor [http://www.yapcwsoft.com/dd/padeldescriptor/] A software to calculate molecular descriptors and fingerprints. The software currently calculates 1875 descriptors (1444 1D, 2D descriptors and 431 3D descriptors) and 12 types of fingerprints (total 16092 bits). The descriptors and fingerprints are calculated using The Chemistry Development Kit with additional descriptors and fingerprints such as atom type electrotopological state descriptors, Crippen's logP and MR, extended topochemical atom (ETA) descriptors, McGowan volume, molecular linear free energy relation descriptors, ring counts, count of chemical substructures identified by Laggner, and binary fingerprints and count of chemical substructures identified by Klekota and Roth.

3. PHARMACOPHORE A Pharmacophore is an abstract description of molecular features that are necessary for molecular recognition of a ligand by a biological macromolecule. The IUPAC defines a Pharmacophore to be "an ensemble of steric and electronic features that is necessary to ensure the optimal supramolecular interactions with a specific biological target and to trigger (or block) its biological response". A Pharmacophore model explains how structurally diverse ligands can bind to a common receptor site. Furthermore, Pharmacophore models can be used to identify through de novo design or virtual screening novel ligands that will bind to the same receptor. In modern computational chemistry, Pharmacophores are used to define the essential features of one or more molecules with the same biological activity. A database of diverse chemical compounds can then be searched for more molecules which share the same features arranged in the same relative orientation. Pharmacophores are also used as the starting point for developing 3D- QSAR models. Such tools and a related concept of "privileged structures", which are "defined as molecular frameworks which are able of providing useful ligands for more than one type of receptor or enzyme target by judicious structural modifications”, aid in drug discovery (Li et al., 2017).

3.1. Pharmer [http://smoothdock.ccbb.pitt.edu/pharmer/] Pharmer is a pharmacophore search technology that can search millions of chemical structures in seconds. Unlike other technologies, the performance of Pharmer scales with the complexity of the query, not the size of the library being searched. Pharmer powers ZINCPharmer, an online pharmacophore search engine for a multi- conformer library of the ZINC database.

3.2. PharmaGist [http://bioinfo3d.cs.tau.ac.il/pharma/index.html] Predicting molecular interactions is a major goal in rational drug design. Pharmacophore, which is the spatial arrangement of features that is essential for a molecule to interact with a specific target receptor, is important for achieving this goal. PharmaGist is a freely available web server for pharmacophore detection. The employed method is ligand based. It does not require the structure of the target receptor. Instead, the input is a www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 59 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Bioinformatics set of structures of drug-like molecules that are known to bind to the receptor. We compute candidate pharmacophores by multiple flexible alignments of the input ligands. The main innovation of this approach is that the flexibility of the input ligands is handled explicitly and in deterministic manner within the alignment process. The method is highly efficient, where a typical run with up to 32 drug-like molecules takes seconds to a few minutes on a standard PC. Another important characteristic of the method is the capability of detecting pharmacophores shared by different subsets of input molecules. This capability is a key advantage when the ligands belong to different binding modes or when the input contains outliers.

3.3. Catalyst [http://accelrys.com/products/collaborative-science/biovia-discovery- studio/pharmacophore-and-ligand-based-design.html] Pharmacophore Modeling and Analysis; 3D database building and searching; Ligand conformer generation and analysis tools; Geometric, descriptor-based querying; Shape-based screening. Distributed by Accelrys as part of Discovery Studio.

3.4. LigandScout [http://www.inteligand.com/ligandscout/] The LigandScout software suite comprises the most user-friendly molecular design tools available to chemists and modelers worldwide. The platform seamlessly integrates computational technology for designing, filtering, searching and prioritizing molecules for synthesis and biological assessment.

3.5. MOE [https://www.chemcomp.com/MOE-Pharmacophore_Discovery.htm] MOE contains the industry-leading suite of Pharmacophore discovery applications used for fragment-, ligand- and structure-based design projects. Pharmacophore modeling is a powerful means to generate and use 3D information to search for novel active compounds, particularly when no receptor geometry is available. Pharmacophore methods use a generalized molecular recognition representation and geometric constraints to bypass the structural or chemical class bias of 2D methods (Mori et al., 2015).

3.6. Phase [https://www.schrodinger.com/phase] Phase is a complete, user-friendly Pharmacophore modeling solution designed to maximize performance in virtual screening and lead optimization. Fast, accurate, and easy-to-use, Phase includes a novel, scientifically validated common Pharmacophore perception algorithm.

4. TARGET FISHING Computational methods for Target Fishing (TF), also known as Target Prediction or Polypharmacology Prediction, can be used to discover new targets for small-molecule drugs. This may result in repositioning the drug in a new indication or improving our current understanding of its efficacy and side effects. We can a new benchmark to validate TF methods, which is particularly suited to analyze how predictive performance varies with the query molecule. Robust target fishing extends multitude benefits to drug research, such as avoiding unwanted side effects from poly pharmacology of small molecules at clinical stages, to reveal the mode-of-actions of a compound and also to repurpose old drugs for new targets. The rule of ‘one-size-does-not-fit-all’ still holds well in target fishing approaches as well. Therefore, it is important to carefully assemble the available methods and resources such that all levels of biological information, from sequences to structures to pharmacophores, are maximally utilized for fishing out the targets for the design of safer next generation drugs (Santiago et al., 2012). Activity Target fi5hing Rational optimization

Chitinase ~~-~·· .. ..,,. r ~ c':' ,::yJ. J 9 .. Arglfln 8 ' www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 60 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Bioinformatics

4.1. ChemMapper [http://lilab.ecust.edu.cn/chemmapper/] ChemMapper is a free web server for computational drug discovery based on the concept that compounds sharing high 3D similarities may have relatively similar target association profile. ChemMapper integrates nearly 300 000 chemical structures from various sources with pharmacology annotations and over 3 000 000 compounds from commercial and public chemical catalogues. In- house SHAFTS method which combines the strength of molecular shape superposition and chemical feature matching is used in ChemMapper to perform the 3D similarity searching, ranking, and superposition. Taking the user-provided chemical structure as the query, SHAFTS aligns each target compound in the database onto the query and calculates the 3D similarity scores and the top most similar structures are returned. Based on these top most similar structures whose pharmacology annotation is available, a chemical-protein network is constructed and a random walk algorithm is taken to compute the probabilities of the interaction between the query structure and proteins which associated with hit compounds. These potential protein targets ranked by the standard score of the probabilities. ChemMapper can be useful in a variety of polypharmacology, drug repurposing, chemical-target association, virtual screening, and scaffold hopping studies.

4.2. PharmMapper Server [http://lilab.ecust.edu.cn/pharmmapper/index.php] The current release, i.e. version 2017, is based on the contents of PDB officially released by Jan 1st, 2016. This release applies Cavity1.1 to detect the binding sites on the surface of a given protein structure and rank them according to the corresponding druggability scores. A receptor-based pharmacophore modeling program Pocket 4.0 was then used to extract pharmacophore features within cavities. In this approach, a total of 23236 proteins covering 16159 pharmacophore models which are predicted as druggable binding sites and 52431 pharmacophore models with a pKd value higher than 6.0 are picked out, which is currently the largest collection of this kind. Compared to the last release (v.2010), target pharmacophore models included in this release have increased more than six times, from 7302 to over almost 53000.

4.3. TargetHunter [http://www.cbligand.org/TargetHunter/] This web portal implements a novel in silico target prediction algorithm, the Targets Associated with its Most Similar Counterparts, by exploring the largest chemogenomical databases, ChEMBL. TargetHunter also features an embedded geography tool, BioassayGeoMap, developed to allow the user easily to search for potential collaborators that can experimentally validate the predicted biological targets or off targets. TargetHunter therefore provides a promising alternative to bridge the knowledge gap between biology and chemistry, and significantly boost the productivity of chemogenomics researchers for in silico drug design and discovery.

4.4. ChemProt [http://potentia.cbs.dtu.dk/ChemProt/] The ChemProt 2.0 server is a resource of annotated and predicted chemical-protein interactions. The server is a compilation of over 1 100 000 unique chemicals with biological activity for more than 15000 proteins. ChemProt can assist in the in-silico evaluation of small molecules (drugs, environmental chemicals and natural products) with the integration of molecular, cellular and disease-associated proteins complexes.

4.5. SwissTargetPrediction [http://www.swisstargetprediction.ch/] This website allows you to predict the targets of a small molecule. Using a combination of 2D and 3D similarity measures, it compares the query molecule to a library of 280'000 compounds active on more than 2000 targets of 5 different organisms.

4.6. SuperPred [http://prediction.charite.de/] SuperPred, which is a prediction webserver for ATC code and target prediction of compounds. Predicting ATC codes or targets of small molecules and thus gaining information about the compounds offers assistance in the drug development process. The webserver's ATC prediction as well as target prediction is based on a pipeline consisting of 2D, fragment and 3D similarity search.

4.7. PASS [http://www.pharmaexpert.ru/passonline/] PASS Online predicts over 4000 kinds of biological activity, including pharmacological effects, mechanisms of action, toxic and adverse effects, interaction with metabolic enzymes and transporters, influence on gene expression, etc. To obtain the predicted biological activity profile for your compound, only structural formula is necessary; thus, prediction is possible even for virtual structure designed in computer but not synthesized yet.

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5. REVERSE DOCKING In reverse docking, one tries to find protein targets which can bind to a Virtual Screening Reve rse Docking particular ligand. The necessary components are similar to those of forward docking methods; preparing data sets, • e searching for ligand poses, I, ~ and scoring and ranking the complex structures. However, several issues including high . ... computational cost and inter-protein score bias ~ makes reverse docking ,. • process rather complex (Wolber et al., 2008). •

5.1. INVDOCK [http://bidd.nus.edu.sg/group/softwares/invdock.htm] A computer method, and its application software INVDOCK, have been developed for computer- automated identification of potential protein and nucleic acid targets of a small molecule. The 3-D structure of the small molecule being studied is input into the programmer, the software automatically searches a protein and nucleic acid 3-D structure database to identify protein, RNA or DNA molecule that the small molecule can bind to. The identified proteins and nucleic acids are considered potential targets of the molecule.

5.2. idTarget [http://idtarget.rcas.sinica.edu.tw] A web server for identifying biomolecular targets of small chemical molecules with robust scoring functions and a divide-and-conquer docking approach 5.3. AMIDE (Automatic molecular inverse docking engine) Molecular docking is widely used computational technics that allows studying structure-based interactions complexes between biological objects at the molecular scale. AMIDE was developed, a framework that allows performing inverse virtual screening to carry out a large-scale chemical ligand docking over a large dataset of proteins. Its ability to reproduce experimentally determined structures and binding affinities highlighted that AMIDE allows performing better exploration than existing blind docking methods.

5.4. VTS (Virtual Target Screening) Virtual Target Screening (VTS)", a set of small drug-like molecules are docked against each structure in the protein library to produce benchmark statistics. This calibration provides a reference for each protein so that hits can be identified for an MOI. VTS can then be used as tool for: drug repositioning, specificity and toxicity testing, identifying potential metabolites, probing protein structures for allosteric sites, and testing focused libraries for selectivity.

5.5. iRAISE (inverse rapid index-based screening engine) Integrates flexibility of hydrophilic rotatable terminal groups (such as hydroxyl groups) of the active site and the query molecule. iRAISE is an inverse screening tool based on the RApid Index-based Screening Engine (RAISE) technolog.

5.6. ACTP (Autophagic Compound-Target Prediction) Autophagy (macroautophagy) is well known as an evolutionarily conserved lysosomal degradation process for long-lived proteins and damaged organelles. Recently, accumulating evidence has revealed a series of small-molecule compounds that may activate or inhibit autophagy for therapeutic potential on human diseases. However, targeting autophagy for drug discovery still remains in its infancy. In this study, we developed a webserver called Autophagic Compound-Target Prediction (ACTP) that could predict autophagic targets and relevant pathways for a given compound.

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6. CONCLUSION Here reviewed all four method tools that have been developed so far and discussed about their uses and advantages compared to others. Efforts are needed to validate the results of different prediction methods. In QSAR correlation between calculated properties of molecules and their experimentally determined biological activity may be derived. Target fishing, or target identification, is an important start step in modern drug development, which investigates the mechanism of action of bioactive small molecules by identifying their interacting proteins. Reverse or inverse docking is proving to be a powerful tool for drug repositioning and drug rescue. Examined how servers and tools were built and what algorithms were applied and what methods were used in detail. This review covers all available tools for the ligand-based drug discovery, which will be beneficial to the researchers who are working on medicinal and/or natural product chemistry.

7. REFERENCES

Buratto, R. (2015). Exploring Ligand Affinities for Proteins by NMR of Long-Lived States. Cereto-Massagué, A., Ojeda, M. J., Valls, C., Mulero, M., Pujadas, G., & Garcia-Vallve, S. (2015). Tools for in silico target fishing. Methods, 71, 98-103. Chirag N. Patel, J. J. G., ., & et al. (2017). Molecular recognition analysis of human acetylcholinesterase enzyme by inhibitors: An in silico approach. Paper presented at the Proceedings of International Science Symposium on Recent Trends in Science and Technology (ISBN: 9788193347553). Gauravi Trivedi, J. J. G. (2016). Identification of novel drug targets and its Inhibitors from essential genes of human pathogenic Gram positive bacteria. Paper presented at the Proceedings of 9th National Level Science Symposium on Recent Trends in Science and Technology (ISBN: 9788192952123). Georrge, J. J. (2015). DOCKING STUDIES, ADMET PREDICTION OF PHYTOCHEMICAL INHIBITORS FOR ALZHEIMER’S DISEASE. Paper presented at the Proceedings of 8th National Level Science Symposium on Recent Trends in Science and Technology (ISBN: 9788192952116). Georrge, J. J. (2016). A Bioinformatics Approach for the Identification of Potential Drug Targets and Identification of Drug-like Molecules for Ribosomal Protein L6 of Staphylococcus species. Paper presented at the Proceedings of 9th National Level Science Symposium on Recent Trends in Science and Technology (ISBN: 9788192952123). Georrge, J. J., & Umrania, V. (2011). In silico identification of putative drug targets in Klebsiella pneumonia MGH78578. Georrge, J. J., & Umrania, V. (2012). Subtractive genomics approach to identify putative drug targets and identification of drug-like molecules for beta subunit of DNA polymerase III in Streptococcus species. Applied Biochemistry and Biotechnology, 167(5), 1377-1395. John L, J. G., Kholia T. (2012). A Reverse Vaccinology Approach for the Identification of Potential Vaccine Candidates from Leishmania spp. Applied Biochemistry and Biotechnology, 167(5), 1340-1350. Joseph, V., Georrge, J., Pandya, J., & Jadeja, R. (2015). O-Vanillin and Some of its Novel Schiff Bases: A Cheminformatic Approach to Identify their Biological Functions. J Theor Comput Sci, 2(136), 2. Kharkar, P. S., Warrier, S., & Gaud, R. S. (2014). Reverse docking: a powerful tool for drug repositioning and drug rescue. Future medicinal chemistry, 6(3), 333-342. Kotadiya, R., & Georrge, J. J. (2015). In silico approach to identify putative drugs from natural products for Human papillomavirus (HPV) which cause cervical cancer. Life Sciences Leaflets, 62, 1-13. Lavecchia, A., & Cerchia, C. (2016). In silico methods to address polypharmacology: current status, applications and future perspectives. Drug discovery today, 21(2), 288-298. Lee, A., Lee, K., & Kim, D. (2016). Using reverse docking for target identification and its applications for drug discovery. Expert opinion on drug discovery, 11(7), 707-715. Li, G.-B., Yu, Z.-J., Liu, S., Huang, L.-Y., Yang, L.-L., Lohans, C. T., & Yang, S.-Y. (2017). IFPTarget: A Customized Virtual Target Identification Method Based on Protein–Ligand Interaction Fingerprinting Analyses. Journal of chemical information and modeling, 57(7), 1640-1651. Mori, M., Cau, Y., Vignaroli, G., Laurenzana, I., Caivano, A., Vullo, D., . . . Botta, M. (2015). Hit recycling: discovery of a potent carbonic anhydrase inhibitor by in silico target fishing. ACS chemical biology, 10(9), 1964-1969. Nishita NV, P. S., John J. Georrge. (2016). Modeling mutations, docking, primer and probe designing of Cytochrome P450 2D6, a drug metabolizing enzyme. Paper presented at the Proceedings of 9th National Level Science Symposium on Recent Trends in Science and Technology (ISBN: 9788192952123). Patel, C. N., Georrge, J. J., Modi, K. M., Narechania, M. B., Patel, D. P., Gonzalez, F. J., & Pandya, H. A. (2017). Pharmacophore-based virtual screening of catechol-o-methyltransferase (COMT) inhibitors to combat Alzheimer’s disease. Journal of Biomolecular Structure and Dynamics, 1-20.

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Rutvi Chovatiya, J. J. G. (2016). Identification of potential phytochemical inhibitors for the treatment of allergic asthma from the medicinal plants. Paper presented at the Proceedings of 9th National Level Science Symposium on Recent Trends in Science and Technology (ISBN: 9788192952123). Santiago, D. N., Pevzner, Y., Durand, A. A., Tran, M., Scheerer, R. R., Daniel, K., . . . Brooks, W. H. (2012). Virtual target screening: validation using kinase inhibitors. Journal of chemical information and modeling, 52(8), 2192-2203. Soni, R., Durgapal, S. D., Soman, S. S., & Georrge, J. J. (2016). Design, synthesis and anti-diabetic activity of chromen-2-one derivatives. Arabian Journal of Chemistry. doi:https://doi.org/10.1016/j.arabjc.2016.11.011 Vyas, N., & Georrge, J. J. (2015). 2DQSAR, DOCKING AND ADME STUDIES OF PTP1B INHIBITORS: A CURE OF DIABETES. Paper presented at the Proceedings of 8th National Level Science Symposium on Recent Trends in Science and Technology (ISBN: 9788192952116). Wolber, G., Seidel, T., Bendix, F., & Langer, T. (2008). Molecule-pharmacophore superpositioning and pattern matching in computational drug design. Drug discovery today, 13(1-2), 23-29.

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STATISTICAL ANALYSIS OF INDUSTRIALLY IMPORTANT THERMOPHILIC ORGANISMS PRODUCING ALPHA-AMYLASE, DNA POLYMERASE AND PROTEASE Rija George1, John J Georrge2* 1,2 Department of Bioinformatics, Christ College, Rajkot-360 005, Gujarat *Corresponding author: [email protected] & [email protected]* ABSTRACT: Enzymes derived from thermophilic organisms can perform industrial processes even under harsh conditions, under which conventional proteins are completely denatured which makes it superior to the traditional catalysts. Statistical analysis of industrially important thermophilic organisms and the organism producing thermostable Alpha-amylase, DNA polymerase and Protease was carried out. The databases used to complete this analysis are NCBI and UniProt. The thermophilic organisms that produce thermophilic Alpha-amylase, DNA polymerase and Protease are 97, 125 and 74 respectively. These data have substantial implications for the completion of the genome sequencing of rest of the organisms, protein stability studies, protein engineering, mutational studies etc.

1. INTRODUCTION The organism that has its optimal temperature, pH, salt concentrations, and pressure for their survival outside of ‘normal’ environment is known as extremophiles (Kristjansson et al., 1995) (Cavicchioli, 2002). To help the extremophile organism to survive in ecological niches, they have established a variety of molecular strategies. As a result, Metabolic systems and enzymes even function at temperatures between –5 and 130 °C, pH 0–12, salt concentrations of 3–35%, and pressures up to 1000 bar (Bertoldo et al., 2009). Extremophiles have different classes like hyperthermophile (Tmax 121°C High-temperature growth), psychrophile (-17°C Cold temperature), acidophile (pHopt 0.7 High acid), halophile (Saturated salt (up to 5.2 M) High salt), alkaliphile (pHopt. 10 High alkaline), radiation-tolerant (High, g, UV, x-ray radiation), toxitolerant (Toxicity), barophile or piezophile (High pressure), oligotroph (Low nutrients), xerophile (Low water activity) (Cavicchioli, 2002). A thermophile is an entity, a type of extremophile that thrives at relatively high temperatures, between 41 and 122 °C (106 and 252 °F). The organism that grow optimally above 80°C are Extremothermophiles and are also termed as “hyperthermophiles” (de Miguel Bouzas et al., 2006) (Stetter, 2007). There are many classifications for thermophiles among which more eminent classification criteria are Moderate thermophiles (45–64 °C), Extreme thermophiles (65–79 °C) and Hyperthermophiles (>80 °C). The group of organisms that grow in extreme environments were named by Carl Woese as archaea- a group with diverse features and hypothesized the archaea as the third domain of life on earth, different from bacteria and Eukarya (Bertoldo et al., 2009). There are bacteria found universally in soil and volcanic habitats which are grouped as thermophiles (Zeikus, 1979). 2. THERMOSTABILITY OF THE ORGANISMS There are several mechanisms used by thermophiles and hyperthermophiles to maintain the stability. Historically, the growth temperature has a significant relation with the lipid composition of the membrane. The organisms growing at elevated temperatures contain lipids with higher melting points (Bělehrádek, 1931). More resistance is observed in monolayer membrane composed of phytanyl chains connected to glycerol with ether links (Comita et al., 1984). The adaptations are looked-for to retain the membrane in a liquid crystalline state (Russell et al., 1990) and to bound the proton permeation rates which helps Bacteria and Archaea to respond to changes in ambient temperature. This can be done in Bacteria at higher temperatures by increasing the chain length of the lipid acyl chains, the ratio of iso/anteiso branching and/or the degree of saturation of the acyl chain (Albers et al., 2000). In some of the Archaea the degree of cyclization of the C40 isopranoid in the tetraether lipids is increased at higher growth temperatures. The lipids can be crammed more tightly by the intensification of the cyclization of the C40 isopranoid chains, as a result a more restricted motion of the lipids and prevents that the membrane becomes too fluid (De Rosa et al., 1988).

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A particular type of DNA topoisomerase is produced by all thermophiles, which is called DNA reverse gyrase, which at the expense of ATP introduces positive supercoils in the DNA molecule, this confers a greater stability and renders it more resistant to thermal denaturation. Some histone-like proteins that help to preserve the duplex structure of DNA, such proteins increase the fusion temperature of DNA up to 40ºC above normal values this is not present in all hyperthermophiles (de Miguel Bouzas et al., 2006).

3. REASON FOR THERMAL STABILITY OF PROTEIN The resistance of material to the changes in physical structure or chemical irreversibility, or spatial structure stability of polypeptide chains at high temperatures is defined as Thermal stability. The sequential and structural properties of protein are the basic reason for thermal stability of thermophilic proteins. To understand the thermal stability of thermophilic proteins the amino acid compositions’ difference had been studied in mesophilic and thermophilic proteins. Alpha-helix the protein secondary structure stability is considered as a necessary factor for thermal stability. Studies suggested that thermal-stability is increased by the characteristics of proteins like increased number of hydrogen bonds, salt bridges, ion pairs, aromatic clusters, sidechain- sidechain interactions, electrostatic interactions of charged residues and hydrophobic interactions (Mahmoudi et al., 2016). 4. AMYLASE Amylase is an enzyme that catalyzes the process of hydrolysis of starch into sugars. The presence of which is in the saliva of humans and other mammals, where it begins digestion (Hill, 1970). Amylase is classified as α- Amylase, β-Amylase and γ-Amylase. 4.1 Alpha-amylase α-Amylase is a protein enzyme EC 3.2.1.1 that hydrolyses alpha bonds of long-chain carbohydrates, such as starch and glycogen and produces glucose and maltose (Pugh, 2000). α-amylase inclines to be faster-acting than β-amylase, because it can work anywhere on the substrate. In human physiology, α-amylases is the type of amylase found in both the saliva and pancreas. It is the chief form of amylase present in humans and other mammals (Voet, 2005). 4.2 Genome and proteome Thermophilic organisms producing Alpha-amylase are 97 in number, among which 39 are Archaea and 58 are Bacteria. The organisms whose whole sequence is available among these 97 are 78, among which 33 are Archaea and 45 Bacteria (NCBI). These 97 organisms are from 34 families and the maximum number of

Moderate Thermophiles(45- Hyperthermophiles 64℃) Archaea, 4 (>80℃) Bacteria, 8 Moderate Thermophiles(45- 64℃) Bacteria, 19

Extreme Thermophiles(65- Hyperthermophiles 79℃) Archaea, 0 (>80℃) Archaea, 35

Extreme Thermophiles(65- 79℃) Bacteria, 31

Figure 1: Thermophilic Alpha-amylase producing organisms classified based on three different temperature classes

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 66 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Bioinformatics organisms are from Thermococcaceae family. The organisms of this family are isolated from hydrothermal vents. They are adapted to an environment with temperature ranging from 70°C – 100 °C (Amenábar et al., 2013). There are 199 sequences of thermophilic Alpha-amylase. Moderate thermophiles, Extreme thermophiles and Hyperthermophiles. On classifying the thermophilic organisms producing Alpha-amylase into the three classes: Moderate thermophilic archaea are 4 and moderate thermophilic bacteria are 19, no organism come under the class Extreme thermophilic archaea and Extreme thermophilic bacteria are 31, Hyperthermophilic archaea are 35 and Hyperthermophilic bacteria are 8 (Figure 1).

70 64 60 60 50 50 40

30 21 20 10 4 0 0 Moderate- Thermophilic Extremophilic Hyper thermophilic ■ Archaea ■ Bacteria

Figure 2: The number of sequences available for thermostable Alpha-amylase.

There are 4, 60, 64, 50 and 21 Alpha-amylase protein sequences available for Moderate Thermophilic Archaea, Moderate Thermophilic Bacteria, Extreme Thermophilic Bacteria, Hyper Thermophilic Archaea and Hyper Thermophilic Bacteria respectively (Figure 2). 5. DNA POLYMERASE (EC 2.7.7.7) In molecular biology, DNA polymerases are enzymes that synthesize DNA molecules from deoxyribonucleotides, the building blocks of DNA (Bollum, 1960). These enzymes are essential for DNA replication and usually work in pairs to create two identical DNA strands from a single original DNA molecule (Falaschi et al., 1966). The primary role of DNA polymerases is to accurately and efficiently replicate the genome to ensure the maintenance of the genetic information and its faithful transmission through generations. DNA polymerase are classified as Bacterial DNA polymerase and Archaeal DNA polymerase. 5.1 Bacterial DNA Polymerase 5.1.1 Pol I Prokaryotic Family A polymerases include the DNA polymerase I (Pol I) enzyme (Ulrich et al., 2010). Pol I is the most abundant polymerase, accounting for >95% of polymerase activity in E. coli. 5.1.2 Pol II DNA Pol II is an 89.9 kD protein, composed of 783 amino acids, that is encoded by the polB (dinA) gene (Banach‐Orlowska et al., 2005). 5.1.3 Pol III DNA polymerase III holoenzyme is the primary enzyme involved in DNA replication in E. coli and belongs to family C polymerases (Olson et al., 1995). 5.1.4 Pol IV Function of Pol IV is to perform translation synthesis (Goodman, 2002). 5.1.5 Pol V DNA polymerase V (Pol V) is a Y-family DNA polymerase that is involved in SOS response and translation synthesis DNA repair mechanisms (Patel et al., 2010).

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Moderate Thermophiles(45- Hyperthermophiles( 64℃) Archaea, 11 Hyperthermophiles( >80℃) Bacteria, 9 >80℃) Archaea, 20

Extreme Thermophiles(65- Moderate 79℃) Bacteria, 30 Thermophiles(45- 64℃) Bacteria, 53

Extreme Thermophiles(65- 79℃) Archaea, 2

Figure 3: Thermophilic DNA polymerase producing organisms classified based on three different temperature classes

5.2 Archaeal DNA Polymerase 5.2.1 Polymerases β, λ, σ and μ (beta, lambda, sigma, and mu) Family X polymerases contain the well-known eukaryotic polymerase pol β (beta), as well as other eukaryotic polymerases such as Pol σ (sigma), Pol λ (lambda), Pol μ (mu), and Terminal deoxynucleotidyl transferase (TdT) (Yamtich et al., 2010). 5.2.2 Polymerases α, δ and ε (alpha, delta, and epsilon) Pol α (alpha), Pol δ (delta), and Pol ε (epsilon) are members of Family B Polymerases and are the main polymerases involved with nuclear DNA replication (Peet, 2012). 5.2.3 Polymerases η, ι and κ (eta, iota, and kappa)

100 90 80 63 60 48 40 19 20 14 5 0 Moderate Thermophilic Extremophilic- Hyper thermophilic- ■ Archaea ■ Bacteria

Figure 4: The number of sequences available for thermostable DNA polymerase.

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Pol η (eta), Pol ι (iota), and Pol κ (kappa), are Family Y DNA polymerases involved in the DNA repair by translation synthesis and encoded by genes POLH, POLI, and POLK respectively (Ohmori et al., 2009). 5.2.4 Polymerases Rev1 and ζ (zeta) Pol ζ another B family polymerase, is made of two subunits Rev3, the catalytic subunit, and Rev7, which increases the catalytic function of the polymerase, and is involved in translation synthesis (Gan et al., 2008). 5.3 Genome and proteome The thermophilic DNA polymerase producing organisms whose whole genome sequences are available are 48, among which 24 are archaea and 24 are bacteria (NCBI). There are 125 organisms producing thermophilic DNA polymerase which includes 92 bacteria and 33 archaea (NCBI and UniProt). On classifying the thermophilic organisms producing DNA polymerase into the three classes: Moderate thermophilic archaea are 11 and moderate thermophilic bacteria are 53, Extreme thermophilic archaea are 2 and Extreme thermophilic bacteria are 30, Hyperthermophilic archaea are 20 and Hyperthermophilic bacteria are 9 (Figure 3). There are 19, 90, 5, 63, 48 and 14 DNA polymerase protein sequences available for Moderate Thermophilic Archaea, Moderate Thermophilic Bacteria, Extreme Thermophilic Archaea, Extreme Thermophilic Bacteria, Hyper Thermophilic Archaea and Hyper Thermophilic Bacteria respectively (Figure 4).

6. PROTEASES Generally, Proteases are the enzymes that catalyze the splitting of proteins into smaller peptide fractions and amino acids by the process of proteolysis. Proteases can be found in Animalia, Plantae, Fungi, Bacteria, Archaea and viruses. Proteases can be classified into seven broad groups based on their catalytic residue like Serine proteases, Cysteine proteases, Threonine proteases, Aspartic proteases, Glutamic protease, Metalloproteases and Asparagine peptide lyases (Oda, 2011). 6.1 Genome and Proteome In a total there are 58 thermophilic organisms whose whole genome sequencing is completed and are producing proteases. Among which 34 are Archaea and 24 are Bacteria (NCBI). Thermophilic Protease producing

Moderate Hyperthermophiles( Thermophiles(45- >80℃) Bacteria, 3 64℃) Archaea, 4

Hyperthermophiles( Moderate >80℃) Archaea, 29 Thermophiles(45- 64℃) Bacteria, 26

Extreme Thermophiles(65- 79℃) Bacteria, 9 Extreme Thermophiles(65- 79℃) Archaea, 3 Figure 5: Thermophilic protease producing organisms classified based on three different temperature classes

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 69 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Bioinformatics organisms are 74 in total. These organisms were divided into 3 class on the basis of the temperature range that is, Moderate thermophiles, Extreme thermophiles and Hyperthermophiles. Moderate thermophilic archaea are 4 and moderate thermophilic bacteria are 26, Extreme thermophilic archaea are 3 and Extreme thermophilic bacteria are 9, Hyperthermophilic archaea are 29 and Hyperthermophilic bacteria are 3. Collectively, the class moderate thermophile, Extreme Thermophiles and Hyperthermophiles includes 30, 12 and 32 organisms respectively (Figure 5). There are 19, 68, 4, 16, 38 and 3 protease protein sequences available for Moderate Thermophilic Archaea, Moderate Thermophilic Bacteria, Extreme Thermophilic Archaea, Extreme Thermophilic Bacteria, Hyper Thermophilic Archaea and Hyper Thermophilic Bacteria respectively (Figure 6).

80 68 70 60 50 38 40 30 19 20 16 10 4 3 0 Moderate Thermophilic Extremophilic Hyper thermophilic

■ Archaea ■ Bacteria

Figure 6: The number of sequences available for thermostable protease.

7. CONSENSUS ORGANISMS Organisms producing Alpha-amylase and also DNA polymerase are 18 in number, among these 15 of them are having their whole genome sequence available. The number of organisms producing both Alpha-amylase and Protease are 25 out of these 21 of the organisms’ whole genome sequence is available. There are 14 organisms producing DNA polymerase as well as protease; 9 organisms among these 14 have their whole genome sequence and 19 organisms that produces all these three proteins have their whole genome sequence too.

8. CONCLUSION The organisms producing industrially important proteins have to be explored more by sequencing their whole genome preliminarily and analyzing it. The yield and activity of the organisms and the proteins can improve by many computational studies. Biological experiments have been done in abundance which has generated a lot of genomic and proteomic information which are left unanalyzed. Stability studies of protein is to be done to explore more about their industrial applications. To improve the stability and activity of the protein, mutational studies has to be done through protein engineering. These studies if done biologically they are time consuming which can be streamlined through bioinformatics analysis, as currently so many bioinformatics tools are available to analysis the data generated.

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REFERENCES Albers, S.-V., Van de Vossenberg, J., Driessen, A., & Konings, W. N. (2000). Adaptations of the archaeal cell membrane to heat stress. Front Biosci, 5, 813-820. Amenábar, M. J., Flores, P. A., Pugin, B., Boehmwald, F. A., & Blamey, J. M. (2013). Archaeal diversity from hydrothermal systems of Deception Island, Antarctica. Polar biology, 36(3), 373-380. Banach‐Orlowska, M., Fijalkowska, I. J., Schaaper, R. M., & Jonczyk, P. (2005). DNA polymerase II as a fidelity factor in chromosomal DNA synthesis in Escherichia coli. Molecular microbiology, 58(1), 61-70. Bělehrádek, J. (1931). Le mecanisme physico-chimique de l'adaptation thermique. Protoplasma, 12(1), 406- 434. Bertoldo, C., & Antranikian, G. (2009). THERMOACTIVE ENZYMES IN BIOTECHNOLOGICAL APPLICATIONS. EXTREMOPHILES-Volume I, 3, 294. Bollum, F. (1960). Calf thymus polymerase. J biol chem, 235(8), 2399-2404. Cavicchioli, R. (2002). Extremophiles and the search for extraterrestrial life. Astrobiology, 2(3), 281-292. Comita, P. B., Gagosian, R. B., Pang, H., & Costello, C. E. (1984). Structural elucidation of a unique macrocyclic membrane lipid from a new, extremely thermophilic, deep-sea hydrothermal vent archaebacterium, Methanococcus jannaschii. Journal of Biological Chemistry, 259(24), 15234-15241. de Miguel Bouzas, T., Barros-Velázquez, J., & Gonzalez Villa, T. (2006). Industrial applications of hyperthermophilic enzymes: a review. Protein and peptide letters, 13(7), 645-651. De Rosa, M., & Gambacorta, A. (1988). The lipids of archaebacteria. Progress in lipid research, 27(3), 153- 175. Falaschi, A., & Kornberg, A. (1966). Biochemical studies of bacterial sporulation II. Deoxyribonucleic acid polymerase in spores of Bacillus subtilis. Journal of Biological Chemistry, 241(7), 1478-1482. Gan, G. N., Wittschieben, J. P., Wittschieben, B. Ø., & Wood, R. D. (2008). DNA polymerase zeta (pol ζ) in higher eukaryotes. Cell research, 18(1), 174. Goodman, M. F. (2002). Error-prone repair DNA polymerases in prokaryotes and eukaryotes. Annual review of biochemistry, 71(1), 17-50. Hill, R. (1970). The chemistry of life: Eight lectures on the history of biochemistry: CUP Archive. Kristjansson, J., & Hreggvidsson, G. (1995). Ecology and habitats of extremophiles. World Journal of Microbiology and Biotechnology, 11(1), 17-25. Mahmoudi, M., Arab, A., Zahiri, J., & Parandian, Y. (2016). An Overview of the Protein Thermostability Prediction: Databases and Tools. J Nanomed Res, 3(6), 00072. Oda, K. (2011). New families of carboxyl peptidases: serine-carboxyl peptidases and glutamic peptidases. The Journal of Biochemistry, 151(1), 13-25. Ohmori, H., Hanafusa, T., Ohashi, E., & Vaziri, C. (2009). Separate roles of structured and unstructured regions of Y-family DNA polymerases. In Advances in protein chemistry and structural biology (Vol. 78, pp. 99- 146): Elsevier. Olson, M. W., Dallmann, H. G., & McHenry, C. S. (1995). DnaX Complex of Escherichia coli DNA Polymerase III Holoenzyme THE χ· ψ COMPLEX FUNCTIONS BY INCREASING THE AFFINITY OF τ AND γ FOR δ· δ′ TO A PHYSIOLOGICALLY RELEVANT RANGE. Journal of Biological Chemistry, 270(49), 29570-29577. Patel, M., Jiang, Q., Woodgate, R., Cox, M. M., & Goodman, M. F. (2010). A new model for SOS-induced mutagenesis: how RecA protein activates DNA polymerase V. Critical reviews in biochemistry and molecular biology, 45(3), 171-184. Peet, A. (2012). Marks' Basic Medical Biochemistry: Lippincott Williams & Wilkins. Pugh, M. B. (2000). Stedman's Medical Dictionary (27 Ed.): Baltimore, Maryland, USA: Lippincott Williams & Wilkins. www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 71 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Bioinformatics

Russell, N., & Fukunaga, N. (1990). A comparison of thermal adaptation of membrane lipids in psychrophilic and thermophilic bacteria. FEMS Microbiology Letters, 75(2‐3), 171-182. Stetter, K. O. (2007). History of Discovery of the First Hyperthermophiles. Paper presented at the Proceedings of International Symposium on Extremophiles and Their Applications International Symposium on Extremophiles and Their Applications 2005. Ulrich, H., Giovanni, M., & Giuseppe, V. (2010). DNA Polymerases: Discovery, characterization and functions in cellular DNA transactions: World Scientific. Voet, D., & Voet, J. G. (2005). Biochimie. (2 ed.): Bruxelles: De Boeck. Yamtich, J., & Sweasy, J. B. (2010). DNA polymerase family X: function, structure, and cellular roles. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1804(5), 1136-1150. Zeikus, J. (1979). Thermophilic bacteria: ecology, physiology and technology. Enzyme and Microbial Technology, 1(4), 243-252.

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IN SILICO PROTEIN ENGINEERING: METHODS AND TOOLS Sneha Thomas, John J Georrge* Department of Bioinformatics, Christ College, Rajkot-360 005, Gujarat, India Email ID: [email protected], [email protected]* ABSTRACT: Protein engineering, the process by which novel proteins with desired properties are developed, has grown by leaps and bounds. Important objective of protein engineering is the design of peptides that can bind selectively to target proteins. Protein engineering strategies aimed at constructing enzymes with novel or improved activities, specificities, and stabilities greatly benefit from in silico methods such as: (i) bioinformatics, (ii) molecular modelling, and (iii) de novo design. Computational approaches offer significant potential for engineering protein structure and function, and can be combined with experimental testing to gain new insights into the fundamental properties of proteins. It is based on altering the affinity of a protein by replacing some amino-acid residues with another to identify the most important residues and their specific involvement to the binding activity. This chapter focuses on the detailed information of the methods and tools of protein engineering and will be beneficial for the researchers pursuing in this field.

1. INTRODUCTION Protein structure design and engineering is a research endeavour in which proteins with predicted structure and function are constructed in the laboratory through rational design, combinatorial selection or combination of both approaches. It is built upon our knowledge about the structure and function of proteins and can be accomplished either from scratch (de novo design) or based on native scaffolds (redesign) (Berry et al., 2011).Protein engineering is the design of new enzymes or proteins with new or desirable functions. It is based on the use of recombinant DNA technology to change amino acid sequences. The ability of proteins to recognize other molecules in a highly selective and specific manner and to create supramolecular complexes has many biological implications. For example, interactions between receptor-ligand, antigen-antibody, DNA- protein, lectin-sugar are involved in many biologically important processes including transcription of genetic information, enzyme catalysis, transmission of nervous and hormonal signals, host recognition by microbes etc. Therefore, characterizing the structure and energy profile of such supramolecular complexes appears as a key factor in understanding biological function. Protein engineering is useful not only in the characterization of a protein's binding abilities, but also has applications in bioanalysis and biotechnology. Protein engineering is typically performed in vitro, with in vivo consequences and applications. In some cases, it may be very efficient to perform computer modelling and simulations before starting wet laboratory experiments. In such cases in silico protein engineering is used, and the main goal is to design appropriate mutations in a much faster and cheaper way. Protein engineering differs from protein design. In protein engineering an existing protein is changed chemically or genetically so that its function can be changed as per desire. In protein design, the aim of the process is to fulfil a desired function. Protein engineering is a multidisciplinary science. The engineering and design flow chart requires the interaction of physicists, biophysicists, molecular biologists, organic chemists and computer scientists. 2. STEPS OF PROTEIN ENGINEERING The first describes methods for 3D structure prediction, a necessary step to perform any In silico engineering, but not involved in the engineering itself. We further describe various approaches for in silico mutagenesis. Afterwards we introduce many techniques which enable the prediction of the preferred orientation of the ligand in the binding pocket, as well as the calculation of the binding free energy, again a technique not directly included in protein engineering itself, but necessary to perform it. The whole process is schematically shown in the flowchart in Fig. 1.

2.1 THREE DIMENSIONAL STRUCTURE AS THE KEY PREREQUISITE The initial point in the in-silico protein engineering process is the 3D structure of a receptor. Current experimental methods for protein structure determination are very well established. If the experimental structure is not available, computational approaches are used to model the 3D structure of the receptor such as X-ray crystallography, NMR spectroscopy, homology modelling and threading.

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2.1.1 X-ray crystallography X-ray crystallography is used to Structure NO Homologous Protein Fold determine the arrangement of Available Proteins libraries atoms in a crystal lattice. The YES procedure of the 3D structure obtaining is composed of four key YES Threading steps such as: (1) to crystallize the protein, (2) to collect the J Structure Protein diffraction, (3) to calculate the Database Structure electron density map, (4) to refine and validate the model of the structure of the protein. A number of factors contributes to the final In silica ------1 Protein/ligand Complex quality of an X-ray structure. The Mutagenesis first factor relates to the crystal characteristics and its diffraction properties, and is evaluated in terms of resolution. Here, the term resolution refers to the level of Mutant Structure detail that can be inferred from the electron density map. Most errors Fig 1: Flowchart of steps performed within in Silico protein engineering result from highly disordered areas in the electron density maps, like flexible loops of proteins. The electron density of atoms with high residual disorder is smeared in the electron density map, and is no longer detectable (Giacovazzo, 2002).

Templalle sequence Target sequence ATQCVTTLPAJITllFCVTAF AX'SSCTQTVllVJ.\llffllT, J'.Ul)SOTSDrU.carst,PHTDrllIPPIJI.U.J:QQHDa.PlQ)S TPSCQSTIDlAVICTQVLIJSCSSCRVQVQVSVllC~JISI OIPUTHKL TT l!tlOP R.lltl.H SQP.;IG RJ'J:VSVWGKP'S.A. TOU U.Pl.SQO(Sl)C.S P7TV>il7CIW1J.IDQIDCIVVL4.VPIC VSAQVlLTillLNFIJ.VCSIDCTl>lll)VIIDAVVVDIIIP y t 2) Sequence aHgnment 1) Templall! structure

lWOC' lia.T- ·•-COl't'flJ'Alf"'9.fOffUAM9C..~WN-'tuA't'H

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Fig 2 Four main steps to solve a protein structure by X-ray crystallography

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2.1.2 NMR spectroscopy NMR spectroscopy is often the only way to obtain high resolution information on protein dynamics as well as on the protein structure in a solvent. For solving a protein structure by NMR in solution it is needed: 1) to know the amino acid sequence, 2) to measure the multidimensional spectra 3) to calculate the distances by NOE and J-coupling effects and 4) to refine and validate the 3D structure of the protein. The molecule of interest is placed in a strong magnetic field, and each of these nuclei is characterized by a unique resonance frequency, depending on the electron density of the local chemical environment. The most important method for protein structure determination utilizes NOE (Nuclear Overhauser effect) experiments to measure the distances between pairs of atoms within the molecule that are not connected via chemical bonds (Wüthrich, 2003). The goal is to assign the observed chemical shifts from multidimensional spectra to their specific atoms (nuclei) in the protein. All the values are then quantified and translated into angle and distance restrains. These restraints are subsequently used to generate the 3D structure of the molecule by solving a distance geometry problem (Saccenti et al., 2008).

NMR

2) 2D spectrum of prote 1) amino acid sequence • • Rl H R.2 -· • • • I I I ;' .. CH- C- N- CH ,; . I b • n • " " ··--- ~ 31 Interactions in spac

H- 0-' 1 H'-H .,I ' H 0 - eI- ii,c-c .~ 4) Final structure I • Fig 3 Protein structure by NMR in solution

2.1.3 Homology modelling In case of X-ray crystallography and NMR spectroscopy, the structures of many biotechnologically and therapeutically relevant proteins remain undiscovered for various reasons. In such circumstances, homology modeling can be used to obtain their 3D structure. Homology modeling is a purely computational procedure that consists of building a protein model using a structural template, normally coming from proteins with a known structure. Homology modeling consists of: 1) Identification of the template, 2) Alignment of the target sequence with the template sequence, 3) Building the target protein backbone, loops and side chains and 4) Refining and evaluating the final model.

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Te, 111:.t..-;e ~1:11;.0qoool~ Ta~gct :stqu f!! r,,,c h TQQVJ'TI. P ..U.TRFSGtSSHAAU.1SIPPlitt>U.U P P.lll .U:tocarni., lCOS )l!l>l,..i.vu:l(l.'TTll\/ll>f:'U .TLllltt:llr.Jtlllf\'.11q-':l((>HAV J.~l'IJV 1M ::;1,,::;:si;.te1f>T TVMri:l\lV\' lt:>Cwt>ft>\"'ltDOt VVUIIUllC VSAOVtLTN8LW'ALVCSICCTDJmYJ.1)AV'1VDI\IPlC y t 2) 3cquc nc4!! ot:ig nment 1} Template slructure•

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;r.,x f'OIIU{~.V.'7~$1oUPK'hl.f"-- ····ilJJf.l~ ,..,._: a,,~-QQUr~&-.ltOAIU."t11Q,»;D0:Ur~~...... ,...... ·-·"•- -lffl~~•...... ,.q

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Fig 4 Steps included in Homology modeling

2.1.3.1 Template selection and sequence alignment The first step for comparative modeling is to check whether there is any protein in the current PDB database having a similar sequence as the protein of interest. If so, the structure of this protein will be used as a template. The search for the template has to proceed using a sequence comparison algorithm that is able to identify the global sequence similarity (Roessler et al., 2008). The sequence of the protein with unknown structure is aligned against the sequence of the template protein, meaning that the sequences are arranged in such a way that the regions which contain the same amino acids in both proteins are superimposed. Then the Cα coordinates of the aligned residues from the template are copied over to the target protein in order to form the skeletal backbone. Commonly used alignment techniques are: standard pairwise sequence alignment, where only 2 sequences are compared at a time, or multiple sequence alignment, where more sequences are compared at a time and which is generally used when the target and template sequences belong to the same family. There are complex sequence alignment algorithms that optimize a score based on a substitution matrix and gap penalties (Nayeem et al., 2006). 2.1.3.2 Loop building Loops participate in many biological events and contribute to functional aspects such as enzyme active sites formation or ligand-receptor recognition. Databases of loop conformations or modeling by ab initio methods are used in order to determine the proper structure of loops. The ab initio loop prediction approach relies on a conformational search guided by various scoring functions and is used for longer loops (di Luccio et al., 2011). 2.1.3.3 The side-chain positioning problem Most of the side-chain positioning methods are based on rotamer libraries with discrete side-chain conformations. Rotamer libraries contain a list of all the preferred conformations of the side-chains of all twenty amino acids, along with their corresponding dihedral angles. Side chain prediction techniques choose the best rotamer for each residue of the protein based on a score that includes both geometric and energetic constraints (combinatorial problem). The combinatorial problem is solved by heuristic techniques such as mean field theory, derivatives of the dead-end elimination theorem or Monte Carlo techniques (Lovell et al., 2000).

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2.1.3.4 Refinement and validation of the final model The final step to determine the structure of a protein by homology modeling is refining the model. However, it was shown that refining a structural model by energy minimization only many times leads to structures that are different compared to those obtained by X-ray crystallography. To avoid such problems, several approaches can be applied including evolutionary derived distance constraints, the combination of molecular dynamics and statistical potentials, adding a differentiable smooth statistical potential or considering the solvent effects (Misura et al., 2006) (Zhu et al., 2008). For the model validation step, scoring functions are used. These are functions based on statistical potentials, local side-chain and backbone interactions, residue environments, packing estimates, solvation energy, hydrogen bonding, and geometric properties (di Luccio et al., 2011). Generally, the quality of the homology model is dependent on the quality of the sequence alignment and of the template structure. A variety of software such as GeneMine, DS MODELER, ICM, SWISS-MODEL are currently in use for homology modeling of protein structures. 2.1.4 Threading Threading is used to model the structure of a protein when no homologs with a known 3D structure are available. It works by comparing a target sequence against a library of potential fold templates using energy potentials and/or other similarity scoring methods. The template with the lowest energy score (or highest similarity score) is then assumed to best fit the fold of the target protein. Three main steps of threading are: The construction of a structure target database based on templates, Calculation of the quality of each model and Selecting the best model. Threading improves the sequence alignment sensitivity by introducing structural information (the secondary or tertiary structure of the targets) into the alignment. Threading methods are not able to give a good sequence–structure alignment. The first reason is that the structure information has many approximations. Most of the threading methods use a ‘frozen’ approximation. It means that the target residues are in the same environments as the template residues if they belong to the same structural fold. But, especially in loop regions, two homologous structures can have slightly different environments. Therefore, only conserved regions are used in threading (Madej et al., 1995). Variety of threading softwares such as GenTHREADER, 3D-PSSM, Phyre2 is available now. 2.2 IN SILICO MUTAGENESIS OF PROTEINS The ultimate goal of protein engineering is to design a protein with novel properties, starting from existing proteins. Protein engineering in the field of recognition has been particularly successful in changing ligand specificity and binding affinity. The availability of computational and graphical tools, which allow to display and explore the three-dimensional structures of proteins, has made in silico mutagenesis easier and more feasible. Basically, two approaches are available - mutation of a single, or of multiple residues. 2.2.1 Performing in silico mutagenesis Performing in silico protein mutagenesis basically means changing the lines of the text that encode the information about the residue being mutated, followed by a set of additional operations meant to properly integrate the mutated residue into the structure. The mutation of one residue to another does not change anything in the backbone atoms. In addition, the protein side chains all start by the β carbon atom, which is the same for all the amino acids except for the glycine. Therefore, the single amino acid mutation is straightforward, since only the side chain atoms need to be changed. Computational chemistry tools are able to include all the possible side chain conformations by using rotamer libraries. Several molecular modeling platforms facilitate single point mutation using the concept of rotamers. Some of commonly used software packages to perform single point or multiple point mutations at selected positions are Swiss-Pdb Viewer, Pymol, MODELLER, Triton. 2.2.2 Alanine scanning mutagenesis Alanine scanning mutagenesis is a method usually used to determine the contribution of a particular residue to protein function by mutating that residue into alanine. Alanine scanning involves substituting of a larger group of atoms with a smaller one. Alanine is the residue of choice because it removes the side chain beyond the β carbon of the amino acid in question, and, most importantly, because it does not alter the main-chain conformation (Wells, 1991). 2.3 QUALITATIVE AND SEMI-QUANTITATIVE APPROACHES TO EVALUATE THE RECOGNITION ABILITY OF PROTEINS A necessary prerequisite for any in silico protein engineering approach is the ability to evaluate how strong the recognition is. In biological systems, the process of recognition, governed by non-covalent interactions, results in the formation of a complex, where one biomacromolecule interacts with another biomacromolecule or a small molecule. Modern computer modeling and simulation methods, such as docking or free energy calculations, make it possible to study the molecular recognition process between two molecules in silico. Evaluation of the recognition ability of biomacromolecules is performed in two steps: (i) docking the small www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 77 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Bioinformatics molecule into the biomacromolecule and (ii) analyzing the interactions and factors that determine the binding affinity. 2.3.1 Principles of molecular docking Molecular docking is a widely-used computational tool for the study of molecular recognition, which aims to predict the preferred binding orientation of one molecule to another when bound together in a stable complex. Docking can be performed between two proteins, a protein and a small molecule, a protein and an oligonucleotide or between an oligonucleotide and a small molecule (Huang et al., 2010). 2.3.2 Receptor site characterization In the process of docking, the first issue is where to dock the ligand, i.e., how to define a search space on the receptor where the search will be performed. If the 3D structure and the binding site of the receptor is known, the search space is defined within and around this binding site (Huang et al., 2010). 2.3.3 Sampling protein and ligand conformational flexibility in docking The main docking operations focus on the ligand. However, during the docking, several preliminary assumptions need to be made about the receptor flexibility. About 85% of proteins undergo conformational changes upon ligand binding, mainly movements in the essential binding site residues. Therefore, performing accurate molecular docking is quite difficult, because of the many possible conformational states of both the biomacromolecule, and the ligand flexible areas. Flexible docking is further categorized into two types: flexible ligand docking, where only the conformation of the ligand changes during the docking, and flexible receptor docking, where both the conformation of the ligand and the conformation of the receptor can change (Najmanovich et al., 2000). 2.3.4 Sampling conformational and configurational space Search space where we sample the structural arrangement of two molecules without changing the conformation of any of the molecule is called configurational search space. The configurational and conformational search is done via a set of algorithms that sample all the desired degrees of freedom of the ligand in order to find the correct binding mode. The set of operations performed to improve a binding mode is often referred to as optimization (Brooks et al., 1983). 2.3.5 Scoring ligand poses To recognize the true binding modes from all the geometries, the binding affinity is scored using scoring functions, i.e., each binding mode is analysed by a set of equations and compared to the other binding modes. Numerous scoring functions developed and evaluated so far can be grouped into three basic categories such as Force field based, Empirical scoring functions and Knowledge based scoring Functions. 2.3.6 Techniques to improve the performance of scoring functions Consensus scoring: This is a combination of the information obtained from different scores. The approach is helpful in balancing out the error of individual scoring functions, thus improving the probability of finding an appropriate solution (Terp et al., 2001; Wang et al., 2002). Clustering: RMSD based clustering of all the docking solutions can be performed. To get the correct pose, the best energy conformation from the most populated cluster should be chosen. 2.4 FREE ENERGY CALCULATION However, the empirical scoring functions, which are based on a single receptor/ligand structure, do not provide accurate enough predictions of the binding free energy (ΔG), the key quantity characterizing the strength of the receptor/ligand interaction. To tackle this problem, molecular dynamics (MD) or Monte Carlo (MC) based methods for free energy calculation were developed (Jorgensen et al., 1985). To evaluate the binding free energy between the receptor and the ligand, namely Free Energy Perturbation (FEP), Thermodynamic Integration (TI), and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA).

3. TOOLS 3.1 CAVER (http://www.caver.cz/) The software tool CAVER is used to analyse tunnel dynamics in trajectories obtained by molecular dynamic simulations and complemented with an analysis of products egressing from buried active sites using Random Accelerated Molecular Dynamics (RAMD)(Chovancova et al., 2012). 3.2 SABER (Selection of Active/Binding Sites for Enzyme Redesign) It is mainly used to analyse the functional sites of the proteins stored in the Protein Data Bank. The tool identifies the active sites amendable to computational redesign by locating potential catalytic residues in pre-defined spatial arrangements(Nosrati et al., 2012). 3.3 POCKETOPTIMIZER (https://omictools.com/pocketoptimizer-tool) The tool can be used to modify the residues making up the protein binding pocket to improve or newly establish the binding of a small ligand(Malisi et al., 2012). 3.4 WISDOM

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Protein WISDOM is a web-based tool integrating methods for various protein design problems, including de novo design. The tool enables searching for templates, designing optimized sequences with stability, analysing fold specificity and binding affinity, and quantitative assessment of the designs by ranking of sequences as well as structures(Smadbeck et al., 2013). 3.5 EVODESIGN It is another web-based tool for designing optimal protein sequences of given scaffolds while predicting multiple sequence and structure-based features for design ranking. The tool uses Metropolis Monte-Carlo search of profiles constructed for homologous structure families in the Protein Data Bank(Mitra et al., 2013). 3.6 PYROSETTA It provides a graphical user interface for preparing and running protocols of ROSETTA and for data analysis.

Meta-tools such as the HotSpot Wizard offer a complete workflow to assess promising mutation sites by combining a variety of methods such as Catalytic Site Atlas, CASTp, CAVER, BLAST, MUSCLE, as well as sequence and structure databases such as UniProt, NCBI GenBank, and PDB. 4. CONCLUSION Computational approaches offer significant potential for engineering protein structure and function, and can be combined with experimental testing to gain new insights into the fundamental properties of proteins. It designs appropriate mutations in a much faster and cheaper way. REFERENCES Berry, S. M., & Lu, Y. (2011). Protein structure design and engineering. eLS. Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan, S. a., & Karplus, M. (1983). CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. Journal of computational chemistry, 4(2), 187-217. Chovancova, E., Pavelka, A., Benes, P., Strnad, O., Brezovsky, J., Kozlikova, B., . . . Medek, P. (2012). CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLoS computational biology, 8(10), e1002708. di Luccio, E., & Koehl, P. (2011). A quality metric for homology modeling: the H-factor. BMC bioinformatics, 12(1), 48. Giacovazzo, C. (2002). Fundamentals of crystallography (Vol. 7): Oxford university press, USA. Huang, S.-Y., & Zou, X. (2010). Advances and challenges in protein-ligand docking. International journal of molecular sciences, 11(8), 3016-3034. Jorgensen, W. L., & Ravimohan, C. (1985). Monte Carlo simulation of differences in free energies of hydration. The Journal of chemical physics, 83(6), 3050-3054. Lovell, S. C., Word, J. M., Richardson, J. S., & Richardson, D. C. (2000). The penultimate rotamer library. Proteins: Structure, Function, and Bioinformatics, 40(3), 389-408. Madej, T., Gibrat, J. F., & Bryant, S. H. (1995). Threading a database of protein cores. Proteins: Structure, Function, and Bioinformatics, 23(3), 356-369. Malisi, C., Schumann, M., Toussaint, N. C., Kageyama, J., Kohlbacher, O., & Höcker, B. (2012). Binding pocket optimization by computational protein design. PloS one, 7(12), e52505. Misura, K. M., Chivian, D., Rohl, C. A., Kim, D. E., & Baker, D. (2006). Physically realistic homology models built with ROSETTA can be more accurate than their templates. Proceedings of the National Academy of Sciences, 103(14), 5361-5366. Mitra, P., Shultis, D., & Zhang, Y. (2013). EvoDesign: de novo protein design based on structural and evolutionary profiles. Nucleic acids research, 41(W1), W273-W280. Najmanovich, R., Kuttner, J., Sobolev, V., & Edelman, M. (2000). Side‐chain flexibility in proteins upon ligand binding. Proteins: Structure, Function, and Bioinformatics, 39(3), 261-268. Nayeem, A., Sitkoff, D., & Krystek, S. (2006). A comparative study of available software for high‐accuracy homology modeling: From sequence alignments to structural models. Protein Science, 15(4), 808-824. Nosrati, G. R., & Houk, K. (2012). SABER: A computational method for identifying active sites for new reactions. Protein Science, 21(5), 697-706. Roessler, C. G., Hall, B. M., Anderson, W. J., Ingram, W. M., Roberts, S. A., Montfort, W. R., & Cordes, M. H. (2008). Transitive homology-guided structural studies lead to discovery of Cro proteins with 40%

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sequence identity but different folds. Proceedings of the National Academy of Sciences, 105(7), 2343- 2348. Saccenti, E., & Rosato, A. (2008). The war of tools: how can NMR spectroscopists detect errors in their structures? Journal of biomolecular NMR, 40(4), 251-261. Smadbeck, J., Peterson, M. B., Khoury, G. A., Taylor, M. S., & Floudas, C. A. (2013). Protein WISDOM: a workbench for in silico de novo design of biomolecules. Journal of visualized experiments: JoVE(77). Terp, G. E., Johansen, B. N., Christensen, I. T., & Jørgensen, F. S. (2001). A new concept for multidimensional selection of ligand conformations (MultiSelect) and multidimensional scoring (MultiScore) of protein− ligand binding affinities. Journal of medicinal chemistry, 44(14), 2333-2343. Wang, R., Lai, L., & Wang, S. (2002). Further development and validation of empirical scoring functions for structure-based binding affinity prediction. Journal of computer-aided molecular design, 16(1), 11-26. Wells, J. A. (1991). [18] Systematic mutational analyses of protein-protein interfaces. In Methods in enzymology (Vol. 202, pp. 390-411): Elsevier. Wüthrich, K. (2003). NMR studies of structure and function of biological macromolecules. Biosci Rep, 23(4), 119-168. Zhu, J., Fan, H., Periole, X., Honig, B., & Mark, A. E. (2008). Refining homology models by combining replica‐exchange molecular dynamics and statistical potentials. Proteins: Structure, Function, and Bioinformatics, 72(4), 1171-1188.

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NONCODING RNAS: DATABASE AND TOOLS Rachel Jani, John J Georrge* Department of Bioinformatics, Christ College, Rajkot-360 005, Gujarat *Corresponding author: [email protected] & [email protected]*

ABSTRACT: Non-coding RNAs contribute to diseases including cancers, autism, and Alzheimer’s. This paper comprehensively reviews on the available databases and tools for ncRNA. The tools, which predict the non-coding RNA are classified based on the algorithms its work and the maximum available databases are also included. The Database have completed sequenced data of organism like human, mouse, cow, rat, chicken, fruitfly, zebrafish, celegans, yeast, Arabidopsis, chimpanzee, gorilla, orangutan, rhesus macaque, opossum platypus and pig. For the prediction on Noncoding RNAs variety of the tools is available for eukaryotes and prokaryotes. Keywords: Non-coding RNAs, Diseases, Databases

1. INTRODUCTION Although a considerable portion of eukaryotic genomes is transcribed as long noncoding RNAs (lncRNAs), the vast majority are functionally uncharacterised. The rapidly expanding catalogue of mechanistically investigated lncRNAs has provided evidence for distinct functional subclasses, which are now ripe for exploitation as a general model to predict functions for uncharacterised lncRNAs. By utilising publicly- available genome-wide datasets and computational methods, we present several developed and emerging in silico approaches to characterise and predict the functions of lncRNAs. We propose that the application of these techniques provides valuable functional and mechanistic insight into lncRNAs, and is a crucial step for informing subsequent functional studies[1].

Protein Codirc Protein RNA

Transcription microRNAs siRNAs

DNA Non Coding Classes of ncRNA .....i..~mall RNAs :_] RNA ....,. snoRNAs, smRNAs, piRNAs & ochers Medium& 7 Large RNAs

Figure 1: An example of the transcription process to produce protein with coding and non-coding RNA genes A noncoding RNA (ncRNA) is a functional RNA that is transcribed from a DNA but does not encode a protein. According to transcriptomic there are thousands of ncRNAs classified into different categories based on their functions and lengths including transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), and long ncRNA (lncRNA) and many more[1]. These ncRNAs has important roles in various cellular processes. For example, rRNA catalyzes the peptide bond formation between amino acids in translation process ,miRNA is important in transcription process and performs miRNA, PIWI-interacting RNAs (piRNAs), small

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 81 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Bioinformatics nucleolar RNAs (snoRNAs), transcribed ultraconserved regions (TUCRs), and large intergenic noncoding RNAs (lincRNAs) and a few diseases including tumorigenesis and neurological cardiovascular, developmental, and other diseases.discussed the roles of lncRNAs in critical biological processes and human diseases like various cancers, diabetes, and AIDS[2]. Noncoding RNAs have emerged as important key players in the cell. Understanding their surprisingly diverse range of functions is challenging for experimental an computational biology. Here, we review computational methods to analyze noncoding RNAs. The topics covered include basic and advanced techniques to predict RNA structures, annotation of noncoding RNAs in genomic data, mining RNA-seq data for novel transcripts and prediction of transcript structures, computational aspects of microRNAs, and database resources[3]. Due to the important roles of ncRNAs in cellular processes and disease development, many experimental and bioinformatics methods have been developed to predict ncRNAs and their functions. As for experimental methods, enzymatic and chemical RNA sequencing, parallel cloning of ncRNAs by specialized cDNA libraries, microarray analysis, and genomic SELEX are among the most popular ones. The readers are referred to a review paper for the details of these methods. However, the experimental methods are expensive and time-consuming, and thus hundreds of computational methods have also been developed to prioritize highly confident ncRNA candidates for further experimental validation. In this paper, we present a comprehensive review on these computational methods. We are fully aware that there have already been several review articles on this hot topic. However, they either focus on a specific ncRNA category or have been out-dated and could not present a panoramic view of the field. Table 1 list out the available Databases for the Non-Coding RNAs[4, 5].

Table 1: Available Databases for the Non Coding RNAs Sr. Name of the Description no tool 1 C-It-Loci A tool to explore and to compare the expression profiles of conserved loci among various tissues in three organisms 2 LNCipedia A comprehensive compendium of human long non-coding rnas 3 lncRNAdb The Reference Database for Functional Long Noncoding rnas 4 LncRNAWiki A wiki-based, publicly editable and open-content platform for community curation of human long non-coding rnas (lncrnas) 5 lncRNome A comprehensive searchable biologically oriented knowledgebase for long noncoding rnas in Humans. 6 MiTranscript A catalog of human long poly-adenylated RNA transcripts derived from ome computational analysis of high-throughput RNA-Seq data from over 6,500 samples spanning diverse cancer and tissue types 7 MONOCLdb The mouse noncode Lung database provides the annotations and expression profiles of mouse long non-coding rnas (lncrnas) involved in Influenza and SARS-cov infections. 8 NONCODE An integrated knowledge database dedicated to ncrnas, especially lncrnas 9 NRED A database of long noncoding RNA expression. 10 slncky This site contains alignments and evolutionary metrics of conserved lncrnas. Evolution Browser

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2. REVIEW OF COMPUTATIONAL INTELLIGENCE TECHNIQUES There has been no clear review article about cl techniques for finding ncRNA and miRNA genes, according to our knowledge, that focuses on CI methods from 2001 to 2016. This methodological review about CI techniques is presented in Table 1 and summarized in Table 2. The primary aim of this review article is to attract both computer and bioinformatics researchers and to make a key reference for further study. In the literature from 2001 to 2016, the CI techniques are developed for finding ncRNA and miRNA genes by using neural networks (NNs), support vector machines (SVMs), Bayesian Networks (BNs), Hidden Markov model (HMM), genetic algorithm (GA), and deep learning. These computational approaches are described in detail in the following sub-sections. The identification of ncRNA functions are an emerging trend in the investigation of human diseases such as cancer, neurological, or cardiovascular disorders. As a result, there has been an increasing interest in the prediction of ncRNA genes.

3. PREDICTING NON-CODING RNAs The studies that used early systematic approaches did not focus on computational methods to predict the function of RNA genes. In, the authors developed a CI approach for identification of functional RNA genes. They used SVM and NN for prediction of RNA genes. They achieved 80%–90% classification accuracy using the NN approach and 90%–99% classification accuracy using the SVM classifier. Afterwards, the authors developed many systems for prediction of ncRNA by utilizing the computational algorithms. Whereas in the SVM algorithm was implemented in graphics processing units (GPUs) based parallel technology to classify ncRNA genes. Large-scale genomic sequence data was utilized to detect these ncRNA sequences. In fact, the authors reported that the detection rates of ncRNA genome sequences are fast using GPU and parallel based hardware implementation. By using data mining algorithms, a new web-based interface was developed in to detect ncRNAs that are not transcribed into proteins. They named this project “ncRNAclass” (https://biotools.ceid.upatras.gr), which can select efficient features and describe effectively the class of ncRNAs compared to other online tools[6]. The ncRNA class tool is used to differentiate between Well-known classes and to target predicted classes of mRNA. developed a positive sample only learning algorithm to identify ncRNA. Due to a lack of appropriate negative training samples they developed a positive sample-only learning algorithm to identify noncoding and coding RNAs. Using a supervised machine learning SVM, the authors classified transcripts according to their features. Introduced a coding or non-coding (CONC) method to differentiate non-coding and coding RNAs. They trained an SVM on eukaryotic ncRNA from RNAdb and NONCODE databases. The SVM predicted that coding RNAs were 97% and non-coding were 94%, where means of F-measures were obtained from cross-validation and with a range of 96.66%–98.2% and a standard deviation of 0.6. The motivation of this study was from the above mentioned CONC method proposed by coding potential calculator (CPC), which employed an SVM to identify ncRNA using six features that have meaningful biological sequence extracts from the transcript’s nucleotide sequence. The dataset used Rfam and RNAdb for noncoding and European Molecular Biology Laboratory for coding sequence (EMBL CDS) developed a boosted genetic programming method to automatically discover a sequence pattern to predict ncRNAs. The main advantages of this method are that it can use the DNA sequence directly as input, works well with larger sequences, is robust with noise training data, can predict ncRNAs, and does not rely on sequence conservation. On the other hand, provided an overview of context sensitive Hidden Markov Models (csHMMs) to predict ncRNA genes. The csHMMs can serve as an efficient framework for these purposes; they also provided an overview of the role of csHMMs in the RNA secondary structure analysis and the prediction of ncRNA genes[7].

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The State-of-the-art computational intelligence (CI) techniques for finding non-coding RNA (ncRNA) genes from 2001 to 2016 and A brief summary of CI techniques with respect to classification algorithms were reviewed in detailed by the Abbas et al.,[5]

4. FINDING MicroRNAs In 2005, Chenghai et al. [8] defined a method to classify real and pseudo mRNA by applying SVM using local structure sequence features. They achieved 90% accuracy on human data. Similarly, in a multilayer ANN classifier was proposed by training 17 parameters to predict a real pre-miRNA or a pseudo pre-miRNA. On average, a sensitivity of 97.40% and a specificity of 95.85% were obtained. This approach was also compared with another four state-of-the-art classification methods: MiPred , MiPred, miRabela, microPred, and Triplet- SVM classifier. A new classifier was developed in to predict the regulation of miRNA. In that study, they showed that the state-of-the-art methods are adequate for determining the pre-miRNA. However, the author developed a system to improve the precision performance of pre-miRNA that can handle a new multiple-stem and loop-secondary structure features by using neural networks. The real pre-miRNAdataset was utilized to successfully construct this new classifier to manage these class imbalance problems. The 5-fold cross-validation method was also used to evaluate the performance of the proposed classifier. In [48] authors have used a hybrid approach to predict small noncoding RNAs genes. In, a supervised NN machine learning approach was developed to predict new miRNA known as pre-miRNAs on a set of coding sequence (CDS) human regions. In that research, the obtained results (99.9% of accuracy (ACC), 99.8% of sensitivity (SN), and 100% of specificity (SP)) provided a more reliable prediction. The experimental results indicated that the miRNA achieves better results than other approaches and declares it to be the most effective tool to predict novel miRNAs. A miRNA target prediction algorithm was proposed in by contrast relaxing and CNN methods. In that study, the input dataset was artificially generated by CNN for the prediction of the target of miRNA when this mechanism is poorly known. To avoid inaccurate prediction, they used the contrast relaxing method to construct a balanced training dataset. The obtained results indicate that they achieved higher values of SN of 88%, SP of 94%, and ACC of 90%[9]. In, the classification of miRNAs was proposed to differentiate between normal and tumor tissues by using a multi-objective evolutionary-optimization technique. In that optimization strategy, the automatic selection of the classifier, its parameters, and feature combination steps were performed. This approach was divided into two steps. Firstly, they used a multi-objective algorithm with four classifiers such as random tree (RT), random forest (RF), sequential minimal optimization (SMO), and logistic regression. Afterwards, the multi-objective algorithm was automatically determined using the classifier, its parameters, and feature sets. In that study, the authors implemented a multi-objective evolutionary method to examine the search capability of non-dominated sorting genetic algorithm (NSGA)-II. The obtained results were also compared with several state-of-the-art methods on mRNA and miRNA datasets. An automatic miRNAs target prediction (deepTarget) algorithm was developed in by using the deep NN based approach to reduce manual selection of features. They showed that many computational tools have been developed to solve this problem, but the false positive rate high. In that study, the performance of the deepTarget algorithm delivered more than a 25% increase in the F-measure compared to that of the state-of-the-art target prediction algorithms[10].

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5. ONLINE TOOLS AND DATA SOURCES The online tools and data sources are provided for researchers to develop new studies based on new CI approaches[11]. These databases are useful to test various annotation and gene-finding techniques. The Rfam database was one of the first. It integrated various new and existing curated structural alignments into a common structure-annotated format. It also uses covariance modeling and automated sequence annotation software. The NONCODE database brought together most publicly available information about experimentally confirmed or computationally predicted ncRNAs with the exception of transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs). It also introduced a classification system termed process function class (PfClass) based on the cellular processes and functions associated with the ncRNA[12]. The RNAdb was launched as a sequence repository for experimentally supported regulatory mammalian ncRNAs (miRNAs, small nucleolar RNAs (snoRNAs), but not tRNAs, rRNAs, and spliceosomal RNAs). Apart from bioinformatics analyses, it also was meant to facilitate microarray chip characterization experiments. This database also includes a large number of commonly accepted ncRNAs from reputable complementary DNA (cDNA) libraries. The authors described the computational methods to identify genes and presented a brief technical reference for future studies. One of the first programs for searching a sequence database for homologs of a single RNA molecule on the basis of secondary structure was RSEARCH. It relies on a local alignment algorithm. The latter are a series of base pair and single nucleotide substitution matrices for RNA sequences. The web-based tool RNALOSS was developed to provide information about the distribution of locally optimal secondary structures[13]. FastR was applied to the discovery of riboswitches, a class of RNA domains, which regulate metabolite synthesis. Given an RNA sequence with known secondary structure, FastR efficiently computes all structural homologs in a genomic database. The tool relies heavily on filter design and optimization as well as the actual filtering algorithms and computation[14].

6. CONCUSION The number of Databases and tools are available. Now a days noncoding RNAs is need to be predict and the functions of noncoding RNAs can be find through the Bioinformatics tools and available literature evidences. The sequencing of noncoding RNAs is possible.

REFERENCES 1. Ban, N., et al., The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science, 2000. 289(5481): p. 905-20. 2. Calin, G.A., et al., Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A, 2002. 99(24): p. 15524-9. 3. Candeias, M.M., et al., P53 mRNA controls p53 activity by managing Mdm2 functions. Nat Cell Biol, 2008. 10(9): p. 1098-105. 4. Cheng, J., et al., Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution. Science, 2005. 308(5725): p. 1149-54. 5. Abbas, Q., et al., A Review of Computational Methods for Finding Non-Coding RNA Genes. 2016. 7(12): p. 113. 6. Dong, X.Y., et al., Implication of snoRNA U50 in human breast cancer. J Genet Genomics, 2009. 36(8): p. 447-54.

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7. Hu, Z., et al., Genetic variants of miRNA sequences and non-small cell lung cancer survival. J Clin Invest, 2008. 118(7): p. 2600-8. 8. Xue, C., et al., Classification of real and pseudo microRNA precursors using local structure-sequence features and support vector machine. BMC Bioinformatics, 2005. 6: p. 310. 9. Mourtada-Maarabouni, M., et al., GAS5, a non-protein-coding RNA, controls apoptosis and is downregulated in breast cancer. Oncogene, 2009. 28(2): p. 195-208. 10. Murayama, S., et al., Pulmonary infection in patients with cyclosporine, azathioprine, and corticosteroids after cardiac transplantation. Clinical and radiographic assessment. Nihon Igaku Hoshasen Gakkai Zasshi, 1991. 51(7): p. 780-9. 11. M. Padariya, U.K., John J. Georrge. Easy access tool for Small Interfering RNA (siRNA) data. in Proceedings of 8th National Level Science Symposium on Recent Trends in Science and Technology (ISBN: 9788192952116). 2015. Christ Publications, Rajkot. 12. Poole, A., D. Jeffares, and D. Penny, Early evolution: prokaryotes, the new kids on the block. Bioessays, 1999. 21(10): p. 880-9. 13. Zhu, Y., et al., Sequence analysis of RNase MRP RNA reveals its origination from eukaryotic RNase P RNA. RNA, 2006. 12(5): p. 699-706. 14. Toda, T., [Ultrasonographical study on luteinized unruptured follicle]. Nihon Sanka Fujinka Gakkai Zasshi, 1990. 42(9): p. 1195-202.

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 86 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Bioinformatics TOOLS FOR ASSEMBLY AND ANNOTATION Mishal John, John J Georrge* Department of Bioinformatics, Christ College, Rajkot, Gujarat E-mail ID: [email protected], [email protected]* ABSTRACT: Due to the development of sequencing techniques, several sequences are being sequenced rapidly. But are mostly being left as it is, either not knowing what can be done further, or not knowing how to analyze, etc. In such circumstances, a guidance to go further can be of great help. Sequence assembly is one way of utilizing the sequence, where it merges the fragmented sequences to form a complete genome. The obtained sequence is further annotated to find the gene locations and all the coding regions in that particular sequence. The sequence is compared for a thorough study of the sequence, so as to find similarities and uses of the sequences, finding the gene responsible for a disease, protein family and domain identification, etc. Now that the possible ways of dealing with a sequence is known, it is also important to choose an appropriate tool. A software analysis of Ge- nome assembly and annotation is specified in this chapter, which might be helpful for the researchers working in this field. Keywords: Assembly, Annotation

1. INTRODUCTION The entire set of genetic information present in the genetic material of an organism (DNA, RNA), including all the coding and non-coding regions is none as the Genome of that organism[1]. The human genome is made up of approximately 3 billion nucleotides. The DNA is composed mainly of four components (nucleotides)i.e, Adenine, Thymine, Guanine and Cytosine (A, T, G, and C). Determining the order in which these components are arranged is known as Genome/DNA sequencing. Various techniques starting from the like Sanger sequencing, leading to the introduction of 454 pyrosequencing, followed by Solexa, SOLiD and Helicos, and also the development of Nanopore technology are used for this purpose[2].Sequences are sequenced independently to a particular length, as per the technology used. Certain computer algorithms are helpful in aligning and merging these fragments (sequence reads) to form contigs (longer continuous stretches) in the process of de novo assembly[3]. Marking specific features of the Genome sequence, briefing out information about its structure and function, is a highly significant process which is known as Annotation. Structural annotation identifies structural elements or segments in the genome using only the characteristics of the sequence relying on its pattern recognition[4]. Collecting information about genes and recounting their biological identity, molecular function, biological role, subcellular location and their expression domains within the organism is done by Functional annotation[5].An assembly with annotation, in the past was considered as build[6].The assessment of similarities and differences (DNA sequence, genes, gene order, regulatory sequences) between the genomes of diverse organisms reveals the relationship between the individuals[7]. Researchers use various computational tools in order carefully compare the characteristics that define various organisms, thus pinpointing the regions of similarity and differences. Comparative genomics distinguishes conserved regions from divergent and functional from non-functional DNA, and also, contributes to the identification of the general functional class of particular DNA segments, like coding regions and non-coding regions and some gene regulatory regions[8-10].

2. DE NOVO ASSEMBLY As it is not easy or possible for the sequencing techniques to sequence a whole genome continuously, the genome is broken down into several fragments in order to make it simpler to sequence. But after sequencing, these have to be put back to make the complete original sequence. Thus there is a need to align and merge these fragments, for which the process called “Assembly” is used[11]. In other words, the reconstruction of the unknown contiguous DNA sequence correctly by inferring it with the help of a number of fragments is said to be Assembly[12].Adapter trimming, quality filtering, error correction, creation of contigs, and verification of contigs by mapping reads to the assembly and the creation/verification of scaffolds are the basic steps involved[13].It is already evident that sequencing is a highly delicate work. Similarly, it is even harder to assemble. The chief difficulty in assembling is the genomic repeats. The struggle of assembly relies on the number of reads that are being assembled. Computational algorithms are being developed to master over such issues, but still the assembly is not close to be the complete solution. In biological research, assembly softwaremust familiarize to the recent applications of the DNA sequencing. There are certain challenges by the sequencing technology as well that impact the assembly which are mentioned below[2]. ■ The presence of short reads and the absence of mate-pair cause difficulty in assembling the repeats. ■ Rising of new types of errors which demands for modification of the existing software and incorporate technology specific features in assembly software. ■ Repetitive property of DNA, leading to the fault-tolerant and alternative seeking algorithms

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 87 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Bioinformatics ■ Huge amount of data leads to the difficulty in the efficiency and requirement of parallel implementations or specialized hardware when practiced in large genomes. None of the assembly approaches available now can reconstruct a genome completely from read data alone.

2.1. Features of Genome Assemblers None of the assembly approaches available now can reconstruct a genome completely from read data alone. As there are more than 200 tools available for Assembling, the top ten Assemblers which are widely being used are described below table 1. SL. NAME FEATURES ADVANTAGES LIMITATIONS NO. 1 Mira • Allows hybrid assemblies of • Contains a comparable combina- • May not be advisable Sanger, 454, Solexa, IonTorrent tion of algorithms For both Ge- for large genomes. and PacBio (CCS &ecCLR) nomic and Transcript data[14] • Use of preprocessed data • Works very well on bacterial ge- data incorrectly leads • Can use paired-end and / or un- nomes to case-error probabil- paired data • Produces large contigs ities and functions to • Supports ancillary data in • Produces contigs containing detect the resolve pos- TRACEINFO format (from reads sible misassem- NCBI) blies[14]. • Marks places of interest with tags so that these can be found quickly in finishing programs • Has an SNP analysis pipeline for sequencing data of viruses and prokaryotes. • Available at http://www.chevreux.org/mira_ downloads.html 2 RS_HGAP • Three steps by first preassem-• Incorporates a 10-fold speed im- • Mapping and trim- _Assem- bling reads, assembling the pre- provement for microbial assem- ming parameters bler.3 assembled reads using Celera bly. might need to be opti- Hierar- Assembler and finally polish us-• Consists of pre-assembly, de mized. chical Ge- ing Quiver. novo assembly with PacBio'sAs- nome As- • Can support up to 100 Mb from sembleUnitig, and assembly pol- sembly SMRT Portal ishing with Quiver[15]. Process • Does not require accurate raw reads to correct errors. 3 A5 miseq• Has a 5-step procedure • Automated adapter trimming,• Following its publica- Pipeline • Substantially revises steps more full-length genes assem- tion, assembly pipe- • As compared to A5 pipeline, in- bled, NCBI-ready outputs and lines might be inad- stead of discarding reads in the production of base-call quality vertently tuned to pro- initial step, only the contami- scores duce high scores spe- nated portion of the read gets• A5-miseq should be particularly cifically on that da- trimmed. useful for researchers with lim- taset. This could result • In many instances, A5-miseq as- ited bioinformatics experience or in artificially high semblies have had higher computing resources scores that do not ac- NGA50 values, fewer misas- curately reflect the ex- semblies and fewer base-calling pected performance errors than A5 pipeline[13]. on other datasets[13]. 4 Assembly • Pioneer of the Representation of• Assembles very large datasets• To correct assembly By Short a de Bruijn graph[16]. produces by sequencing human assessing breakpoint Sequenc- • Highly contiguous genome as- genome. metric has to be man- ing semblies f long reads were ob-• Able to parallelize the assembly ually done. (ABySS) tained from human and other or- of billions of short reads over a• Contig length range is ganisms whose contiguity cluster of commodity hard- still limited to tens of ranges in megabase[17]. ware[16] kb which is shorter • Could assemble 3.5 billion than the megabases paired-end reads. obtained by the long- • ~2.76 million contigs (>100 read sequences[17]. bps) were created, which

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 88 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Bioinformatics represented the reference human genome by 68%[16]. 5 ALL- • 2 concepts – • All the assemblies are highly• Terminate at worst PATHS Finding all paths across given complete and contiguous. case, so to fail to re- read pair • Coverage >96% in all cases turn any terminations And localization • good accuracy, short-range conti- at all. Leading to holes • Presented as a graph retaining guity, long-range connectivity, in the final assembly, ambiguity and coverage of the genome[18]. thus prone to er- • Available at ftp://ftp.broadinsti- ror[19]. tute.org/pub/crd/ALL- PATHS/Release-LG/ 6 CAP 3 • Uses forward – reverse con-• Often produces less errors as• With the use of for- straints to correct errors and join compared with PHRAP ward – reverse con- contigs • Scaffold construction is easier straint as some of • In construction of consensus se- than PHRAP them are not correct quences, CAP3 uses redundant• Poor regions and false overlaps due to the lane track- coverage are identified and removed ing and cloning er- • Freely available at rors[20] [email protected]. 7 Ray Meta • Assembly based on distributed• Produces longer contigs and• Loss of information MPI more bases due to the construc- • Longer contigs as compared• Widespread application tion of graph with many others like velvet • Provide precise taxonomic profu-• Produce excessive • Accurate in assembling and pro- sions number of misassem- filing a 3 billion red meta-• Can run on multiple comput- blies[23] genomic experiment on bacte- ers[22] • Processing of large rial genome, in 15 hours with• Can be written in C++ and can and complex datasets 1,024 processor cores, by using run in parallel on numerous inter- can be facilitated by only 1.5GB per core[21]. connected computers. the software. • Open source available on http://denovoassembler.sf.net. 8 Meta Vel-• Uses supervised machine learn-• Generate assemblies with higher• Velvet scaffolding is vet-SL ing to improve performance N50 scores and higher qual- error-prone • Classifies every node from the ity[25]. • Exclusive access to a graph • Fast computer with large • For very short read (25-50 bp)• Widely used amount of available datasets and high coverage, it is memory for a single highly preferred[24]. MetaVelvet assembly • https://www.ebi.ac.uk/~zerb- is required (minimum ino/velvet/s 128 GB)[22]. • Produces only consensus sequences • May not work as well on gappy error models (e.g. 454, IonTorrent, PacBio) 9 TruSPAdes• Long and accurate virtual reads• Produces long and accurate con-• Multiple input librar- TSLA are generated from an assembly tigs ies are not sup- (TruSeq of barcoded pools of short reads.• Works with many data types (e.g. ported[27]. Synthetic • Instead of the entire meta- NanoporeMinION, PacBio) • Produces only consen- Long genome, it inherits the repeat• Supports RNA and metagenome sus sequences Reads) structure of a TSLR barcode data [23]. • Doesn't work with low from an individual coverage • 6.2Gb, 28M reads, 2x100bp, in- • Not designed for large sert size ~ 215bp (stdE.coli iso- genomes late) • 6.3 Gb, 29M reads, 2x100bp, insert size ~ 270bp (E.coli single cell)[26].

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 89 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Bioinformatics • Can be downloaded from http://cab.spbu.ru/software/spades/. 10 PHRAP • Uses a banded version of Smith-• If the sequence on both the• More error rate as Waterman-Gotoh algorithm to strands are covered or not, if the compared with compare the input sequences reads are sequenced by more than CAP3[20]. pairwise. one chemistry and the quality • PHRED quality score infor- values of the base in each read, mation is used to create accurate factors like this contribute the and high quality contig se- calculation of the PHRAP score quences • Able to balance between the dis- • Cloning vectors which would crepancies and preventing of interfere with the read align- stacking repeat sequences[28]. ments in the assembly are masked by Cross match/Swat algorithm Table. 1. Properties of Widely used Assemblers in brief

Given below certain other tools for genome assembly- 1. TriMetAss 1.2 The Trinity-based Iterative Metagenomics Assembler. Only select regions surrounding interesting features in metagenomic data can be assembled. Used in very common and well-conserved genes[29]. 2. OMWare 1.0 Efficient Assembly of Genome-wide Physical Map. Aims to help scientists in using optical map data. As they exist in a range of formats, it summarizes the optical maps with their most common manipu- lations[30]. 3. LightAssembler Lightweight Resources Assembly Algorithm. For high-throughput assembly reads[31]. 4. QUAST 4.1 Quality Assessment Tool for Genome Assemblies. Can work irrespective of the presence of reference genome[32]. 5. DNA Baser 4.36 DNA Sequence Assembly & Analysis. Revolutionary in automatic DNA sequence assembly, DNA sequence analysis, contig editing, file format conversion and mutation detection[33, 34]. 6. COCACOLA A general outline of Binning MetagenomicContigs using Sequence COmposition, Read CoverAge, CO-alignment, and Paired-end Read LinkAge. Is able to construct species from highly complicated environmental samples besides handling strain-level variations[35]. 7. MaxBin 2.2 Binning Assembled Metagenomic Sequences. Bins assembled metagenomic sequences based on Ex- pectation-Maximization algorithm[36]. 8. GAML 0.1 Genome Assembly by Maximum Likelihood. A prototype genome assembly tools based on maximum likelihood of the assembly. Covers error rate, insert length and other features of individual sequencing technologies[37, 38]. 9. NanoMark DNA Assembly Benchmark for Nanopore long reads. Based on third generation sequencing[39]. 10. ARC 1.1.4-beta Assembly by Reduced Complexity. Pipeline that facilitates iterative, reference guided de novo assemblies. Capable of breaking large, complex problems in to smaller manageable chunks. [http://ibest.github.io/ARC/] 11. TransPS 1.1.0 Transcriptome Post Scaffolding. Pipeline to post-process the pre-assembled transcriptomes with the help of reference-based method. An align-layout-consensus consisting of 3 major stages is applied[40]. 12. assemblyManager Computing the Robotic Commands for 2ab Assembly[41]. 13. BinPacker 1.1 Packing-Based De Novo Transcriptome Assembly from RNA-seq Data. Novel de novo assembler. Transcriptome assembly problem is modeled as tracking a set of trajectories representing coverage based on sizes of their corresponding isoforms and solves a series of bib-packing problems[42]. www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 90 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Bioinformatics 14. FermiKit 0.13 De novo Assembly based Variant Calling pipeline for Illumina Short Reads. Variant calling pipeline for deep Illuminaresequencing data based on de novo assembly. The assembly retains long deletion, novel sequence insertions, translocations and copy number besides encoding SNPs and short IN- DELs. It is also considered a better long insertion caller[43]. 15. REPdenovo A tool to Construct Repeats directly from Raw Reads. Designed to construct repeats directly from the sequence reads. Provides much functionality and can generate much longer repeats. Its main functionalities are Assembly and Scaffolding[44]. 16. Xander Gene-targeted Metagenomic Assembler. Novel method to target assembly of specific protein-coding genes with the help of a graph structure with a combination of Bruijn graph and HMMs[45]. 17. SWAP-Assembler 2 A scalable and fully parallelized Genome Assembler. Intended for massive sequencing data. A multistep bi-directed graph is adopted with which the standard genome assembly becomes equivalent to the edge merging operations in a semi-group[46]. 18. TGNet Visualization and Quality Assessment of de novo Genome Assemblies. A visualization and quality assessment of de novo genome assemblies based on a Cytoscape. It is capable of detecting incon- sistencies between a genome assembly and an independently derived transcriptome assembly[47]. 19. misFinder v0.4.05.05 Identify Mis-assemblies in an unbiased manner using Reference and Paired-end Reads. Destines to identify the assembly errors with high accuracy in an unbiased way. Their mis-assembled positions are corrected to improve the assembly accuracy for downstream analysis[48]. 20. Scaffold_builder v2.2 Contigs generated by draft sequencing along a reference sequence are ordered by this software. N’s help in filling the gaps and Muscle help in aligning he small overlaps. It is possible not only to assemble but also annotate the genomes[49]. 21. Rnnotator 3.5.0 Pipeline which generates models by de novo assembly. Full-length transcripts are reconstructed in the absence of a complete reference genome.producese highly accurate contigs[50]. 22. SATRAP 0.2 SOLiD Assembler TRAnslation Program. It adopts an efficient strategy to translate into bases the color space assembly. It can also be used as a stand –alone program so to perform color space translation[51]. 23. Bandage v0.7.1 Main purpose is to visualize de novo assembly graph. It opens up new possibilities for analyzing de novo assemblies by displaying connections which are not present in the contig file[52]. 24. HapCol Haplotype Assembly from Long Gapless Reads. For each single nucleotide polymorphism position it is exponential in the maximum number of corrections, hence reducing the overall error-correction score. Fast and efficient in memory for haplotypes from long reads that are gapless[53]. 25. REAGO 1.1 REconstruct 16S ribosomal RNA Genes from MetagenOmic data. Approaches the challenges by with a combination of secondary structure – aware homology search, properties of zRNA genes and de novo assembly[54]. 26. FGAP 1.8.1 Automated Gap Closing tool It merges alternative assemblies or incorporates alternative data in order to improve the genome sequences, analyses the gap region to indicate the best sequence to close the gap[55]. 27. DETONATE 1.10 DE novo TranscriptOmerNa-seq Assembly with or without the Truth Evaluation. consists of two component packages, RSEM-EVAL and REF-EVAL. Both packages are mainly intended to be used to evaluate de novo transcriptome assemblies, although REF-EVAL can be used to compare sets of any kinds of genomic sequences[56]. 28. Trinity 2.1.1 Trinity represents a novel method for the efficient and robust de novo reconstruction of transcriptomes from RNA-Seq data. Trinity combines three independent software modules: Inchworm, Chrys- alis, and Butterfly, applied sequentially to process large volumes of RNA-Seq reads. Trinity partitions the sequence data into many individual de Bruijn graphs, each representing the transcriptional complexity at at a given gene or locus, and then processes each graph independently to extract full-length splicing isoforms and to tease apart transcripts derived from paralogous genes[57]. www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 91 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Bioinformatics 29. IsoSCM 2.0.11 IsoSCM (Isoform Structural Change Model) is a new method for transcript assembly that incorporates change-point analysis to improve the 3′ UTR annotation process[58]. 30. IVA 1.0.3 – Iterative Virus Assembler IVA is a de novo assembler designed to assemble virus genomes that have no repeat sequences, using Illumina read pairs sequenced from mixed populations at extremely high and variable depth[59].

3. GENOME ANNOTATION Gene annotation is been widely misunderstood as gene prediction. But in reality, a gene prediction is a prediction of the intron–exon structure of a gene based on a mathematical model, while gene annotation is the synthesis of intron–exon structure from multiple lines of evidence including gene prediction, expression data (often in the form of mRNA-seq data), protein homology, and repetitive elements[60] involving two steps i.e., evidence generation and synthesis[61].Evidence generation combines repeat masking, transcript and protein alignments, gene predictions, and whole genome alignment of closely related species are commonly used to provide evidence in genome annotation[62]. Once the evidence is generated, the annotator begins its next step of synthesizing the information into gene annotations[63].

Prediction of General structural database Statistical gene features search prediction

Gene/protein/ RNA set

Specialized database search D Predicted gene functions

Co11text a11a(v.,i.

Figure 1: generalized flow chart of genome annotation[64].

Table2: Certain widely used tools for genome Annotation is listed. Sl. No. Name Description Advantages Limitation 1 Blast2GO A universal tool for annotation, Accuracy is 65–70% Accuracy visualization and It has successful in ex- Difficulty in analy- analysis in functional genomics tracting relevant func- sis of poorly char- research tional features of the se- acterized organ- quences based on theuse isms of the predicted annotation[65]. 2 Rast Rapid Annotations using Sub- Able to identify protein - Time consuming systems Technology. encoding. Accuracy not satis- rRNA and tRNA genes. factory. Assigns functions to the genes Predicts which subsystems are represented in the genome. Reconstructs the metabolic network[66].

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 92 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Bioinformatics 3 KAAS An automatic genome annota- Gives three views in out- Time and accuracy server tion and pathwayreconstruction put server. Gives the pathways involved[67]. 4 BASYs a web server for automated bac- Compares favorably with Accuracy does not terial other automated systems. exceed 60% genome annotation Permits high throughput, detailed and fully automated annotation of bacterial genomes[68].

Annotation tools rarely being used Sl No Description 1. Prokka Rapid prokaryotic genome annotation Decreases running time in multi-core computers. Requires certain features like High multicore computers, multiple single CPU threads, etc., 2. GenDB Open source genome annotation system for prokaryote genomes Its flexible and extensible 3. SVM Support Vector Machine. One advantage of this is its scalability. 4. eggNO-Mapper Fast Genome-Wide Functional Annotation through Orthology Assignment. Based on fast Orthology mapping. 5. CDART A protein homology by based ob Domain Architecture. 6. FTG A web-server for analyzing nucleotide sequences to predict the genes using Fourier transform techniques. 7. EGPred Prediction of Eukaryotic Genes Using Ab Initio Methods After Combining with Sequence Similarity Approaches. 8. SOBA Sequence Ontology BI analysis. and includes both a bug tracker and a feature request tracker for continued development and maintenance of the tool. 9. VIGOR An annotation program for small viral genomes. User friendly and is used for five different virus gene prediction. 10. FLAN Short for FLu ANnotation. A web server for influenza virus genome annotation.

4. CONCLUSION Although there are many tools for both Assembly and Annotation, based on the three-parameter analysis, out of the tools available for Assembly, the best can be Mira and RS_HGAP, and in the case of Annotation, it can be Blast2GO, K AAS server and Rast server.

5. REFERENCES

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www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 95 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Bioinformatics 62. Campbell, M.S. and M. Yandell, An Introduction to Genome Annotation. Current protocols in bioinformatics, 2015: p. 4.1. 1-4.1. 17. 63. Adams, M.D., et al., The genome sequence of Drosophila melanogaster. Science, 2000. 287(5461): p. 2185-2195. 64. Koonin, E.V. and M.Y. Galperin, Principles and methods of sequence analysis, in Sequence—Evolution— Function. 2003, Springer. p. 111-192. 65. Conesa, A., et al., Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics, 2005. 21(18): p. 3674-3676. 66. Aziz, R.K., et al., The RAST Server: rapid annotations using subsystems technology. BMC genomics, 2008. 9(1): p. 75. 67. Moriya, Y., et al., KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic acids research, 2007. 35(suppl_2): p. W182-W185. 68. Van Domselaar, G.H., et al., BASys: a web server for automated bacterial genome annotation. Nucleic acids research, 2005. 33(suppl_2): p. W455-W459.

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Section 5

BIOTECHNOLOGY

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Aspergillus flavus - A MENACE TO FARMERS Avani Ranipa1, Anju Shrilal1, Akash Nimavat1, Jalpa Rank1, Ramesh Kothari1*, John J. Georrge2* 1, Department of Biosciences, Institute of Biotechnology, Saurashtra University, Rajkot – 360005 2 Department of Bioinformatics, Christ College, Rajkot – 360005 (GUJ.) INDIA *Corresponding authors: [email protected], [email protected]

ABSTRACT: Aspergillus flavus is a globally distributed filamentous, saprophytic fungus that frequently infects oil-rich seeds of various crop species such as oilseed crops maize, peanuts, cottonseed and tree nuts. The colonization of peanuts (Arachis hypogaea L.) and other oilseeds by the fungal pathogen Aspergillus flavus results in the contamination with carcinogenic mycotoxins known as aflatoxins leading to economic losses and potential health threats to humans. Aflatoxin contamination of peanut is a major problem of rain-fed agriculture in India. In human and animal consumption of aflatoxin contaminated seeds cause cancer and liver disease. Hence, it is the need of the hour to explore the possibility of identification of novel drug targets and designing of drugs against A. flavus. Keywords: A. flavus, aflatoxin, peanut, drug target

1. INTRODUCTION Aspergillus flavus is a is a globally distributed filamentous, saprotrophic and pathogenic fungus with a cosmopolitan distribution. Members of the genus Aspergillus are ubiquitous filamentous fungus found anywhere on earth. To date, over 185 Aspergillus species have been identified, 20 of which have been reported to cause harmful infections in humans, animals, and plants. Among the Aspergillus species, the Aspergillus flavus may be the most infamous because it causes direct infections and systematic diseases in humans (Cleveland TE et al.2009). Aspergillus flavus (A. flavus) is a fungus that frequently infects oil-rich seeds of various crop species such as oilseed crops maize, peanuts, cottonseed and tree nuts during pre- and post-harvest with subsequent production of mycotoxins such as cyclopiazonic acid, aflatrem, and the well-known Aflatoxin (Duran et al., 2014; Kenneth C. Ehrlich 2014). It is a common fungus with a typical yellow green in appearance. Its population increases during hot dry weather, drought stress, and extreme temperatures in the geocarposphere (area surrounding peanut pod that influences microbial complement of the soil) (F. Hamidou et al., 2014; A.M. Torres et al., 2014). A. flavus has a wide range of temperature tolerance (10–15/35–40°C) with about 28–30°C optimum for growth and about 25–30°C for AF production (J.C. Fountain et al. 2014) depending on the strain. However, optimal growth of A. flavus is at 37°C (Schmidt- Heydt et al., 2009). The ability of A. flavus to produce a broad spectrum of degrading enzymes and to infect a wide variety of plant or animal hosts, and to use non-living substrates suggests it is an opportunistic pathogen capable of subsisting on a diverse range of nutritional sources (Mellon et al., 2007; J.C. Fountain et al., 2014). It is well known that the genus Aspergillus can survive in a wide range of environments; moreover, the genus can also inhabit extreme environments for survival, including oxidative stress, osmotic changes, heat shock (Sirot et al., 2013), nutrients limitation, pH changes and chemical stresses. When exposed to stress conditions (Yu J et al. 2011), the fungal cell responds by modifying several genes expression to be able to

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 97 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Biotechnology endure the adversities. The major key defense of fungi to withstand environmental aggressions is their cell wall and cell membrane. The cell wall represents a dynamically forming exoskeleton which protects the fungal protoplast. Under stress conditions cell wall is damaged, but cell wall is repaired and remodeled through cell wall biosynthesis which requires an intact cell wall integrity pathway. In addition to being an entry barrier for antifungal components, the cell membrane dictates the entry of nutrients and the exit of metabolites and represents a selective barrier for their translocation (Bandyopadhyay S et al. 2010). The sequencing of A.flavus whole genome has been completed. Initial annotation of the sequence revealed that there are about 13,071 genes in the A. flavus genome. Genes which potentially encode for enzymes involved in secondary metabolite production in the A flavus genome have been identified (Osbourn, 2012).

2. PLASMA MEMBRANE COMPOSITION Plasma membrane of fungi contains sterols that are essential for the organization and functions of the cellular structure. The major sterol in filamentous fungi is ergosterol instead of cholesterol in mammalian cells and phytosterols in plants. The amount of ergosterol varies between Aspergillus species and depends on the age of the culture, the developmental stage and the growth environment. Each of these sterols is the end-product of a long multistep biosynthetic pathway that derives from a common initial pathway (acetyl CoA to squalene epoxide) (Fig.1). An extensive literature reports the fundamental contribution of sterols to fluidity, permeability, micro domain formation, protein functionality and membrane activities (Jacobson et al. 2007; Guan et al. 2009; Tyler et al. 2009; Lingwood et al. 2010).

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ERGOSTEROL Fig.1 Ergosterol biosynthetic pathway www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 98 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Biotechnology 3. CELL WALL COMPOSITION The fungal cell wall is a complex cross-linked network of chitin, glucans, other polysaccharides as well as integral proteins. The central core consists of glucans cross linked to chitin, with various decorating polysaccharides depending on the species. Many of the components of the cell wall are fungal-specific, thus inhibition of these components represents a logical target for antifungal agents. This approach has been validated by the echinocandin class of antifungal that target β-1, 3 glucan synthesis (Pfaller MA et al., 2013; Mukherjee PK et al, 2011). However, the fungal cell wall is a dynamic and developmentally plastic construction, capable of compensating for loss of β-1, 3-glucan by increased chitin deposition (L.V. Roze et al., 2013).

3.1 Chitin Chitin is long linear homo polymer of B-1, 4-linked N-acetyl glucosamine. Chitin synthases are responsible for chitin synthesis & use UPD-N-acetyl glucosamine as a substrate. The enzyme is integrally located in the membrane. Chitin accounts for only 1-2% of the dry weight of the yeast cell wall; however, the cell wall of Aspergillus species contains 10-20% chitin. It contributes enormously to the overall integrity of the cell wall because when it’s synthesis is disrupted, the wall becomes malformed & unstable (Latge J.P., and Beauvais A, 2014). Multiple chitin synthases reported to be involved in septum synthesis, lateral wall biosynthesis & spore formation were identified in Aspergillus species (Fortwendel JR et al. 2010). This multiplicity is certainly due to the different roles that the enzymes play in the physiology of the organism.

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Fig.2: Cell wall structure in Aspergillus sp. Chitin Synthase: Aspergillus flavus has eight chitin synthase encoding gene in its genome, which are as follows; ChsA, ChsB, ChsC, ChsD, ChsF, ChsG, CsmA, CsmB. Chitin synthase has been localized in the membranes of Golgi complexes and intracellular vesicles, as well as in plasma membranes, it may be concluded that the enzyme follows an exocytotic pathway, accumulating in cytoplasmic vesicles during its transport to the cell surface (Dolezal AL et al., 2013). Chitin synthesis is inhibited when microtubules are disrupted by cytoskeletal poisons such as colchicine or vinblastine. In fungal systems, substantial data have accumulated indicating that chitin synthase activity of at least one chitin synthase isoform (CHS3p) is associated with specialized intracellular micro vesicles, known as chitosomes, which exhibit a special lipid and protein composition. Chitin exists in the cell wall of several zygomycetes species in its deacetylated form referred to as chitosan (Cardoso et al. 2012). The Biosynthesis of chitosan in fungal proceeds by the coordinated action of chitin synthase and chitin de-acetylase. www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 99 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Biotechnology 4. AFLATOXIN Aflatoxin are a group of carcinogenic mycotoxins produced mainly by Aspergillus flavus and A. parasiticus. They are considered to be a threat to human health, global food safety, and security (J.I. Pitt et al., 2013; Luciano Pinotti, 2016). Aflatoxin has been rated as class 1A carcinogens by the International Agency for Research of Cancer (IARC, 2012). A. flavus commonly produces B1 and B2 Aflatoxins, while A. parasiticus produces two additional aflatoxin, G1 and G2. The B prefix refers to the blue fluorescence and the G refers to the yellowish-green fluorescence under ultraviolet light. These toxins are largely associated with food commodities produced in the humid tropics and subtropics, such as cereals (maize, sorghum, pearl millet, rice, and wheat), oilseeds(peanut, soybean, sunflower, and cotton), spices (chilies, black pepper, turmeric, coriander, and ginger), nuts (almond, Brazil nut, pistachio, walnut, and coconut), and milk(K.R.N. Reddy et al., 2010). Aflatoxins are not digestible by animals and thus end up in the meat. They are also heat and freeze stable and thereby, remain indefinitely in the food. AFB1is the most toxic and potent carcinogen (Y. Liu and F. Wu, 2010). In addition to being a common natural contaminant of food and feed supplies, A. flavus is one of the leading causes of aspergillosis (Brown GD et al., 2012), Several studies have linked these toxins directly to liver disease, tumor development, stunted development in children and other medical syndromes (reviewed in Wu et al. 2014). Daily intake of low doses of aflatoxin over time causes chronic aflatoxicosis, resulting in impaired food consumption, stunted growth, immune suppression, and possible liver cancer development. On the other hand, acute aflatoxicosis is caused by high concentrations of aflatoxin intake in just one or a few exposures. Aflatoxin B1 is considered to be the major cause of liver cancer along with the chronic infection with hepatitis B and C viruses and acute aflatoxicosis (Wild et al. 2009). The term "Aflatoxin" is derived from the name of one of the molds that produce it, Aspergillus flavus. It was coined around 1960 after its discovery as the source of "Turkey X disease". Aflatoxin form one of the major groupings of mycotoxins.

5. DISEASES CAUSED IN GROUNDNUT Maize (Zea mays L.) and groundnut (Arachishypogaea L.) are the most susceptible crops to Aflatoxin contamination and serve as the main source of Aflatoxin exposure for humans. Groundnuts tend to be colonized and contaminated by Aspergillus spp. at pre-harvest, during harvest, post-harvest drying, in storage and also during transport covering the whole value chain (M.C. Willcox et al., 2013; Waliyar, et al., 2015). Three types of resistance mechanisms namely in-vitro seed colonization resistance (IVSC), resistance to pre-harvest Aflatoxin contamination (PAC) and resistance to Aflatoxin production in seeds have been reported in groundnut (Spurthi N Nayak et al, 2017). However, the Aflatoxin is produced only in the cotyledons of groundnut kernels after fungal infection (M.C. Willcox et al., 2013). In groundnut, fungi cause seed rots and seedling diseases such as root rot, stem rot, wilts, blight, pod rot; and foliar diseases such as rust and early and late leaf spots. Seed rots and seedling diseases. Many soil inhabiting fungi infect and damage the seed and germinating seedlings of groundnut (MP 1and MP 2). They may be identified by fungal spores that give characteristic colorations to the seed, e.g., gray spores indicate Rhizopus arrhizus, and black spores are Aspergillus Niger. The fungus, Aspergillus flavus, produces Aflatoxin and causes aflaroot of groundnut. A. flavus has the potential to infect seedlings by sporulation on injured seeds. In grains, the pathogen can invade seed embryos and cause infection, which decreases germination and can lead to infected seeds planted in the field. The pathogen can also discolor embryos, damage seedlings, and kill seedlings, which reduces grade and price of the grains. The incidence of A. flavus infection increases in the presence of insects and any type of www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 100 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Biotechnology stress on the host in the field as a result of damage. Stresses include stalk rot, drought, severe leaf damage, and/or less than ideal storage conditions. Generally, excessive moisture conditions and high temperatures of storage grains and legumes increase the occurrence of A. flavus Aflatoxin production (Matthew Atongbiik Achaglinkame et al, 2017).

Fig. 3 Contamination of Aspergillus flavus in groundnut

6. EFFECT OF ASPERGILLUS FLAVUS ON ANIMALS This filamentous fungus can produce natural infection in animals including birds. However, the infection is contained to cattle, dog, buffalo, goat, and poultry (Pal, 2007). A.flavus is one of the filamentous fungi involved in the etiology of mycotic abortion, and mastitis of dairy animals (Pal and Jadhav, 2013).The birds affected with aflatoxicosis exhibit the signs of anorexia, loss of body weight, retarded growth, weakness, dullness, depression, paralysis, and de-creased egg production (Pal, 2007). Aflatoxin M1 is less mutagenic and carcinogenic than B1, it exhibits high genotoxic activity.

The other effects of AFTs-M1 include liver damage, decreased milk production, immunity suppression and reduced oxygen supply to tissues due to anemia (Frantisek Malir et al., 2016), which reduces appetite and growth in dairy cattle. Several studies have shown the detrimental effects of Aflatoxin exposure on the liver (Sharmila Banu G et al., 2009), epididymis, testis (Faisal et al., 2008), kidney and heart (Mohammed A. M. and Metwally N. S., 2009; Panahirad S et al, 2014). It has been found that Aflatoxin presences in post-mortem brain tissue, suggested that its ability to cross the blood brain barrier (Qureshi H. et al., 2015). AFTs also cause abnormalities in the structure and functioning of mitochondrial DNA and brain cells. The effects of Aflatoxin on brain chemistry have been reviewed in details by (Bbosa et al. 2013). Furthermore, few reports have described the effects of AFTs-B1 administration on the structure of the rodent central nervous system (Laag E. M. and Abdel Aziz H. O., 2013). When contaminated food is processed, Aflatoxin enters the general food supply where they have been found in both pet and human foods, as well as in feed stocks for agricultural animals. Animals fed contaminated food can pass aflatoxin transformation products into eggs, milk products, and meat. For example, contaminated poultry feed is suspected in the findings of high percentages of samples of aflatoxin-contaminated chicken meat and eggs in Pakistan (Iqbal et al. 2014).

7. EFFECT OF ASPERGILLUS FLAVUS ON HUMAN Some Aspergillus species cause serious disease in humans. The liver toxicology of Aflatoxin is also a critical issue (Iqbal et al., 2014). Limited doses are not harmful to humans or animals; however, the doses that do cause-effects diverse among aflatoxin groups. A positive correlation has been established between the consumption of Aflatoxin-contaminated foods and the increased incidence of liver cancer (Nogueira L et al., www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 101 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Biotechnology 2015) worldwide. The symptoms include fever, cough, chest pain or breathlessness, which also occur in much other illness so, diagnosis can be difficult. Usually, only patients with already weakened immune systems or who suffer other lung conditions (Iqbal et al., 2014) are susceptible. aflatoxin cause reduced efficiency of immunization in children that lead to enhanced risk of infections. The hepato carcinogenicity of aflatoxin is mainly due to the lipid peroxidation and oxidative damage to DNA. AFTs-B1 in the liver is activated by cytochrome p450 enzymes, which are converted to AFTs-B1-8, 9-epoxide, which is responsible for carcinogenic effects in the kidney. Among all major mycotoxins, aflatoxin creates a high risk in dairy because of the presence of their derivative, AFTs-M1, in milk, posing a potential health hazard for human consumption. AFTs-B1 is rapidly absorbed in the digestive tract and metabolized by the liver, which converts it to AFT-M1 for subsequent secretion in milk and urine. Most commonly, it causes serious threats to human and animal health by causing various complications such as hepatotoxicity, teratogenicity, immunotoxicity (L.V. Roze et al., 2013), chronic pulmonary aspergillosis (CPA), aspergilloma or allergic broncho pulmonary aspergillosis (ABPA), allergic disease in immune competent host (Goel, Ayush, 2015)

Fig. 4 Pulmonary Aspergillosis

8. FOOD SAFETY Food safety is one of the major problems currently facing the world; accordingly, a variety of studies have been conducted to discuss methods of addressing consumer concerns with various aspects of food safety (A.M. Torres et al., 2014). Since 1985, the United States Food and Drug Administration (USFDA) have restricted the amount of mycotoxins permitted in food products. The USDA Grain and Plant Inspection Service (GPIS) have implemented a service laboratory for inspection of mycotoxins in grains. Additionally, the Food and Agricultural Organization (FAO) and World Health Organization (WHO) have recognized many toxins present in agricultural products. When mycotoxins are contaminated into foods, they cannot be destroyed by normal cooking processes. However, there have been many recent advances in food processing developed to keep final food products safe and healthy, such as hazard analysis of critical control points (HACCP) and good manufacturing practices (Lockis VR et al., 2011; Cusato S et al., 2013; Maldonado-Siman E.et al., 2014).

9. ANTIFUNGAL DRUG AND ITS TARGETS The essential role of sterols in maintenance of cell membranes make ergosterol and its biosynthetic pathway essential for fungal growth, and a primary target for most, currently available, antifungal drugs to treat severe human fungal infections. The antifungal drugs that are commonly used to treat invasive aspergillosis belong to three classes (Terry Roemer and Damian J. Krysan, 2014). with distinct mechanisms of action: the polyene www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 102 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Biotechnology amphotericin B, which acts on the cell membrane; the azoles voriconazole, which acts on ergosterol biosynthesis; and the echinocandins, which act on the cell wall. The oldest class of antifungal drugs is the polyenes, of which amphotericin B is the only example used to treat systemic infections. The polyene amphotericin B (AMB) has represented, for more than 30 years, the standard antifungal therapy for invasive aspergillosis (IA). Amphotericin B binds to ergosterol, a membrane sterol that is unique to fungi, as part of its mechanism of action (Gray et al. 2012). Amphotericin B is fungicidal and is the broadest spectrum antifungal available. One of the primary drawbacks of polyenes is their significant toxicity, although the development of lipid for mulations of Amphotericin B has reduced this problem significantly (Hamill RJ 2013). Finally, the main category of antifungal agents used against Aspergillus is azoles drugs, such as voriconazole (VCZ) and itraconazole, and a new generation of azoles that includes posaconazole, isavuconazole, and albaconazole. Azoles revolutionized medical mycology due to their broad spectrum and reduced toxicity compared to AMB (Kaneko Y et al., 2013). Azoles block the ergosterol biosynthesis pathway via inhibition of 14-α sterol demethylase (Cyp51/Erg11), a key enzyme that removes the methyl group at position C-14 of precursor sterols. Inhibition of ergosterol synthesis at this biochemical level results in toxic sterol accumulation and cell death. Azoles inhibit ergosterol biosynthesis and, in general, are fungi static; an important exception is that voriconazole is fungicidal toward A. fumigatus (Lewis K. 2013). Azoles are extremely well tolerated, although they interfere with the metabolism of a number of other drugs owing to their ability to inhibit cytochrome P450. The allylamine group (eg.Terbinafine) of antimycotics interferes at the early-stage of ergosterol biosynthesis by inhibiting the enzyme squalene epoxidase (Erg1). Allylamine are not used to treat IA, however potential use in combination with azoles, polyenes, or echinocandins in the management of severe drug-resistant or refractory mycoses has been proposed (Krishnan-Natesan, S., 2009).

UniProt Target DrugBank Drug Name Type Mechanism Target ID ID organism P08684 CYP450- DB01152 Candicidin Inhibitor Binds and Various Fungus dependent inhibits Species 14-alpha DB00646 Nystatin Binder ergosterol Fungi sterol DB00681 Amphotericin Inhibitor synthesis Various Fungus demethylase B Species DB11633 Isavuconazole Inhibitor Aspergillis, Candida & other fungi

DB01263 Posaconazole Inhibitor Aspergillis, candida & other fungi Inhibitor Fungi, yeast DB01167 Itraconazole and protozoans DB12073 Albaconazole Inhibitor Aspergillus spp. DB00826 Natamycin Inhibitor Various Fungus Species DB00196 Fluconazole Inhibitor Fungi

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 103 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Biotechnology Voriconazole Inhibitor Yeast & other DB00582 fungi P50859 Sterol-14- DB00251 Terconazole Inhibitor Fungi alpha DB09040 Efinaconazole Fungi demethylase A2QLK4 1,3-beta- Inhibits Aspergillis, glucan synthesis of Candida and synthase DB01141 Micafungin Inhibitor 1, 3-D- other fungi glucan, Aspergillis, essential Candida and content of other fungi DB00520 Caspofungin Inhibitor fungal wall Aspergillis, Candida and DB00362 Anidulafungin Inhibitor other fungi DB03632 Argifin Inhibitor Inhibit - Q873X9 Chitinase chitinase - DB04350 Argadin Inhibitor enzyme Inhibits Aspergillis, chitin chitin Candida and

synthase DB12939 Nikkomycin Z Inhibitor biosynthesis other fungi - Polyoxin Inhibitor - Table 1: Antifungal drug and its target

10. ADME (ABSORBTION DISTRIBUTION METABOLISM EXCRETION) OF AVAILABLE DRUGS In pharmacokinetics and pharmacology, this describes the disposition of a pharmaceutical compound within an organism (Bellmann R, et al, 2017).The four criteria all influence the drug levels and kinetics of drug exposure to the tissues and hence influence the performance and pharmacological activity of the compound as a drug. (Roemer T and Boone C., 2013). The selection of a therapeutically effective and safe dose is crucial for drug development and requires assessment of intrinsic and extrinsic factors that influence drug pharmacokinetics (concentration and duration of drug exposure) with proper disposal from body without creating toxicity. Absorption deals with Routes of administration and drug’s bioavailability. Factors such as poor solubility, gastric emptying time, intestinal transit time, chemical instability in the stomach, and inability to permeate the intestinal wall can all reduce the extent to which a drug is absorbed after oral administration. Hence can be done in via intravenously or by inhalation. Distribution means distribution into muscles and organs, thus drug is subjected which leads to lower its plasma concentration. Compounds begin to metabolized as soon as they enter the body and majority of small-molecule called metabolites are carried out in the liver by redox enzymes, termed cytochrome P450 enzymes. When metabolites are pharmacologically inert, metabolism deactivates the administered dose of parent drug and this usually reduces the effects on the body. Metabolites may also be pharmacologically active, sometimes more so than the parent drug eg prodrug (Murrell D, et al, 2017).Compounds and their metabolites need to be removed from the body via excretion, which can be through kidney, fecal excretion or biliary excretion and lastly by lungs. Unless excretion is complete, accumulation of foreign substances can adversely affect normal metabolism.

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 104 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Biotechnology Drug Metabolism Half life Excretion Toxicity Amphotericin B Kidney 24 hours 40% in urine Anaemia, chills, metabolism hypomagnesaemia. etc Nystatin In GI Dependent on 100% 0% GI transit Isavuconazonium Hepatic 80-130 hours 45% both in Its active moiety avoided metabolism urine and feces in pregnant women Itraconazole Liver 21 hours 3-18% in feces Upper respiratory tract metabolism 40% in urine infection. etc Voriconazole Hepatic 2% unchanged Nausea, abdominal pain, metabolism in urine rash..etc Terbinafine Liver Highly variable 70-90% Abdominal cramps, metabolism absorbed anorexia, gastritis..etc Anidulafungin Hepatic 27 hours 30% in feces & Infection, infestation metabolism 1% in urine NOS, mediastinal disorder.etc Caspofungin Hepatic 9-11 hours Urine (41%) & Infection, infestation feces (35%) NOS, GI disorder.etc Micafungin Liver 11-17 hours 40% in feces & GI disorder, metabolism 15% in urine malnutrition, infection.etc Efinaconazole 29.9 hours Allergic like rash, hives..etc Teruconazole Systematically 6.9 hours 32-56% in urine Allergic like rash, metabolized & 47-52% in hives..etc fecal Posaconazole Hepatic ~35 hours ~17% in urine & Diarrhoea, headache..etc metabolism fecal Table 2: Pharmocology and Pharmakinetics of antifungal drugs

11. ROLE OF BIOINFORMATICS In silico studies considered as a back bone of drug industry. Bioinformatics provide wide range of drug related databases and softwares, which can be used for various purposes, related to drug designing and development processes. The prolonged use of the antibiotics over the years has transformed many organisms resistant to multiple drugs. This has made the field of drug discovery of vital importance in curing various infections and diseases. The drugs act by binding to a specific target protein of prime importance for the cell’s survival. Several drugs like Natamycin, Filipin, Effinaconazole etc. having many side-effects, are reported to be ineffective against Aspergillus Flavus when used alone. Hence, it is the need of the hour to explore the possibility of identification of novel drug targets and designing of drugs against A. Flavus. In silico subtractive/differential genome analysis (Barh D et al., 2011) is a powerful approach for identifying genus- or species-specific genes, or groups of genes that are responsible for a unique phenotype. It can be possible now due to the availability of whole proteome sequences of the desired organism from NCBI (https://www.ncbi.nlm.nih.gov) and UniProt (http://www.uniprot.org). In the present study, the proteome of the A. Flavus was analyzed. The computational approach, based on subtractive genomics, has been used successfully in earlier studies for finding putative drug targets and potential drug. In this work we used different tools such as BLASTp, KEGG (http://www.genome.jp/kegg/) and database such as NCBI, PubChem (https://pubchem.ncbi.nlm.nih.gov), PubMed (https://www.ncbi.nlm.nih.gov/m/pubmed/), DrugBank (https://www.drugbank.ca), TTD (Therapeutic Target Database) etc. The search for novel drug targets is relying on the genomics data. Comparative genomics

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 105 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Biotechnology approach can be used for selecting non-homologous genes coding for proteins which are present in pathogen but not in the host. For identifying such genes, Basic Local Alignment Search Tool (BLAST) against the human using BLASTP program can be performed. This will eliminate homologous genes present in the human. The genes and their products which can be used as a potential drug targets can be identified by analyzing these genes with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database

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Pfaller MA, Messer SA, Motyl MR, Jones RN, Castanheira M. (2013). In vitro activity of a new oral glucan synthase inhibitor (MK-3118) tested against Aspergillus spp. By CLSI and www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 108 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Biotechnology EUCAST broth micro dilution methods. Antimicrob Agents Chemother 57: 1065–1068. Qureshi H., Hamid S. S., Ali S. S., Anwar J., Siddiqui A. A., Khan N. A. (2015). Cytotoxic effects of aflatoxin B1 on human brain micro vascular endothelial cells of the blood– brain barrier. Med. Mycol.53 409–416. 10.1093/mmy/myv010 Roemer T, Boone C. (2013). Systems-level antimicrobial drug and drug synergy discovery. Nat Chem Biol 9: 222–231. Sharmila Banu G., Kumar G., Murugesan A. G. (2009). 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Section 6

MICROBIOLOGY

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THE FUTURE OF OUR ENVIRONMENT Dr. Chetana Rajyaguru M.V.M.Science & Home science college, Rajkot ABSTRACT: The only life-bearing planet, our home, Earth was formed around 4 billion years ago. A unique environmental balance is what supported the unicellular and now us. Human existence spans over 2,00,000 years in the midst of natural resources. The Environment is the gift to our existence and development. We, Human beings are at the centre of concerns for sustainable development and are entitled to a healthy and productive life in harmony with nature. There is growing evidence of the impacts of global environmental changes in ecosystems especially in last 70 years because of people, and a renewed consciousness among peoples and nations need to act quickly to protect the planet’s ecological and climatic systems. There are now 7.6 billion of us on Earth. As our numbers continue to grow, we continue to increase our need for more water, food, land, transport and energy. As a result, we are accelerating the rate at which we are changing our climate. In fact, our activities are not only completely interconnected with but now also interact with, the complex system we live on: Earth. It is important to understand how all this is connected and what are major environmental concerns. The planet needs a mandate for change. Being the super species, we have taken earth as granted. Our thoughts affect our environment through our actions. The only solution left to us is to change our behaviour, radically and globally, on every level. In short, we urgently need to consume less. A lot less and we need to conserve more. To accomplish such a radical change in behaviour would also need radical government action. Today our planet and our world are experiencing the best of times, and the worst of times. The world is experiencing unprecedented prosperity, while the planet is under unprecedented stress. Over the course of the age, the relationship between the human world and the planet that sustains it has undergone a profound change. when the century began neither human numbers nor technology had the power to radically alter planetary systems. As the century closes, not only do vastly increased human numbers and their activities have that power, but major, unintended changes are occurring in the environment, in soils, in waters, among plants and animals, and in the relationships among all of these. The rate of change is outstripping the ability of scientific disciplines and our current capabilities to assess and advice.

ENVIRONMENTAL STRESS The Stress on Environment leads to many issues like survival of human race, greenhouse effects, depletion of ozone layer, air pollution, toxic waste accumulation, desertification, loss of forests, species loss and acidification. The satisfaction of human needs and aspirations is the major objective of development. the essential needs of vast number of people in developing countries for food, clothing, shelter, jobs are not being met, beyond their basic needs there is a need these people to have legitimate aspirations for an improved quality of life. The living standards that go beyond the basic minimum are sustainable only if consumption standards everywhere have egad for long term sustainability. The growth has no set limits in terms of population or resource use beyond which lies ecological disaster. There are different limits hold for the use of energy, materials, water and land, but each is unwilling to assume that others will behave in this socially desirable fashion, and hence all continue to peruse narrow self-interest. It is frustrating the attempting of political and economic institutions, which evolved in a different, more fragmented world, to adopt and cope. For this to happen, we started to understand better the symptoms of stress that confront us, identifying the causes, designing new approaches to managing environmental resources and to sustaining human development. All types of misuse of nature have unforeseen effects on the environment. Today's environmental challenges arise both from the lack of development and from the unintended consequences of some forms of economic growth. The environmental stress has often been seen as the result of the growing demand on scarce resources and the pollution generated by the rising living standards of the relatively affluent. But the poverty itself pollutes the environment, creating environmental stress in a different way. Those who are poor and hungry will often destroy their immediate environment in order to survive. The poverty is a major global scourge. They will cut down forests, their livestock will overgraze grasslands, they will overuse marginal land, and in growing numbers they will crown into congested cities. The cumulative effect of these changes is so far reaching as to make poverty itself a major global scourge. The over production of products has had unforeseen effects on the environment. Thus, today's environmental challenges arise both from the lack of development and from the unintended consequences of some forms of economic growth. The failures that we need to correct arise both from poverty and from short sighted way in which we have often perused prosperity. Due to growth many of www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 110 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Microbiology the products and technologies that have gone into this improvement are raw material and energy intensive and entail a substantial amount of pollution. Environmental stresses and patterns of economic development are linked one to another. For economically sustainable policy of globe to happen we must understand better the symptoms of stress that confront us, we must identify the causes, and we must design new approaches to managing environmental resources and to sustaining human development. It is not that there is one set of villains and another of victims. All would be better off if each person took into account the effect of his or her acts upon others. But each is unwilling to assume that others will behave in this socially desirable fashion, and hence all continue to peruse narrow self-interest. The communities or governments can compensate for this isolation through laws, education, taxes, subsidies and other methods. Well enforced laws and strict liability legislation can control harmful effects. The more important, effective participation in decision making processes by local communities can help them articulate and effectively enforce their common interests. Future Earth is a global research platform designed to provide the knowledge needed to support transformations towards sustainability. Future Earth seeks to build and connect knowledge to increase the impact of research, to explore new development paths, and to find new ways to accelerate transitions to sustainable development. Future Earth will contribute to achieving goals on global sustainable development. COMMON CONCERNS We have common concerns as the Earth is one but the world is not. We all depend on one biosphere for sustaining our lives. Yet each community, each country, strives for survival and prosperity with little regard for its impact on others. The failures we need to correct arise both from poverty and from short signed way in which we have often pursued prosperity. The people will cooperate to build a future that is more prosperous, more just, and more secure. It is not that there is one set of villains and another of victims. all would be better off if each person took into account the effect of his or her acts upon others. The communities or governments can compensate for this isolation through laws, education, taxes, subsidies and other methods. well enforced laws and strict liability legislation can control harmful effects. The more important, effective participation in decision making processes by local communities can help them articulate and effectively enforce their common interests. For equity and common interest in sustainable development to protect through education, institutional development and law enforcement. but many problems of resource depletion and environmental stress arise from disparities in economic and political power. The traditional social systems recognized some aspects of interdependence and enforced community control over agricultural practices and traditional rights relating to water, forests and land. This enforcement of the common interest did not necessarily impede growth and expansion though it may have limited the acceptance and diffusion of technical innovations. SUSTAINABLE FUTURE The development that meets the needs of the present without compromising the ability of future generations to meet their own needs. It contains within it two key concepts. 1.the concept of needs in particular the essential needs of the world's poor, to which overriding priority should be given. 2. The idea of limitations imposed by the state of technology and the social organization on the environments ability to meet present and future needs. Living standards that go beyond the basic minimum are sustainable only if consumption standards everywhere have regard for long term sustainability. Yet many of us live beyond the world’s ecological means, for instance in our patterns of energy use. perceived needs are socially and culturally determined. and sustainable development requires promotion of values that encourage consumption standards that are within the bounds of the ecological possible and to which all can reasonably aspire. The global scenario of human activity is to be scrutinized through Environmental Impact Assessment. The use of new inventions and technology must be justified in the favour of nature which, otherwise is detrimental for our sustenance on earth. There should be a set of rules for conservation of nature and it follow up to address the critical survival issues like degradation of Natural Resources, Ecosystems, Biodiversity and related environmental issues. The adoption of sustainable maintenance of our environment leads us to the better future. It will be by implementing long term environmental strategies, cooperation among major-minor nations to achieve common and mutually supportive objectives that take account of the interrelationships between people, resources, environment and development. The fulfilment of all the work requires the reorientation of technology-the key link between humans and nature. The vision of Future Earth is for people to thrive in a sustainable and equitable world. This requires contributions from a new type of science that links disciplines, knowledge systems and societal partners to support a more Fragile global innovation system. In all countries, the processes of generating alternative technologies, upgrading traditional ones, and selecting and adapting imported technologies should be informed by commercial organizations is developed to product and process innovations that have market value. The Physical linkages among the countries for the sustainable development and management of ecosystems is demanded to be merged for now.

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POPULATION ISSUE At a faster rate the world population increases without thought of People resource ratio. There are several fundamental steps being taken by all Governments to decrease in population rate, eliminate mass poverty, implementation of Family planning by Mutual understanding. The world population today is 7.5 billion which is estimated to become 8 billion by the year 2023. FOOD ISSUE Today the picture of world food production is heterogeneous as all nations cannot produce the enough amount of food for their people, may be due to geography, climate, poor technology or inefficient agricultural practices. we need to improve on this to provide food to every citizen of the world. The future of the food requires systematic attention to the renewable natural resources. It requires a holistic approach focused on ecosystems at national, regional, and global levels, with coordinated use of land and careful planning of water usage and forest exploitation. SPECIES AND ECOSYSTEMS ISSUE There are 6.5 million on Land and 2.2 million in Ocean- total 8.7 million species are found on world according to latest survey of the earth. So many of the animal and plant species are extinct and many more are endangered. There is still time to save species and their ecosystems which is a prerequisite for sustainable development. ENERGY ISSUE The current scenario of world's energy resources is utility and supply based. The solar energy is 1 % of the total electricity production of the world. In case of marine energy 0.5 GW is being produced and 1.7 GW is under construction. The nuclear energy in 2015 involves 65 nuclear reactors producing 64 GW of power and 44 nuclear reactors are under construction. Hydro power is 71 % of the renewable electricity production in 2015 total 1209 GW. Energy based on oil is 32.9 % of global energy consumption. The energy generation from natural gas is 22 % of the total production. Up to the year 2015 wind power generation reached to 432 GW which is 7 % of the total generation. There is decline in energy production from coal in 2015 but still we get 40 % of total electricity from coal. The output from geothermal energy is 75 TWh for heat and 75 TWh for power. Bio energy is 10 % of the total energy production in world. Out of 18 % renewable energy 14 % is produced by Bio energy. A safe environmentally sound and ecologically viable energy pathway that will sustain human progress into the distant future is clearly imperative and possible. It requires new dimensions of political will and institutional cooperation to achieve it. INDUSTRIALIZATION ISSUE The Industries are made for production of goods useful for mankind. The Explosion of industrialization without any care of the environmental fragility is not sustainable. The ratio of established industries worldwide has been ever increasing to meet the demand of increasing population as well as affluent attitude of people. International trade associations and labour unions should develop special environmental training programmes for developing countries and disseminate information on pollution control, waste minimization, and emergency preparedness plans through local sources. To focus on longer term strategies to preserve and build on the achievements of the existing Treaty system, nations must create the means to foster dialogues among politicians, scientists, environmentalists and industrialists within and outside it. URBANIZATION ISSUE According to the survey done in 2018 the rate of urbanization all over the world is very fast. In North America 81 %, Latin America 80 %, Europe 74 %, Oceania 69 %, Worldwide 54%, Asia 49 % and in Africa it is 41 %. The rush of rural people towards the urban areas is a challenge to be managed. The small urban areas should be supported enough to reduce the big pressures on larger cities. As a result of over urbanization there is a big risk of exposure of rural people to environmental hazards. It also needs to meet the demands of large population to live in. INTERNATIONAL STRATEGIC PLANS The world must quickly design strategies that will allow nations to move from their present, often destructive, processes of growth and development paths. This will require policy changes in all countries, with respect both to their own development and to their impacts on other nations development possibilities. The critical objectives for Environment Development Policies are to revive growth, to change quality of life, to meet essential needs for jobs, food, energy, water and sanitation, to ensure a sustainable level of population, to conserve and enhance the resource base to reorient technology and manage risk, to merge environment and economies in decision making. The developing countries to interrelated concerns lie at the heart of issues on financial flows; one concerns the quantity and other the quality of resource flows to developing countries. The www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 112 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Microbiology idea that developing countries would do better to live within their limited means is a cruel illusion which has to be corrected and hand of cooperation should be given. FOCUS ON HEALING THE DAMAGES The pressure on resources increases when people lacks alternatives. The development policies must widen people's options for earning sustainable livelihood, particularly for resource-poor households and in areas under ecological stress. The ultimate limits of global development are perhaps determined by the availability of energy resources and the capacity of the biosphere to absorb the by-products of used energy. So, enhancing resource base can help decrease this pressure. GREEN FUTURE In Future work will be done with partners in society to co-develop the knowledge needed to support decision- makers and societal change at all scales and in diverse contexts, by focusing on three Research Themes- Dynamic Planet, Global Sustainable Development and Transformations towards Sustainability. These are the steps to be made in future. Deliver water, energy, and food for all, and manage the synergies and trade-offs among them, by understanding how these interactions are shaped by environmental, economic, social and political changes. Decarbonise socio-economic systems to stabilise the climate by promoting the technological, economic, social, political and behavioural changes enabling transformations, while building knowledge about the impacts of climate change and adaptation responses for people and ecosystems. Safeguard the terrestrial, freshwater and marine natural assets underpinning human well-being by understanding relationships between biodiversity, ecosystem functioning and services, and developing effective valuation and governance approaches. Build healthy, resilient and productive cities by identifying and shaping innovations that combine better urban environments and lives with declining resource footprints, and provide efficient services and infrastructures that are robust to disasters. Promote sustainable rural futures to feed rising and more affluent populations amidst changes in biodiversity, resources and climate by analysing alternative land uses, food systems and ecosystem options, and identifying institutional and governance needs. Improve human health by elucidating, and finding responses to, the complex interactions amongst environmental change, pollution, pathogens, disease vectors, ecosystem services, and people’s livelihoods, nutrition and well-being. Encourage sustainable consumption and production patterns that are equitable by understanding the social and environmental impacts of consumption of all resources, opportunities for decoupling resource use from growth in well-being, and options for sustainable development pathways and related changes in human behaviour. Increase social resilience to future threats by building adaptive governance systems, developing early warning of global and connected thresholds and risks, and testing effective, accountable and transparent institutions that promote transformations to sustainability. Several Solutions are also to be put into practice. Open and inclusive platforms for observing and monitoring the status, trends and thresholds of the planet in a timely manner at different scales, including tracking fast-changing sentinel processes and systems. Tailored metrics and evaluation tools for well-being and sustainable development. A new generation of integrated Earth system models to deepen our understanding of complex Earth systems and human dynamics across different disciplines, and to underpin systems-based policies and strategies for sustainable development. Science-based data, tools and resources to support improved resilience of people, communities and economies, including disaster risk reduction. Scenarios for transformative development pathways that enable global sustainability, to help evaluate different strategies and options. Critical contributions to key debates on global sustainability issues, including inputs to scientific assessments and decision-relevant syntheses. Innovations in communicating, engaging and visualising global change and sustainability, fully exploiting the potential of new technologies and overcoming differential access to information across the world. Research area can be made strong by a few decisions like Conducting fundamental and applied research in ways that engage with diverse societal partners across all regions of the world to maximise impact and responsiveness to society’s needs, and monitoring the effectiveness of these new approaches to research. Establishing Future Earth as a globally recognised model for engagement and collaboration in research for global sustainable development, effective in all world regions. Stimulating debate, illustrating good practice and mobilising capacities for solutions-oriented science, technology and innovation for sustainability. Changing international research funding practices to better support interdisciplinary and trans disciplinary research and engagement across and within regions. Fostering collaboration among national and international agencies’ research programmes, to maximise resources for and impacts of research towards sustainability. Contributing to improved modes of sharing data about environmental change and progress towards sustainability in order to support policy and practice at different levels.

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SECURITY AND DEVELOPMENT No single blue print of sustainability will be found, as economic, social and ecological conditions differ widely among countries. Each nation will have to work out its own concrete policy implications. Yet irrespective of these differences, sustainable development should be seen as a global objective. No country can develop in isolation from others. Hence the pursuit of sustainable development requires a new orientation in international relations. The unity of human needs requires a functioning multilateral system that respects the democratic principle of consent and accepts that not only the earth but also the world is one and secure. MULTILATERALISM The multilateralism is now the ultimate need as there are mutual interests involved in many environmental and development issues to be solved forgetting the boundaries of the nations. The multilateralism can cut across the divides of national sovereignty, of limited strategies for economic gain and of separate discipline of science. Public awareness and actions will have the indispensable and crucial role in putting the world onto the path of sustainable development. Together, we will span the globe, and pull together to formulate an interdisciplinary, integrated approach to global concerns and our common future. REFERENCES • Global Future Toward Just and Sustainable Human Development, Approved By the 208th General assembly (1996) Presbyterian Church (U.S.A.) (1996), Developed by the advisory Committee on Social Witness policy, Published by the Office of the General Assembly100 Witherspoon Streetlouisville, Kentucky 402021396 • Our Common Future, The World Commission On Environment and Development, Oxford University Press,Oxford(1987) • Environmental Sustainability: A Definition for Environmental Professionals, John Morelli, Rochester Institute of Technology, (2011), [email protected] • Cooper, P. J., & Vargas, M. (2004). Implementing sustainable development: From global policy to local action. Lanham, MD: Rowman and Littlefield Publishers, Inc. • Dernbach, J. C. (1998). Sustainable development as a framework for national governance. Case Western Reserve Law Review, 1-103. • United Nations Conference on the Human Environment. (1992). Rio Declaration on Environment and Development. Rio de Janiero, Brazil: United Nations • World Headquarters. Global Sustainability Principles. (2007) Web. 30 Oct. 2010. . • Global Survey on world Urbanization by continent 2017

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 114 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Microbiology

BIOACTIVITY GUIDED FRACTIONATION OF PRICKLES OF BOMBAX CEIBA LINN. FOR IN-VITRO ANTIMICROBIAL ACTIVITY WITH REFERENCE TO ACNE VULGARIS. Darshan Vasani, Riddhi Gorasiya, Neha Chohala, Ms. Jigna Vadalia*. Department of Pharmaceutical Sciences, Saurashtra University, Rajkot -360005, Gujarat, India. Email: [email protected] ABSTRACT: Acne is a cutaneous, pleomorphic disorder of pilosebaceous gland. Due to side effect of current therapy and development of resistance by microbes for frequently used antibiotics which leads to treatment failure. Hence develops a need to identify constituent with antiacne properties. This plant is official in ayurvedic pharmacopeia and is recommended for treatment of acne. The prickles were collected and verified by the botanist and then dried, powdered and extracted using various solvents like petroleum ether, chloroform, ethyl acetate, methanol, hydroalcoholic mixture. The In-vitro antimicrobial activity of prickle extracts were preliminarily screened by agar diffusion method and then most potent fraction was selected for bioautography to identify the principle responsible for antimicrobial activity. Based on above mentioned method ethyl acetate extract and its n-butanol fraction was found to be sensitive against Propionibacterium acne than all other extracts and bioautography of n-butanol extract was carried out for anti-microbial activity and compared with standard quercetin to identify active phytoconstituent. The present study was carried out to evaluate the In-vitro anti-microbial activity of extracts of prickles of Bombax ceiba portrayed by different microbial species with reference to acne vulgaris. Keywords: Bombax ceiba, Acne, In-vitro anti-microbial activity, Bioautography.

1. INTRODUCTION Acne is a cutaneous, pleomorphic disorder of pilosebaceous gland. Due to side effect of current therapy which leads to treatment failure. It is traditional drug is recommended for treatment of acne. Main goal was to check antimicrobial activity of active constituent with reference to acne vulgaris. The In-vitro antimicrobial activity of prickle extracts and its fractions were preliminarily screened by agar diffusion method using Propionibacterium acne as it is one of the causative agents for acne. And then most potent fraction was bioautographed to identify Rf value of active phytoconstituent. 2. DATA ANALYSIS 1) Antimicrobial activity Propionibacterium acne (MTCC NO. 1951) was activated by incubation kept for 72 hours at 37°C. Nutrient agar media was prepared and inoculated with strain by pour plate method. Agar plates were allowed to solidify. Wells were made using sterile cup borer (diameter 6mm). 100µL of test extracts of concentration 5,10, and 20 mg/ml was introduced into the wells and plates were incubated for 24 hours at 37°C in incubator. Inhibitory activity was identified by comparing diameters of zone of inhibition (mm). Azithromycin was used as positive control (reference standard). Experiment was done in triplicates and results were expressed as diameter of zone of inhibition ± SEM. Extract with maximum zone of inhibition was further fractioned with other solvents like hexane, dichloromethane, butanol and water and again, antimicrobial activity was done as mentioned above using fractions obtained using concentrations of 5,10 and 15 mg/ml. Results were expressed as diameter of zone of inhibition ± SEM. 2) Bioautography Contact bioautography: Most potent fraction was resolved using TLC using suitable solvent. These resolved plate was flipped and placed in inoculated agar layer and left for 45 minutes for diffusion subsequently plate was removed and agar plates were incubated for 72 hours at 37°C. Immersion bioautography: Fractions were applied on silica plate, develop with toluene: ethyl acetate: methanol: formic acid (3:7:1:0.5). Developed chromatogram was placed in prepared plate and then incubated at 37°C for 24 hours. Then sprayed with aqueous solution of tetrazolium salt. Area of inhibition shows purple coloured background. Rf value was measured and compared with standard quercetin.

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3. RESULT AND DISCUSSION Zone of inhibition for extracts (in mm) Conc. of extracts Pet ether Chloroform Ethyl acetate Methanol Hydroalcohol (mg/ml) Azithromycin 14.33±0.33 13.66±0.33 17.33±0.33 15.66±0.33 15.33±0.33 (100µg/ml) 5 - 7.33±0.33 6.66±0.33 11.33±0.33 11.33±0.33 10 5±0.00 11.33±0.33 12.66±0.33 17.33±0.33 11.66±0.33 20 8.33±0.33 11.33±0.33 19.66±0.33 15.66±0.33 14.66±0.33 Zone of fractions for extracts (in mm) Conc of extracts Hexane Dichloromethane n- butanol Water (mg/ml) Azithromycin 14.33±0.33 15.66±0.33 16.33±0.33 15±0.00 (100µg/ml) 5 7.33±0.33 8.33±0.33 8.66±0.33 7.33±0.33 10 10.33±0.33 12±0.00 14.66±0.33 10.33±0.33 15 14.66±0.33 15±0.00 24±0.00 14.±0.00 Bioautography of extracts shows zone of inhibitions for P.acne pathogen at Rf value between 0.89 to 0.93 in contact as well as immersion method was almost found nearer to standard quercetin . 4. CONCLUSION From performed practical and its proven activities prickles of Bombax ceiba extracts (n-butanol fraction of ethyl acetate extract) was found to have antimicrobial activity with reference to acne vulgaris due to presence of phenols, flavonoids, tannins and quercetin which were identified by bioautography. 5. ACKNOWLEDGEMENT I earnestly wish to express sincere thanks and gratitude to my guide Ms. Jigna Vadalia, Assistant professor, Department of Pharmaceutical sciences, Saurashtra University, Rajkot, for her keen interest, valuable guidance and suggestions, constant encouragement and advice throughout the tenure of this study. In addition, she was always accessible and willing to help me. As a result, this dissertation work became smooth and rewarding for me. And would like to especially thank Ms. Nikunja Bavishi to include us in her dissertation work My Special thanks also to our principal Dr. Navin Sheth, Dr. Ashwin Dudhrejiya and also Dr. Mihir Raval for their valuable suggestion, constructing criticism and the unending inspiration that have given a shape to this work. REFERENCES 1. Burkhart C.G., Burkhart C.N., Lehmann P.F., 1999, Acne: a review of immunologic and microbiologic factors, Journal of Postgraduate Medicine, 75; 328-331. 2. Verma, V., Jalalpure, S., Sahu, A., Bhardwaj, L. & Prakesh, Y. 2011. Bombax ceiba Linn: pharmacognostical, phytochemistry, ethnobotany, and pharmacology studies. International Pharmaceutical Sciences, 1, 62-68. 3. Betina et al. 1973. Bioautography in paper and TLC and its scope in antibiotic field. Journal of Chromatography A, 78.41-51.

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Botany

Section 7

BOTANY

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Botany

EFFECT OF INCREASING CONCENTRATION OF SEAWATER IN SOIL ON GROWTH OF PLANTS (RGR, NAR, LAR) OF PEARL MILLET CROP OF SAURASHTRA REGION Rasik C.Viradia Department of Botany, H. & H. B. Kotak Institute of Science, Rajkot, 360001, Gujarat E-mail: [email protected] ABSTRACT: The basic objective of the plant growth (RGR, NAR, LAR) of millet crop in different concentration of seawater in soil is to give farmers for increasing agricultural production. The western region of Gujarat state in India can be divided into three zones. Intensive agriculture is restricted to the central zone of Gujarat, which is characterized by semi-arid eco-climate. Plants are not incompatible to soil salinity, but most of the plants do not grow in the presence of high concentration of salts. The most common effect of salinity on plants is suppression of growth, which is associated with reduction in crop yields. Three major constraints which have been recognized on the growth of plants in saline habitats are as below (i) Water stress arising from the more negative water potential (elevated osmotic potential) of the rooting medium, (ii) Specific ion toxicity usually associated with excessive intake of chloride. In the coastal area and the saline desert, Plant growth and crop production is generally poor due to the concentration of salt in soil. Moreover, in coastal area, salt concentration is increasing in ground water due to ingression of Arabian Sea. Eventually, ground water containing high salt-content is used for irrigation of crops. Present investigation is to study the responses of pearl millet (Pennisetum glaucum, L) to soil salinisation with respect to plant growth. Key words: Salinity; Soil type; Seawater concentration; Water potential; RGR, NAR, LAR; Plant growth.

1. INTRODUCTION One of the major obstacles to increasing plant growth and crop production in arid and semi-arid regions is the lack of fresh water resources. Waters with salinities higher than 3 dSm-1 can be used to irrigate salt tolerant crops, but should be used judiciously for salt sensitive crops (FAO, 1992). If some fresh water is available, it can be used to reduce the salinity of water by mixing before irrigation (FAO, 1992). Plant growth and crop that are relatively salt sensitive can be irrigated with saline water during the less sensitive stages of growth (Rhoades, 1984). Specifically, fresh water should be used in early stages and saline water in later stages (Hamdy et al., 1993). Salt tolerant crops are often grown with water containing salt using surface, sprinkler and drip irrigation methods (Ayers, 1977; FAO, 1992). However, these methods usually result in increased soil salinity, soil degradation and nutrient imbalances (Maas and Hoffman, 1977; Pasternak et al., 1986). In arid areas, even fresh water irrigation could result in severe salinity problems due to the high evaporation rates at the soil surface (Gupta ad Abrol, 1990). 2. MATERIAL AND METHODS Total 200 polyethylene bags were each filled with 2 kg black-cotton soil and arranged in 10 sets so that each set contained 20 bags. Soils contained in bags for 10 different sets were then separately watered with 600 ml water containing 50%, 40%, 33%, 25%, 20%, 17%, 14%, 12%, 10% and 0% seawater. Seawater was mixed with tap water in above proportions to prepare different concentrations of seawater. Tap water without addition of seawater was treated as control or as 0% seawater content. Thus, there were a graded series of soils containing ten concentrations of seawater. Watering with 600 ml water was estimated to raise the soil moisture up to field capacity (30% moisture). Thereafter, soils were allowed to dry. After five days soil in each bag was raked with fingers and 10 seeds of perl millet were sown at the depth of 8-12 mm. Immediately after sowing soil was irrigated with tap water. Subsequently, soil in each bag was irrigated with tap water at the intervals of 8 days. After the germination, two seedlings that emerged first were left in each bag and others were uprooted. During growth period seedlings were watered at the intervals of 8 days to keep the soil moist. Three weeks after the emergence, seedlings in five bags at each salinity level were washed to remove the soil particles adhered with roots. Morphological characteristics of each plant were recorded. Shoot height and root length were measured. Leaf area was marked out on graph paper. Dry weight of leaves (leaf blades), stems (along with leaf sheaths) and roots were determined. At the last three harvests, i.e. at 6, 9 and 12 weeks, inflorescences were separated from the plants and their dry weight was determined. Values of dry weight of leaf, stem, inflorescence and root components of plants together with leaf area were used to calculate RGR, NAR and LAR as follow: www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 117 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Botany

log W" – log W′ Relative growth rate(RGR) = , Where W" and W' are plant dry weights at time t" and t' t" – t′ Log L" – log L′ (W" – W′) Net assimilation rate (NAR) = , Where, W', L' and W", L" are plant dry weight and leaf (L" – L′) (t" – t′ ) area, respectively at time t" and t'

Leaf area in cm2 Leaf area ratio (LAR) = Plant dry weight in mg 3. RESULTS AND DISCUSSION 3.1 PEARL MILLET ELONGATION OF STEM AND ROOT AND EXPANSION OF LEAF Height of shoots of control plants consistently increased till 12-week period and was maintained constant thereafter (Fig.3A, B & C.). A similar pattern for stem elongation was also obtained for the plants grown in saline soils, however, plant height decreased with increasing soil salinity. Reduction in shoot height of plants grown in saline soils was recorded as compared to the shoot height of control plants since 3-week growth period. Plants grown in soils at 18.4 and 20.0 dSm-1 salinity did not survive after 6-weeks. Elongation of shoot was most rapid between 3-to 6-week period for the plants grown in soils at 1.2, 6.0, 6.3 and 8.5 dSm-1 salinity, whereas most rapid elongation was recorded between 6 to 9-week growth period for plants grown in soils with > 8.5 dSm-1 salinity (except for plants grown in soils at 15.8, 18.0 and 20.0 dSm-1 salinity). Moreover, shoot elongation was least rapid till 3-week growth period for plants grown under both control and saline conditions. There was a negative relationship between shoot height at 12-week growth period and soil salinity according to the following expression: Y = 62.89 - 2.181 X (r = -0.667, p<0.01, df = 23), where Y is shoot height (cm) and X is soil salinity (dSm-1). Pattern of root elongation for control as well as salt –stressed plants was similar to that of shoot elongation. Root length for plants grown in control as well as in saline soils was lower than the shoot height. Root length decreased with increasing soil salinity. Elongation of root was most rapid for control as well as for salt-stressed plants during the first 3-week growth period. Relationship between root length and soil salinity at 12-week growth stage was obtained according to the following expression: Y = 50.91 -1.77 X (r = -0.722, p<0.01, df = 23), where, Y is root length (cm) and X is soil salinity (dSm-1). Leaf area of control as well as of salt-stressed plants increased till 9-week growth period and following this period leaf area decreased. Moreover, leaf area of salt-stressed plants decreased with the increase in soil salinity. Leaf expansion was most rapid between 3-and 6-week growth period for both control and salt-stressed plants. There was a negative relationship between leaf area at 12-week growth stage and soil salinity according to the following expression: Y = 434.98 - 23.39 X (r = -0.778, p<0.01, df = 23), where Y is leaf area (cm2) and X is soil salinity (dSm-1).

400

45 350 C 40 B 300 35 ~ ."1 250 C. -E 30 ~ 25 1 200 {20 ~ 150 .; 8 15 .3 100 "' 10 50 5 0 0 0 3 6 9 12 0 3 6 9 12 week week Fig.3A.Shoot height and B. root length of pearl millet plants over time in response Fig .3C. Leaf area of pearl millet plants over time in response to soil salinity. to soil salinity. (• ), 1.2 dSm·' ; (0 ), 6.0 dSm·':• ),6.3 dSm·': \ll ), 8.5 dSm·': (• ),1.2 dSm·' ; ( O ), 6.0 dSm.' ; ( ■ ),6.3 dSm·' : (D ), 8.5 dSm·': (• ), 9.5 dSm·': (• ), 9.5 dsm·': (" ),10.2 dsm·• : ( ♦ ),13.0 dsm·'and (◊ ),15.0 dsm·• salinity. ( " ),10.2 dsm·•: ( ♦ ),13.0 dsm·'and (◊ ),15.0 dsm·• salinity. 3. 2 PEARL MILLET DRY MATTER ACCUMULATION Shoot weight of control plants increased till 9-week and it declined slightly thereafter (Fig.6A, B & C.). Pattern of dry matter accumulation in shoots of plants grown in saline soils (soils mixed with different concentrations of seawater) was similar to that in shoots of control plants. Initially, dry weight of shoots of salt-stressed plants was almost equal to that of control plants till 3-week growth stage. Following this growth period, dry weight of shoots of salt-stressed plants was lower than that of control plants. Moreover, dry weight of shoots of salt- stressed plants decreased as the salt concentration increased in soil. Plants grown in soils at 18.4 and 20.0 dSm- 1 salinity did not survive after 6-week growth period. There was a negative relationship between shoot weight and soil salinity according to the following expression: Y = 4668.7-275.6 X (r = -0.777, p<0.01, df = 23), where, Y is shoot weight (mg) and X is soil salinity (dSm-1). Pattern of dry matter accumulation in roots of plants growth in control and saline soils was similar to that of shoots. There was a negative relationship www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 118 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Botany between root weight at 12-week growth stage and soil salinity according to the following expression: Y = 2166.9 -14.17 X (r = -0.776, p<0.01, df = 23), where, Y is root weight (mg) and X is soil salinity (dSm-1). Moreover, pattern of dry matter accumulation in whole plant (shoot+root) was similar to that of shoot in both control and salt-stressed plants. Relationship between total plant weight at 12-week growth stage and soil salinity was obtained according to the following expression: Y = 8630.8 -479.6 X ( r = -0.791, p<0.01, df = 23), where, Y is total plant weight (mg) and X is soil salinity (dSm-1). Emergence of inflorescence was recorded on about 30% plants grown in control conditions. Further, about 30% 30%, 30%, 30%, 25% 20% and 10% plants grown in soils at 6.0, 6.3, 8.5, 9.5, 10.2, 13.0 and 15.0 dSm-1 salinity produced inflorescence. Inflorescence weight at 12-week growth stage was added to shoot weight. 7000

6000 C ) 1 1800 - 5000 1600 B

1400 ) 4000 1 - 1200

1000 3000

800 2000 600 Total plant weight plant ( weight plant mg Total

Root weight weight (mg plant Root 400 1000

200 0 0 0 3 6 9 12 0 3 6 9 12 week week l l l l Fig.6A.Shoot weight and B.root weigth of pearl millet plants over time in response Fig.6C. Total plant weight of pearl millet plants over time in response to soil salinity. -1 -1 -1 -1 -1 -1 -1 -1 -1 to soil salinity. ( ),1.2 dSm ; (0 ), 6.0 dSm ;(■ ),6.3 dSm ; ( ), 8.5 dSm ; ( ),1.2 dSm ; ( 0 ), 6.0 dSm ;( ■ ),6.3 dSm ; ( □ ), 8.5 dSm ; ( ), 9.5 dSm ; -1 • -1 -1 -1 • -1 -1 -1 ( ), 9.5 dSm ; ( A ),10.2 dSm ; ( ♦ ),13.0 dSm and (◊ ),15.0 dSm salinity. ( A ),10.2 dSm ; ( ♦ ),13.0 dSm and ( ◊ ),15.0 dSm salinity. 3. 3 PEARL MILLET FUNCTIONAL GROWTH ANALYSIS (RGR, NAR and LAR) The functional growth analysis approach was followed to assess the effect of increasing soil salinity on plant growth. RGR of control plants was consistently greater than that of plants grown in saline soils. Moreover, RGR of plants grown in saline soils decreased with increase in soil salinity. There was maximum RGR at 6- week growth stage and minimum at 12-week growth stage for plants grown in both control and saline soils. Negative RGR values were obtained at 12-week growth state and it can be attributed to advance age of plants. Plants grown in soils at 18.4 and 20.0 dSm-1 salinity did not survive after 9-week growth period. There was a negative relationship between RGR and soil salinity according to the following expression: Y = - 0.0161 + 0.0037 X ( r = - 0.719, p< 0.01, df = 23), where, Y is RGR and X is soil salinity. The NAR of the control plants was also consistently greater than that of salt-stressed plants. Further, NAR of plants grown in saline soils decreased with increasing salt-stress. At 12-week growth stage shoot tissues were metabolically least active and negative NAR was obtained. There was a negative relationship between NAR and soil salinity according to the following expression: Y = - 0.170 + 0.0424 X (r = - 0.632, p< 0.01, df = 23), where, Y is NAR and X is soil salinity. There was a positive relationship between RGR and NAR for plants grown in control and saline conditions. Y = 0.0113 + 11.837 X (r = 0.939, p<0.01, df = 23), where, Y is RGR and X is NAR. LAR was maximum at 3-week growth stage and it consistently decreased thereafter over time for the plants grown in control and saline soils. However, LAR for control plants was consistently greater than that for salt- stressed plants. Increasing soil salinity caused reduction in LAR. 4. CONCLUSION There was reduction in elongation of stem and root, and expansion of leaves of millet plants with increasing soil salinity. These results were primarily due to water stress induced by soil salinity. Dry matter accumulation in leaf, stems, and root tissues of salt-stressed plants decreased. In general, salinity can reduce plant growth through: osmotic effects, (ii) toxic effects of ions and (iii) imbalance of nutrients. Reduction in relative growth rate (RGR) of crop plants was recorded with increase in soil salinity. It can be attributed to the reduced net assimilation rate (NAR) and not to leaf area ratio (LAR). The older leaves of salt-stressed plants were yellow in colour and these leaves might have lower photosynthesis rate and greater respiration rate. Leaf area can limit or promote plant growth may influencing an aspect of NAR, such as photosynthesis or respiration. REFERENCES Akhavan-Kharazian M., Campbell W.F., Jurinak J.J. and Dudley L.M. 1991. Calcium amelioration of NaCl effects on plant growth, chlorophyll and ion concentration in Phaseolus vulgaris. Arid Soil Research and Rehabilitation 5:9-19. Bernstein, L. 1975. Effects of salinity and sodicity on plant growth. Ann. Rev. Photopathol. 13: 295-312.

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Chow, W.S., Ball, M.C., Anderson, J.M. 1990. Growth and photosynthetic responses of spinach to salinity : implications of K nutrition for salt tolerance, Australian Journal of plant physiology, 17 : 563- FAO., 1992. The use of saline waters for crop production. Irrigation and drainage papers, 48. FAO Rome. Francois, LE. Maas, E.V., Danouvan, T. J. and Youngs, V.L. 1986. Effect of salinity on grain yield and quality, vegetative growth, and germination of semi-dwarf and durum wheat. Agron. J. 78 : 1053-1058. Grattan, S. R. Grieve, C. M. 1992. Mineral element acquisition and growth response of plants grown in saline environments. Agriculture Ecosystems and Environment. 38 : 275-300. Greenway, H. 1973. Salinity, plant growth and metabolism. J. Aust. Inst. Agric. Sci. 39 : 24-34. Munns, R. 1993. Physiological processes limiting plant growth in saline soil: some dogwas and hypotheses. Plant cell Environ. 16 : 15 – 24. Ramoliya, P. J. and A. N. Pandey; 2002. Effect of increasing salt concentration on emergence, growth and survival of seedlings of Salvadora oleoides (Salvadoraceae). J. Arid Environment., 51: 121-132. Tozlu, I., Moore, G. A. and Guy, C. L., 2000. Effect of increasing NaCl concentration on stem elongation, dry mass production, and macro-and micro-nutrient accumulation in Poncirus trifoliate. Australian Journal of Plant Physiology, 27 : 35-42.

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ANALYSIS OF PHYSICO CHEMICAL PARAMETERS OF DIFFERENT SOIL SAMPLE OF SURENDRANAGAR Dr. Rutva H. Dave1 and Dr. Jignasa B. Joshi2 Department of Botany1, H. & H. B. Kotak Institute of Science1, Rajkot, 360 001, Gujarat Department of Botany2, Christ college2, Rajkot, 360 005, Gujarat E-mail: [email protected] ABSTRACT: Soil is a natural body of mineral and organic material differentiated into horizons, which differ among themselves as well as from underlying materials in their morphology, physical make – up, chemical composition and biological characteristics. Plants always depend on the soils for their nutrients, water and minerals supply, the soil type is a major factor to determine what types of plants will grow in any area. Analysis of soil was carried out for studies of various parameters like PH, Electrical Conductivity (EC), Total Organic Carbon, available Phosphorus (P2O5), available Potassium (K2O) and Sodium (Na) for the Surendranagar. From this study we get overall information about soil quality of this region. This information will help farmers to solve the problems related to soil nutrients amount of which fertilizers to be added to soil to increase the yield of crops. Key words: Physicochemical, Conductivity, Organic carbon, pH, EC, Phosphorus, Soil analysis. INTRODUCTION Soil is one of the world's most important natural resources. Together with air and water it is the basis for life on planet earth. It has many important functions which are essential for life. Not only does it play the major part in allowing us to feed the world's population, but it also plays a major role in the recycling of air, water, nutrients, and maintaining a number of natural cycles, thereby ensuring that there will be a basis for life in generations to come. Without soil, the world's population neither would nor could survive. Soil consists of a mixture of mineral grains that come from the rock deposits and sediments beneath them. This mixture gives the overall texture of the soil, namely whether the soil is mainly sandy, loamy or clayey. Importantly, soil also contains organic matter, mainly in the top 20cm. The western region of Gujarat state in India can be divided into three zones (1) the Kutch, a northern saline desert, (2) a coastal area along the shore of the Arabian sea, and (3) a central area.[1] Soil texture can give a guide to: soil water - its retention and release to plants; soil structure - its development and stability; nutrient retention and availability; activity and retention of residual soil-acting herbicides; erodibility by wind and water; stickiness and ease of cultivation; drainage characteristics and suitability for artificial drainage; cropping suitability; soil temperature. The aim of the study is for comparison of soil properties of two different region of Saurashtra. STUDY-AREA Surendranagar District is an important district in Saurashtra region. The district has 10 talukas with 658 villages. Total population of the district is about 13, 85, and 148. Male population is 56% while 45 % of the population consists of female. 8.63% of population belongs to scheduled tribe category. However, the percentage of scheduled cast population is only 12.0%. MATERIAL AND METHODS Study area is Surendranagar region. Halvad is located at 23.02°N 71.18°E. It has an average elevation of 46 meters (150 feet). The average rainfall during last 4 years has been more than 700 mm. Work was started on second week of December 2011. Soil analysis is done in Botany laboratory of H. & H.B. Kotak Science College. Physical characteristic of soil Surface soil was collected from an agriculture field, dried. 3 lots of soil of 10 kg each were spread separately over thick plastic sheets. For soil texture water holding capacity and bulk density. Soil texture is measured by standard technique where gravel, coarse sand, fine sand, silt ,clay content is separated by passing through 2.0 mm , 0.2 to 2.0 , 0.02 to 0.2, 0.002 to 0.02 & below 0.002 mm mesh respectively oven dried soil of each sample is weighed. Water holding capacity (WHC) is measured using Gooch crucible. The formula of water holding capacity is as under.

퐚퐦퐨퐮퐧퐭 퐨퐟 퐰퐚퐭퐞퐫 퐢퐧 퐭퐡퐞 퐬퐨퐢퐥 퐱 ퟏퟎퟎ Water holding capacity = 퐖퐞퐢퐠퐡퐭 퐨퐟 퐨퐯퐞퐧 퐝퐫퐲 퐬퐨퐢퐥

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Soil texture

Textural group Size in mm

Gravel Above 2.0 Coarse sand 0.2 to 2.0 Find sand 0.02 to 0. 2 Silt 0.002 to 0.02 Clay Below 0.002

Bulk density: 10×10 cm area soil is randomly taken than their weight &volume is measured.

Bulk Density(gm/cm3) =Weight of soil(gm) Volume of soil Chemical characteristic of soil The pH value is measure by Ph meter method. EC (electrical conductivity) is measured with the help of electrical conductivity meter. Organic carbon is measured by colorimeter with 660 millimicron filter of red color. [2] Phosphorus is measured by Olsen method with use of photoelectric colorimeter (with red colorfilter) and potassium is measure byflame photometer method. Arithmeticmean and standard error is calculated. [3] RESULT Soil texture of Surendranagar is black cotton soil with good fertility. As shown in given table, water holding capacity of the place is medium.

TABLE 1: Physical properties of soil of Surendranagar region Parameter S1 S2 S3 S4 Sand(%) 46.3 ± 0.04 46.1 ± 0.07 45.1 ± 0.15 44.1 ± 0.20 Silt (%) 13.3 ± 0.08 13.8 ± 0.09 12.9 ± 0.10 14.1 ± 0.05 Clay (%) 7.57 ± 0.04 7..55 ± 0.18 7 ± 0.37 6.89 ± 0.83 Water holding capacity 27.6 ± 0.18 29 ± 0.17 30.7 ± 0.23 28.5 ± 0.21 (%) Bulk density (gm/cm3) 1.35 ± 0.01 1.01 ± 0.02 1.23 ± 0.01 1 ± 0.01

TABLE 2: Chemical properties of soilof Surendranagar region Parameter S1 S2 S3 S4 pH 8.4 ± 0.01 7.5 ± 0.02 8.1 ± 0.02 8.4 ± 0.02 E.C. 1.2 ± 0.1 1.7 ± 0.09 1.3 ± 0.10 1.2 ± 0.08 O. C (%) 0.96 ± 0.012 0.61 ± 0.01 0.71 ± 0.008 01 ± 0.005 P (kg / hector) 41 ± 0.44 51.3 ± 0.6 41 ± 0.8 55.4 ± 0.7 K (kg / hector) 722 ± 1.8 777 ± 4.7 761 ± 5.9 539 ± 4.2 DISCUSSION The result of soil analysis onmajority of the sample collected fromvarious areas of the district reveal (Tab. 1&2) that the soil is sandy, having medium to lower WHC. Thesoil has pH value in range of neutral toslightly alkaline. It is good from the point of view of soil health. Electricityconductivity is low. The content oforganic Carbon, Nitrogen andPhosphorus in the soil is low. Fertilizers should be added of contain nitrogen and phosphorus in adequateamount. Strategy should be evolved tobring it in high range. Potash content ishigh. So, overall soil properties areaverage from point of view ofagriculture. ACKNOWLEDGEMENT We are grateful to Dr. RanjanaAgarwal (Principal) and Dr. Yvonne Fernandes (Principal) for arranging such avaluable seminar. The authors are alsothankful to soil testing laboratory staff, government of Gujarat for providing laboratory facility. REFERENCES 1. A.N. Pandey, T.D. Parmar and S.R. Tanna.1999. Desertification: a casestudy from Saurashtra region of Gujarat state of India. Trop. Ecol.,40: 213-220. 2. F.E. Broadbent 1953. The soilorganic fraction.Adv. Agron.5:153-183. 3. C.M. Woolf, 1968., Principles ofBiometry. D. van Nastrand Co.Inc. Princeton, New Jersey, London, Melbourne.

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FUNCTIONAL ABILITIES OF CULTIVABLE RHIZOSPHERIC BACTERIA IN PHYLLANTHUS FRATERNUS AND THEIR POTENTIAL FOR PLANT GROWTH PROMOTION AND PHYTO-PATHOGEN CONTROL Kalpna D. Rakholiya**, Krupali Sureja**, Riddhi Chaniyara**, Kana Parmar**, ** * * Dhrupa Mayatra , Mital J. Kaneria , Satya P. Singh All authors have equal contribution *Department of Biosciences (UGC-CAS), Saurashtra University, Rajkot – 360005, Gujarat **Institute of Biotechnology, Department of Biosciences (UGC-CAS), Saurashtra University, Rajkot – 360005, Gujarat **Corresponding author: [email protected] ABSTRACT: In order to assess the potential of plant growth promoting rhizobacteria (PGPR) to protect plants against saline environments, growth-promoting effects of PGPR and their bio-control activity was evaluated. The isolation was done in four different media (NA, KB, Jenson’s Media, ASM agar). Phosphate solubilization, ammonia, and IAA secretion of rhizospheric strain was tested. Moreover, bio-control (hydrolytic enzyme production and antagonistic effects) activity also measured. The isolate SUPF 2 showed highest percentage inhibition against Rhizoctonia sp. All the studied isolates showed varied level of IAA and NH3 production. The studied isolates appear as effective PGPR inoculants as it possesses a number of traits useful in establishment and proliferation in saline regions. Further studies on its biochemical and molecular identification are in progress. Keywords: Rhizospheric bacteria; Antifungal production; Agricultural applications, IAA, NH3 1. INTRODUCTION Various environmental stresses such as drought, flooding, salinity, heat, cold, and heavy metals adversely affect growth, development and productivity of crop plants (Sapre et al., 2018). Salinity is a major abiotic factor that constrains agricultural productivity. Salt stress retards plant growth by affecting many physiological, biochemical and metabolic processes. The reduced growth of the plants under salinity is due to nutrient disturbances, affecting the availability, transport and partitioning of nutrients. Plant growth promoting rhizobacteria (PGPR) could be applied as an important strategy to improve cultivation in saline soils (Jha et al., 2012). PGPRs are known to promote plant growth and improve nutrition either directly or indirectly. The direct facilitation of plant growth by PGPR includes supplementation essential nutrients such as nitrogen, phosphate, zinc, potassium and iron and production of phytohormones (Jha et al., 2012, Spare 2018). The indirect plant growth promotion occurs when PGPR prevent the deleterious effects of pathogens and pest on plants (Singh et al., 2015). PGPRs are known to alleviate salt stress in many crop plants. Several reports have been known presenting the imperative role of PGPRs in mitigating salt stress in different crop plants tomato, groundnut, wheat, rice and red pepper (Mayak et al., 2004; Shukla et al., 2012; Bal et al., 2013). PGPR can enhance plant growth by employing multiple mechanisms and assuring the availability and uptake of certain macronutrients as well as micronutrients to the plant (Jaisingh et al., 2016; Tabassum et al., 2017). Simultaneously, PGPR facilitate plan protection from various pathogens, including fungi, insects and viruses, thus acting as biocontrol agents. PGPR create stress tolerance enhance the efficiency of fertilizers, can act as biocontrol agents for disease control and bioremediation. PGPR promote plant growth and there are numerous reports of plant growth and yield stimulation by PGPR (Bergottini et al., 2015). Plant growth- promoting rhizobacteria produce various compounds, including growth regulators (phytohormones), siderophores, and organic acids, fix atmospheric nitrogen, solubilize phosphorus and produce antibiotics to suppress harmful rhizobacteria. These substances either directly affect the metabolism of plants or improve the adaptive capacity of plants to absorb other nutrients from the soil. The main objectives of this study were to isolate and characterize the rhizobacteria associated in the rhizosphere of Phyllanthus fraternus medicinal plants. Using a cultivation-dependent approach, a total of eight isolates showing different phenotypic properties were obtained from the rhizosphere region. The isolates were examined for antifungal activity as well as phosphate solubilization, IAA and ammonia production to find out highly efficient bacterial isolates have the capacities to be used as plant growth promoting and bio-control agents.

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Botany

2. MATERIALS AND METHODS 2.1 Collection and isolation The isolates were collected from the rhizospheric region of Phyllanthus fraternus from Saurashtra University, during August 2017. The isolation was done on different agar media along with 5% NaCl. 2.2 Biocontrol activity 2.2.1 In vitro antifungal activity against plant pathogenic fungi The antagonistic effect of the isolates was assessed against four fungal plant pathogens viz., Rhizoctonia sp., A. flavus, Alternaria sp., and Fusarium sp. by disc diffusion assay (Rakholiya et al., 2014). The plates were incubated at 28°C for 5 days. The percent inhibition was calculated as described earlier by Kumar et al. (2017). The experiments were carried ou tin three replicates. 2.2.2 Hydrolytic enzymes production The production of enzymes was determined by spot plate assay as described for catalase (Cappuccino and Sherman 1992), chitinase (Roberts and Selitrennikoff, 1988), Cellulase (Kasana et al., 2008), protease (Pandey et al. 2012), amylase (Kammoun et al., 2008), and lipase (Dastager et al. 2009). Each experiment was performed thrice with three replicates. Colony diameter and a clear zone around the colony was measured and enzymatic activity index was calculated as described by Alp and Arikan (2008). 2.3 Characterization for potential plant growth promoting traits 2.3.1 Phosphorous solubilization The phosphate solubilization was assessed using Pikovskaya agar plate and method performed as described by Pikovskaya (1948). The solubilization efficacy (SE) was determined by according to Edi-Premono et al. (1996). 2.3.2 Indole-3-acetic acid (IAA) production The Indole-3-acetic acid (IAA) production was performed as described in Dinesh et al. (2015). 2.3.3 Ammonia production NH3 production was determined by the method described by Cappuccino and Sherman (1992). 2.4 Statistical analysis Each experiment was performed individually in triplicate. The results are expressed as Mean value (n = 3) ± Standard Error of Mean (SEM). 3. RESULTS AND DISCUSSION 3.1 Broad spectrum antifungal activity The isolated rhizospheric bacteria from medicinal plant Phullanthus fraternus was evaluated for their ability to inhibit the growth phyto pathogens viz. Rhizoctonia sp., Aspergillus flavus, Alternaria sp. and Fusarium sp. using in vitro dual culture assay on potato dextrose agar (PDA). All the isolates were able to inhibit plant pathogens at extent level (Figure 1). The isolates showed inhibition from 29.41%-57.65% inhibition against A. flavus. The isolates showed inhibition from 42.86%-57.14% inhibition against Alternaria sp. The isolates showed inhibition from 16.67%-50% inhibition against Fusarium sp. The isolates showed inhibition from 50%- 80% inhibition against Rhizoctonia sp. SUPF 6 showed highest percentage inhibition (80%) against Rhizoctonia sp. as compared to other isolates. 3.2 Hydrolytic enzyme production The production of hydrolytic enzymes by rhizospheric bacteria is another mechanism by which it can degrade various components of phytopathogens (Ghodsalavi et al., 2013; Rahmoune et al., 2017). The different rhizospheric isolates were screened for production of hydrolytic enzymes in plate assay and enzyme activities were expressed in terms of enzyme index (EI) (Table 1). Out of six isolates, SUPF 6 able to produce all hydrolytic enzymes and it produced lipase in higher amount (EI = 4). SUPF 1 and SUPF 4 only able to produce cellulase and amylase and their enzyme index was 3.3 and 2.8 respectively. Isolate SUPF 2 produce all hydrolytic enzymes except chitinase while SUPF 3 and SUPF 5 did not showed any enzyme production.

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Botany

C 60 ·-.....0 ·-.Q ·-.c C 40 ~

0 ~ ~ ""~ ~ ~ ;;;;> ;;;;> f/1 f/1 Isolates

■A.jlavus II Alterneriasp. ■ Fusariumsp. Rhizoctonia sp. Figure 1: Antagonistic effect of rhizospheric isolates against plant pathogens

Table 1: Hydrolytic enzyme production of rhizospheric isolates from Phyllanthus fraternus

Amylase Protease Cellulase Chitinase Lipase Rhizospheric isolates CD* Z* EI CD* Z* EI CD* Z* EI CD* Z* EI CD* Z* EI

SUPF 1 NA NA NA NA NA NA 1 2.3 3.30 NA NA NA NA NA NA

SUPF 2 0.80 1.62 3.02 0.93 1.62 2.73 1.5 2.5 2.67 NA NA NA 0.85 1.28 2.51

SUPF 3 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

SUPF 4 0.5 0.9 2.8 NA NA NA NA NA NA NA NA NA NA NA NA

SUPF 5 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

SUPF 6 0.5 1 3 0.87 1.6 2.84 1.4 2 2.43 1.2 2.5 3.08 0.7 2.1 4

*: length in centimetre, CD: Colony diameter, Z: Zone of clearance, EI: Enzyme index, NA: No activity

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Botany

3.3 Plant growth promoting traits (PGP) 3.3.1 Phosphate solubilization Phosphate solubilization is one very important trait of plant growth promotion as microorganisms solublize insoluble phosphate making it available for plants (Goswami et al., 2014). The estimation of phosphate solubilization is routinely screened by a plate assay method using Pikovskaya (PVK) agar medium shown in Figure 2. Out of six isolates, two isolates were solubilized phosphate at 7 DAI. The efficiencies of these bacterial strains to solubilize inorganic phosphorus were also monitored quantitatively by determining the percentage solubilizing efficacy. SUPF 6 isolate showed highest percentage solubilization efficacy (130.77%) as compared to other studied isolates followed by isolate SUPF 5 (100%). ~ Ul """" 0 0 """" ...... (,;J ~ t:' (j 1 m ~ ~ (,;J ~ ~ en '!- 0 """" 0 efficacy cm) ( N Ul 0 Diameter """" Solubilization % 0 0 SUPF2 SUPF3 SUPF5 SUPF4 SUPF6 SUPFl ~ ('D tr. tr. """"' 0 l""I" - Figure 2: Phosphate solubilization of rhizospheric isolates from Cardiospermun halicacabum at 7 DAI

3.3.2 Indole acetic acid production Auxin is a class of plant hormone. The most common and well characterized is indol-3-acetic acid (IAA), which is known to stimulate both rapid (e.g., increases in cell elongation) and long term responses in plants. They regulate growth by affecting physiological and morphological processes at very low concentrations (Egamberdieva et al., 2008). The results of colorimetric analysis indicated that all isolates varied greatly in their efficiency for producing IAA in the broth (Figure 3). IAA production was quantified as ranging from 4.49 to 111.67 μg ml- 1. The more IAA production after 96 h of incubation with 111.67 μg ml-1 supplement of L- tryptophan by isolate SUPF 6. Similar observation for IAA production has been reported earlier (Selvakumar et al., 2008, Sharma et al., 2017)

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Botany

150 ------...-.. .:... 111.67 5 111.32 ~ 100 +------,;t$H;'7------)!1-1Jrt~---91Jt~~ ~:..____ '-'= :.:::0 co: .;: =Qj I,,)

=0 I,,) < < ~ 0 LB LB+T LB LB+T LB LB+T

48 hours 72 hom·s 96 hours Incubation duration

~ SUPFl ~ SUPF2 -&-SUPF 3 ~ SUPF 4 ~ SUPFS ~ SUPF6

Figure 3: IAA production by isolates at different time intervals

40 40.00 ' 37.21 ....,-... ] " 33.40 30 ~ :::t '-_,I ....0== ;.l 20 ...~ ;.l Q,,== ~ 13.65 ~~94 ! 0== ~ 10 f") =z 0 48 hours 72 hours 96 hours Incubation ~ s UPF l ~ sUPF2 ~ sUPF3 ~ SUPF4 ~ SUPF S ~ SUPF6 Figure 4: Quantitative estimation of ammonia production of isolates

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Botany

All isolates were able to produce ammonia in peptone water in the range of 11.91 - 40 μM ml-1 at different time intervals. The highest amount of ammonia production was observed by isolate SUPF 6 (40 μM ml-1) followed by isolate SUPF 5 (37.21 μM ml-1) at 96 hours. Similar findings were reported previously from Bacillus amyloliquefaciens isolated from rhizosphere of Zea maize (Passari et al., 2018). 4. CONCLUSIONS AND FUTURE PERSPECTIVES In the present investigation, Total eight isolate were studied from rhizospheric region of Phyllanthus fraternus for their biocontrol and biofetilizer activity. Out of six isolates studied SUPF 6 have potent antifungal properties as well as plant growth promoting activities. PGPR strains isolated under present investigation may be useful as a novel biofetilizer and biocontrol for crop production at saline region. Further studies on its biochemical and molecular identification are in progress. 5. ACKNOWLEDGMENTS The Authors are thankful to the DST- Science and Engineering Research Board (SERB), New Delhi, India for providing financial support under National Postdoctoral Fellowship (NPDF) scheme (File No. PDF/2015/000430/LS, 06 June, 2016). We Thanks to Prof. S. P. Singh, head, Department of Biosciences, Saurashtra University and Dr. R. K. Kothari, Professor and Co-ordinatior of Institute of Biotechnology for providing excellent research facility. 6. REFERENCES

Alp S, Arikan S (2008). Investigation of extracellular elastase, acid proteinase & phospholipase activities as putative virulence factors in clinical isolates of Aspergillus species. J Basic Microbiol. 48:1–7. Bafana A, Lohiya R (2013) Diversity and metabolic potential of culturable root-associated bacteria from Origanum vulgare in sub-Himalayan region. World J Microbiol Biotechnol 29:63–74. Bergottini VM,Otegui MB, Sosa DA, Zapata PD, Mulot M, Rebord M, Zopfi J,Wiss F, Benrey B, Junier P, 2015. Bio-inoculation of yerba mate seedlings (Ilexparaguariensis St: Hill.) with native plant growth- promoting rhizobacteria: a sustainable alternative to improve crop yield. Biol. Fert. Soils 51 (6):749-755. Cappuccino JC, Sherman N (1992). In: Microbiology. A Laboratory Manual, 3rdEdn. Benjamin/ Cummings Publications Co., New York. Dastager SG, Pandey A, Lee JC, Li WJ, Kim CJ (2009). Polyphasic taxonomy of novelactinobacteria showing macromolecule degradation potentials in Bigeum Island. Korea.Curr. Microbiol. 59:21-29. Dinesh R, Anandaraj M, Kumar A, Bini YK,Subila KP, Aravind R (2015). Isolation, characterization, and evaluation of multi-trait plant growth promoting rhizobacteria for their growth promoting and disease suppressing effects on ginger. Microbiological Research 173:34–43. Edi-Premono M, Moawad AM, Vlek PLG (1996). Effect of phosphate-solubilizationPseudomonasputida on the growth of maize and its survival in the rhizosphere. Indonesian Journal of Crop Sciences 11:13–23. Ghodsalavi B, Ahmadzadeh M, Soleimani M, Madloo PB, Taghizad-Farid R(2013). Isolation and characterization of rhizobacteria and their effects on root extracts of Valeriana officinalis. Aust. J. Crop Sci. 7:338-344. Goswami D, Dhandhukia P, Patel P, Thakker JN (2014). Screening of PGPR from saline desert of Kutch: Growth promotion in Arachis hypogea by Bacillus licheniformis A2. Microbiological Research 169:66– 75. Jaisingh R, Kumar A, Dhiman M, (2016). Isolation and characterization of PGPR from rhizosphere of Sesame indicum L. Int. J. Adv. Res. Biol. Sci. 3:238-244. Jha B, Gontia I, Hartmann A, (2012). The roots of the halophyte Salicornia brachiata are a source of new halotolerant diazotrophic bacteria with plant growth promoting potential. Plant Soil 356: 265–277. Kammoun R, Naili B, Bejar S (2008). Application of a statistical design to the optimization of parameters and culture medium for α-amylase production by Aspergillus oryzae CBS 819.72 grown on gruel (wheat grinding by-product). Bioresour. Technol. 99:5602-5609. Kasana RC, Salwan R, Dhar H, Dutt S, Gulati AA(2008). Rapid and easy method for the detection of microbial cellulases on agar plates using gram's iodine. Curr. Microbiol. 57:503-507. Kumar K, Amaresan N, Madhuri K (2017). Alleviation of the adverse effect of salinity stress by inoculation of plant growth promoting rhizobacteria isolated from hot humid tropical climate. Ecological Engineering 102:361-366.

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Botany

Marques APGC, Pires C, Moreira H, Rangel AOSS, Castro PML (2010). Assessment of the plant growth promotion abilities of six bacterial isolates using Zea mays as indicator plant. Soil Biology and Biochemistry 42:1229–1235. Mayak S, Tirosh T, Glick BR, (2004). Plant growth-promoting bacteria confer resistance in tomato plants to salt stress. Plant Physiol. Biochem. 42: 565–572. Mota MS, Gomes CB, Souza Junior IT, Moura AB (2017). Bacterial selection for biological control of plant disease: criterion determination and validation. Brazilian Journal of Microbiology 48:62-70. Pandey S, Rakholiya KD, Raval VH, Singh SP (2012). Catalysis and stability of an alkaline protease from a haloalkaliphilic bacterium under non-aqueous conditions as a function of pH, salt and temperature. J. Biosci. Bioeng. 114: 251-256. Passari AK, Lalsiamthari PC, Zothanpuia, Leo VV, Mishra VK, Yadav MK, Gupta VK, Singh BP (2018). Biocontrol of Fusarium wilt of Capsicum annuumby rhizospheric bacteria isolated from turmeric endowed with plant growth promotion and disease suppression potential. Eur J Plant Pathol 150:831–846 Pikovskaya RI (1948). Mobilization of phosphorus in soil in connection with vital activity of some microbial species. Microbiology 17:362–370. Rahmoune B, Morsli A, Khelifi-Slaoui M, Khelifi L, Strueh A, Erban A, KopkaJ, Prell J, Van Dongen JT (2017). Isolation and characterization of three new PGPR and their effects on the growth of Arabidopsis and Datura plants. J. Plant Interactions 12:1-6. Rakholiya K, Vaghela P, Rathod T, Chanda S(2014). Comparative study of hydroalcoholic extracts of Momordica charantia L. against foodborne pathogens. Indian J. Pharmaceutic. Sci. 76:148-156. Rao, N Venkat; Chandra Prakash, K; Shanta Kumar, SM (2006). Pharmacological investigation of Cardiospermum halicacabum (Linn) in different animal models of diarrhoea", Indian Journal of Pharmacology, 38 (5): 346–349, Roberts WK, Selitrennikoff CP(1988). Plant and bacterial chitinases differ in antifungal activity. J. Gen. Microbiol. 134:169-176. Salla TD, Silva TR, Astarita LV, Santarem ER (2014) Streptomyces rhizobacteria modulate the secondary metabolism of Eucalyptus plants. Plant Physiology and Biochemistry 85: 14-20. Sapre S, Gontia-Mishra I, Tiwari S (2018). Klebsiella sp. confers enhanced tolerance to salinity and plant growth promotion in oat seedlings (Avena sativa). Microbiological Research 206:25-32. Selvakumar G, Kundu S, Gupta AD, Shouche YS, Gupta HS (2008). Isolation and characterization of nonrhizobial plant growth promoting bacteria from nodules of Kudzu (Puerariathun bergiana) and their effect on wheat seedling growth. CurrMicrobiol 56:134–139. Shukla PS, Agarwal PK, Jha B, (2012). Improved salinity tolerance of Arachis hypogaea (L.) by the interaction of halotolerant plant-growth-promoting rhizobacteria. J. Plant Growth Regul. 31: 195–206. Tabassum B, Khan A, Tariq M, Ramzan M, Khan MSI , Shahid N, Aaliya K (2017) Bottlenecks in commercialisation and future prospects of PGPR. Applied Soil Ecology 121: 102-117. Thakur D, Kaur M and Mishra A (2017). Isolation and screening of plant growth promoting Bacillus spp. and Pseudomonas spp. and their effect on growth, rhizospheric population and phosphorous concentration of Aloe vera. Journal of Medicinal Plants Studies 5(1): 187-192.

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Botany

CULTIVABLE PLANT GROWTH-PROMOTING ACTIVITIES OF RHIZOSPHERIC BACTERIA ISOLATED FROM INDIGENOUS MEDICINAL PLANT CARDIOSPERMUM HALICACABUM Kalpna D. Rakholiya**, Kana Parmar**, Krupali Sureja**, Riddhi Chaniyara**, ** * * Dhrupa Mayatra , Mital J. Kaneria , Satya P. Singh All authors have equal contribution *Department of Biosciences (UGC-CAS), Saurashtra University, Rajkot – 360005, Gujarat **Institute of Biotechnology, Department of Biosciences (UGC-CAS), Saurashtra University, Rajkot – 360005, Gujarat Corresponding author: [email protected] ABSTRACT: Soil salinization is increasing steadily in many parts of the world and causes major problems for plant productivity. Under these stress conditions, root associated beneficial bacteria can help improve plant growth and nutrition. In the present investigation, eight salt tolerant PGPR strains were isolated from rhizosphere of Cardiospermum halicacabum from Saurashtra University, Rajkot, Gujarat, India. All the PGPR strains were able to show growth at up to 5% NaCl (w/v). In order to study their in vitro characteristics, SUCH 11strain are able to solubilize phosphate (Pi) on Pikovskaya agar plates 24 mm (5 DAI). All isolates able to produce produced phytohormone indole-3-acetic acid (IAA), ammonia production at different time intervals as well as able to inhibit plant pathogens. The results strongly suggest that the PGPR strains isolated under present investigation may be useful as a novel biofertilizers for crop production at saline region. Keywords: Rhizospheric bacteria; Antifungal production; Agricultural applications, IAA, NH3

1. INTRODUCTION The rhizosphere concept was first introduced by Hiltner to describe the narrow zone of soil surrounding the roots where microbe populations are stimulated by root activities (Hiltner, 1904) and nutrients attract bacteria and fungi, which multiply in the rhizosphere to densities up to and exceeding 100 times those in the bulk soil (Gupta et al., 2016; Hassan, 2017). The study of rhizosphere bacteria from the important medicinal plants is very crucial, as they are well known to have impact on plant growth and also produce industrially important metabolites and improve quality of medicinal product (Bafana and Lohiya 2013; Salla et al., 2014). Medicinal plants support a great diversity of microflora in their rhizosphere including PGPR. Over the years, PGPR have gained worldwide importance and acceptance for agricultural crops. The PGPR are naturally occurring soil bacteria that aggressively colonize plant roots and benefit plants by providing growth promotion. Inoculation of crop plants with certain strains of PGPR at an early stage of development improves biomass production through direct effects on roots and shoots growth and also offers an attractive way to replace chemical fertilizers, pesticides, and supplements (Thakur et al., 2017). Their application increases the availability of nutrients to plants, control the soil borne pathogens and maintain and sustain the soil fertility and will ultimately help in conservation of medicinal plants and their productivity. Cardiospermum halicacabum belongs to the family Sapindaceae. It is generally known as Balloon Vine and it is a climbing plant widely distributed in tropical and subtropical Africa and Asia. It has been examined for antidiarrhoeal as well as homoeopathic medicinal properties (Rao et al., 2006) The main objectives of this study were to isolate and characterize the rhizobacteria associated in the rhizosphere of Cardiospermum halicacabum medicinal plants. Using a cultivation-dependent approach, a total of eight isolates showing different phenotypic properties were obtained from the rhizosphere region. The isolates were examined for antifungal activity as well as phosphate solubilization, IAA and ammonia production to find out highly efficient bacterial isolates have the capacities to be used as plant growth promoting and bio-control agents. 2. MATERIALS AND METHODS 2.1 Collection and isolation The isolates were collected from the rhizospheric region of Andrographis paniculata from Saurashtra University, during August 2017. The isolation was done on different agar media along with 5% NaCl.

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2.2 Biocontrol activity 2.2.1 In vitro antifungal activity against plant pathogenic fungi The antagonistic effect of the isolates was assessed against four fungal plant pathogens viz., Rhizoctonia sp., A. flavus, Alternaria sp., and Fusarium sp. by disc diffusion assay (Rakholiya et al., 2014). The plates were incubated at 28°C for 5 days. The percent inhibition was calculated as described earlier by Kumar et al. (2017). The experiments were carried ou tin three replicates. 2.2.2 Hydrolytic enzymes production The production of enzymes was determined by spot plate assay as described for catalase (Cappuccino and Sherman 1992), chitinase (Roberts and Selitrennikoff, 1988), Cellulase (Kasana et al., 2008), protease (Pandey et al. 2012), amylase (Kammoun et al., 2008), and lipase (Dastager et al. 2009). Each experiment was performed thrice with three replicates. Colony diameter and a clear zone around the colony was measured and enzymatic activity index was calculated as described by Alp and Arikan (2008). 2.3 Characterization for potential plant growth promoting traits 2.3.1 Phosphorous solubilization The phosphate solubilization was assessed using Pikovskaya agar plate and method performed as described by Pikovskaya (1948). The solubilization efficacy (SE) was determined by according to Edi-Premono et al. (1996). 2.3.2 Indole-3-acetic acid (IAA) production The Indole-3-acetic acid (IAA) production was performed as described in Dinesh et al. (2015). 2.3.3 Ammonia production NH3 production was determined by the method described by Cappuccino and Sherman (1992). 2.4 Statistical analysis Each experiment was performed individually in triplicate. The results are expressed as Mean value (n = 3) ± Standard Error of Mean (SEM). 3. RESULTS AND DISCUSSION 3.1 Broad spectrum antifungal activity The isolated rhizospheric bacteria from medicinal plant Cardiospermun halicacabum strains was evaluated for their ability to inhibit the growth phyto pathogens viz. Rhizoctonia sp., Aspergillus flavus, Alternaria sp. and Fusarium sp. using in vitro dual culture assay on potato dextrose agar (PDA). All the isolates were able to inhibit plant pathogens at extent level (Figure 1). The isolates showed inhibition from 29.41- 64.71% inhibition against A. flavus at different time intervals. The isolates showed inhibition from 17.40-71.43% inhibition

100

C 0 :.0 60 :E .c ·-C 40 ~ -C,

0 N ~ •n \0 r-- =- -..-I - - ""..-I - - ..-I u u u u u u u u ;;;;,= ;;;;,= ;;;;,= ;;;;,= ;;;;,= ;;;;,= ;;;;,= ;;;;,= i;f.l VJ. VJ. VJ. VJ. VJ. VJ. i;f.l Isolates

II A.jlavus ■ A lterneria sp. Fusarium sp. ■ Rhizoctonia sp.

Figure 1: Antagonistic effect of rhizospheric isolates against plant pathogens www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 131

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Botany against Alternaria sp. at different time intervals. The isolates showed inhibition from 26.67- 46.67% inhibition against Fusarium sp. at different time intervals. The isolates showed inhibition from 50.00- 80.00% inhibition against Rhizoctonia sp. at different time intervals. SUCH 11 showed highest percentage inhibition (100.00%) as compare to other isolates against Rhizoctonia sp. 3.2 Hydrolytic enzyme production The production of hydrolytic enzymes by rhizospheric bacteria is another mechanism by which it can degrade various components of phytopathogens (Ghodsalavi et al., 2013; Rahmoune et al., 2017). The different rhizospheric isolates were screened for production of hydrolytic enzymes in plate assay and enzyme activities were expressed in terms of enzyme index (EI) (Table 1). Two isolates, SUCH 10 and SUCH 11 were produced amylase and their enzyme index was 2.60. Four isolates SUCH 11, SUCH 13, SUCH 14 and SUCH 15 were produce protease and their enzyme index were 3.20, 2.20, 2.40 and 12.00 respectively. Isolate SUCH 11 was able to produce cellulase, chitinase, lipase and their enzyme index were 2.67, 2.47 and 2.67 respectively. None of the enzyme production was produced by isolate SUCH 16 and SUCH 17.

Table 2: Hydrolytic enzyme production of rhizospheric isolates from Cardiospermun halicacabum Amylase Protease Cellulase Chitinase Lipase Rhizospheric I CD isolates CD* Z* EI CD* Z* EI CD* Z* EI CD* Z* EI Z* EI *

SUCH 10 0.5 0.8 2.60 NA NA NA NA NA NA NA NA NA NA NA NA

SUCH 11 0.5 0.8 2.60 1 2.2 3.20 1.5 2.5 2.67 1.5 2.2 2.47 1.5 2.5 2.67

SUCH 12 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

SUCH 13 NA NA NA 1 1.2 2.20 NA NA NA NA NA NA NA NA NA

SUCH 14 NA NA NA 1.5 2.1 2.40 NA NA NA NA NA NA NA NA NA

SUCH 15 NA NA NA 0.5 5.5 12.00 NA NA NA NA NA NA NA NA NA

SUCH 16 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

SUCH 17 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA *: length in centimetre, CD: Colony diameter, Z: Zone of clearance, EI: Enzyme index, NA: No activity

3.3 Plant growth promoting traits (PGP) 3.3.1 Phosphate solubilization Phosphate solubilization is one very important trait of plant growth promotion as microorganisms solublize insoluble phosphate making it available for plants (Goswami et al., 2014). The estimation of phosphate solubilization is routinely screened by a plate assay method using Pikovskaya (PVK) agar medium shown in Figure 2. Out of seven isolates three isolates were solubilize phosphate at 7 DAI. The efficiencies of these bacterial strains to solubilize inorganic phosphorus were also monitored quantitatively by determining the percentage solubilizing efficacy. SUCH 11 isolate showed highest percentage solubilization efficacy (140%) as compared to other studied isolates followed by isolate SUCH 16 (66.67%) and SUCH 10 (33.33%).

3.3.2 Indole acetic acid production

IAA production is dependent on the presence of L-tryptophan in the medium as the organism utilizes the indole moiety from L-tryptophan to produce IAA. Increase in IAA production was in corroboration with an increase in L-tryptophan in the medium (Goswami et al., 2014). The results of colorimetric analysis indicated that all isolates varied greatly in their efficiency for producing IAA in the broth (Figure 3). IAA production was quantified as ranging from 3.86 to 111.67 μg ml- 1. The more IAA production after 96 h of incubation with 111.67 μg ml-1 supplement of L- tryptophan by isolate SUCH 11. Similar observation for IAA production has been reported earlier (Selvakumar et al., 2008).

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30 150

....u ~ ,-.., u 20 100 !.: 8 ~ 8 <.> '-' :i) .=S: ....<.> .... 8 ~ ~ ~ a 10 50 :E -=0 V1

0 0 0 .-; N "'> ..... If) 1,0 r-- .-; .-; .-; .-; .-; .-; .-; .-; ::c: ::c: ::c: ::c: ::c: ::c: ::c: ::c: u u u u u u u u ;;;;i ;;;;i ;;;;i ;;;;i ;;;;i ;;;;i ;;;;i ;;;;i VJ VJ VJ VJ VJ VJ VJ VJ Isolates CD zc _._%SE

Figure 2: Phosphate solubilization of rhizospheric isolates from Cardiospermun halicacabum at 7 DAI

150

...,-.., -+-SUCH 10 ' s ...su CHll 00 -a-SUCH 12 '-'=- 100 ~ SUCH13 .5:.....= ....E -I-SUCH 14 <.> ~ SUCH15 u= ~ SUCH16 0 u= 50 < - SUCH17 ....<

48 hours 72 hours 96 hours Incubation duration

Figure 3: IAA production by isolates at different time intervals

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O'\ 0 ) 1 - mI (µM ,l;o,. 0 N 0 concentl'ation 3 NH 0 SUCHll SUCHlO SUCH12 SUCH13 SUCH15 SUCH17 SUCH14 SUCH16 ~ ~ ~ ~ 0 ,.,, - f f f N \0 ,l;o,. QO --.J O'\ 0 C: 0 C: 0 C: r.,, r.,, r.,, -: -: -: =- =- =-

Figure 4: Quantitative estimation of ammonia production of isolates

3.3.3 Quantitative assay of ammonia (NH3) production Ammonia production is a general characteristic of PGPR and it influence the plant growth indirectly (Mota et al., 2017). Ammonia production also plays an important role in plant growth by accumulating nitrogen and increasing root and shoots growth as well as plant biomass (Marques et al., 2010). All isolates were able to produce ammonia in peptone water in the range of 10.20 – 53.55 μM ml-1 at different time intervals. The highest amount of ammonia production was observed by isolate SUCH 11 (53.55 μM ml-1) at 96 hours. Similar findings were reported previously from Bacillus amyloliquefaciens isolated from rhizosphere of Zea maize (Passari et al., 2018). 4. CONCLUSIONS AND FUTURE PERSPECTIVES In the present investigation, Total eight isolate were studied from rhizospheric region of Cardiospermum halicacabum for their biocontrol and biofetilizer activity. Out of eight isolates studied SUCH 11 have potent antifungal properties as well as plant growth promoting activities. PGPR strains isolated under present investigation may be useful as a novel biofetilizer and biocontrol for crop production at saline region. Further studies on its biochemical and molecular identification are in progress. 5. ACKNOWLEDGMENTS The Authors are thankful to the DST- Science and Engineering Research Board (SERB), New Delhi, India for providing financial support under National Postdoctoral Fellowship (NPDF) scheme (File No. PDF/2015/000430/LS, 06 June, 2016). We Thanks to Prof. S. P. Singh, head, Department of Biosciences, Saurashtra University and Dr. R. K. Kothari, Professor and Co-ordinatior of Institute of Biotechnology for providing excellent research facility. 6. REFERENCES Alp S, Arikan S (2008). Investigation of extracellular elastase, acid proteinase & phospholipase activities as putative virulence factors in clinical isolates of Aspergillus species. J Basic Microbiol. 48:1–7. Bafana A, Lohiya R (2013) Diversity and metabolic potential of culturable root-associated bacteria from Origanum vulgare in sub-Himalayan region. World J Microbiol Biotechnol 29:63–74. Cappuccino JC, Sherman N (1992). In: Microbiology. A Laboratory Manual, 3rdEdn. Benjamin/ Cummings Publications Co., New York. Dastager SG, Pandey A, Lee JC, Li WJ, Kim CJ (2009). Polyphasic taxonomy of novelactinobacteria showing macromolecule degradation potentials in Bigeum Island. Korea.Curr. Microbiol. 59:21-29. www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 134 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Botany

Dinesh R, Anandaraj M, Kumar A, Bini YK,Subila KP, Aravind R (2015). Isolation, characterization, and evaluation of multi-trait plant growth promoting rhizobacteria for their growth promoting and disease suppressing effects on ginger. Microbiological Research 173:34–43. Edi-Premono M, Moawad AM, Vlek PLG (1996). Effect of phosphate-solubilizationPseudomonasputida on the growth of maize and its survival in the rhizosphere. Indonesian Journal of Crop Sciences 11:13–23. Ghodsalavi B, Ahmadzadeh M, Soleimani M, Madloo PB, Taghizad-Farid R(2013). Isolation and characterization of rhizobacteria and their effects on root extracts of Valeriana officinalis. Aust. J. Crop Sci. 7:338-344. Goswami D, Dhandhukia P, Patel P, Thakker JN (2014). Screening of PGPR from saline desert of Kutch: Growth promotion in Arachis hypogea by Bacillus licheniformis A2. Microbiological Research 169:66– 75. Gupta H, Saini RV, Pagadala V, Kumar N, Sharma DK, Saini AK (2016). Analysis of plant growth promoting potential of endophytes isolated from Echinacea purpurea and Lonicera japonica.16: 558-577. Hassan SE (2017). Plant growth-promoting activities for bacterial and fungal endophytes isolated from medicinal plant of Teucrium polium L. Journal of Advanced Research 8: 687-695. Kammoun R, Naili B, Bejar S (2008). Application of a statistical design to the optimization of parameters and culture medium for α-amylase production by Aspergillus oryzae CBS 819.72 grown on gruel (wheat grinding by-product). Bioresour. Technol. 99:5602-5609. Kasana RC, Salwan R, Dhar H, Dutt S, Gulati AA(2008). Rapid and easy method for the detection of microbial cellulases on agar plates using gram's iodine. Curr. Microbiol. 57:503-507. Kumar K, Amaresan N, Madhuri K (2017). Alleviation of the adverse effect of salinity stress by inoculation of plant growth promoting rhizobacteria isolated from hot humid tropical climate. Ecological Engineering 102:361-366. Marques APGC, Pires C, Moreira H, Rangel AOSS, Castro PML (2010). Assessment of the plant growth promotion abilities of six bacterial isolates using Zea mays as indicator plant. Soil Biology and Biochemistry 42:1229–1235. Mota MS, Gomes CB, Souza Junior IT, Moura AB (2017). Bacterial selection for biological control of plant disease: criterion determination and validation. Brazilian Journal of Microbiology 48:62-70. Pandey S, Rakholiya KD, Raval VH, Singh SP (2012). Catalysis and stability of an alkaline protease from a haloalkaliphilic bacterium under non-aqueous conditions as a function of pH, salt and temperature. J. Biosci. Bioeng. 114: 251-256. Passari AK, Lalsiamthari PC, Zothanpuia, Leo VV, Mishra VK, Yadav MK, Gupta VK, Singh BP (2018). Biocontrol of Fusarium wilt of Capsicum annuumby rhizospheric bacteria isolated from turmeric endowed with plant growth promotion and disease suppression potential. Eur J Plant Pathol 150:831–846 Pikovskaya RI (1948). Mobilization of phosphorus in soil in connection with vital activity of some microbial species. Microbiology 17:362–370. Rahmoune B, Morsli A, Khelifi-Slaoui M, Khelifi L, Strueh A, Erban A, KopkaJ, Prell J, Van Dongen JT (2017). Isolation and characterization of three new PGPR and their effects on the growth of Arabidopsis and Datura plants. J. Plant Interactions 12:1-6. Rakholiya K, Vaghela P, Rathod T, Chanda S(2014). Comparative study of hydroalcoholic extracts of Momordica charantia L. against foodborne pathogens. Indian J. Pharmaceutic. Sci. 76:148-156. Rao, N Venkat; Chandra Prakash, K; Shanta Kumar, SM (2006). Pharmacological investigation of Cardiospermum halicacabum (Linn) in different animal models of diarrhoea", Indian Journal of Pharmacology, 38 (5): 346–349, Roberts WK, Selitrennikoff CP(1988). Plant and bacterial chitinases differ in antifungal activity. J. Gen. Microbiol. 134:169-176. Salla TD, Silva TR, Astarita LV, Santarem ER (2014) Streptomyces rhizobacteria modulate the secondary metabolism of Eucalyptus plants. Plant Physiology and Biochemistry 85: 14-20. Selvakumar G, Kundu S, Gupta AD, Shouche YS, Gupta HS (2008). Isolation and characterization of nonrhizobial plant growth promoting bacteria from nodules of Kudzu (Puerariathun bergiana) and their effect on wheat seedling growth. CurrMicrobiol 56:134–139. Thakur D, Kaur M and Mishra A (2017). Isolation and screening of plant growth promoting Bacillus spp. and Pseudomonas spp. and their effect on growth, rhizospheric population and phosphorous concentration of Aloe vera. Journal of Medicinal Plants Studies 5(1): 187-192.

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Zoology

Section 8

ZOOLOGY

Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Zoology

STUDIES OF BIOCHEMICAL PARAMETERS OF OREOCHROMIS NILOTICUS EXPOSED TO CADMIUM CHLORIDE (CDCL2, 2H2O) Hetalben Parekh1, Dr. Shantilal Tank1 1 Department of Bio-Science, Veer Narmad South Gujarat University, Surat, INDIA Email: [email protected]

ABSTRACT: The hostile effects of Cadmium Chloride (CdCl2, 2H2O) on the specimen Nile tilapia (Oreochromis niloticus) were examined, for evaluation as chronic toxicity. In order to assess the effect of prolonged exposure of low concentration of cadmium bio-chemical assessment was made after 24 hour, 7 days and 15 days of exposure. The study of bio-chemical parameters demonstrated significant rise in the levels of SGPT (Serum Glutamate Pyruvate Transaminase) and SGOT (Serum Glutamate Oxaloacetate Transaminase). On the contrary decline in levels of Alkaline Phosphatase was observed. SGPT and SGOT enzyme levels rise due to elevated enzymatic activities followed by damage to hepatic tissues due to toxicant exposure. While Alkaline Phosphatase drop can be linked with indicates disturbances in the structure and integrity of cell organelles, like endoplasmic reticulum and membrane transport system.

INTRODUCTION The impact of heavy metals in the environment has been greater than before in some areas to levels, which jeopardize the well-being of aquatic and terrestrial organisms including human (Honda et al., 2008). Due to increased human activities and interests cadmium has become one of the most menacing heavy metals in aquatic environments. Effluents from such activities are sources of cadmium into aquatic environments and could threaten aquatic organisms including fish. Bio monitoring at the lower levels of biological organisation allows for an “early warning system” whereby stressors can be detected at an early stage, and dealt with, before they exert their effects in higher levels of biological organisation. Fishes are relatively sensitive to changes in their surrounding environment including an upsurge in pollution & have the capability of concentrating metals by feeding and metabolic processes, which can lead to accumulation of high concentrations of metals in their tissues. Fish health, thus reflect the health status of a specific aquatic ecosystem. Nile Tilapia (Oreochromis niloticus) offers a relatively high tolerance for temperature and pH alteration is available throughout the year and is able to easily adapt to laboratory conditions. Therefore, the study on the fish is appropriately justified. For many years, the importance of clinical chemistry assessment in toxicology studies has been recognized. Fish are responding to various stressors by a series of biochemical and physiological stress reactions, so called secondary stress responses analogous to those of higher vertebrates. Sub lethal effects are biochemical in origin as most toxicants put forth their effects at primary level of the organism by reacting with enzymes or metabolites and other functional components of the cell. The estimation of blood chemistry parameters is an accustomed and authoritative tool providing the vital information on the physiological status of organism (Chen et al., 2003). To measure the effects these substances could have, on fish species and subsequently on human populations, biochemical tests produce meaningful and useful results. In this study, the effect of heavy metal, cadmium on various biochemical parameters of freshwater fish species, Oreochromis niloticus, was investigated. MATERIALS AND METHODS Oreochromis niloticus as test organism: The reasons for selection of the fish are availability throughout the year, ease of care and acclimation and high tolerance to toxicant and other ambient conditions. The fish were obtained from a local breeder in a village Napad, Dist. Anand, approximately four weeks prior to each experiment. They were acclimatized for two weeks in common tanks and then transferred to experiment aquariums and allowed to acclimatize for one more week. Estimation of Bio-Chemical effects after chronic exposure: The specimens in four groups of 30 each (10 X 3 to avoid oxygen depletion, high levels of waste production per tank and stress induced by crowding) were exposed to the decided concentration of Cadmium after primary and secondary acclimation. The fish were fed on a daily basis throughout the period of exposure. Out of the four groups one group was exposed to no cadmium. i.e. was kept as control with reference to other groups. While rest exposure groups each of thirty specimens, were exposed to the respective cadmium concentration of 0.1mg/L, 0.2mg/L and 0.3mg/L respectively. In our acute study, we derived the NOEC value to be 1.2017mg/L. Exposure concentrations being lower than required to cause lethal effects no death was observed in any of the groups. After exposure of 24 hours, 7 days and 15 days nine fishes from each group were sacrificed to collect blood samples from them. The collected blood samples were analyzed for measurement of different

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Bio-Chemical parameters like Serum Glumate Pyruate Transaminase (SGPT), Alkaline Phosphatase and Serum Glumate Oxaloacetate Transaminase (SGOT). SGOT and SGPT were measured using IFCC NADH- kinetic UV methods while Alkaline Phosphatase estimation was carried out using p-NPP kinetic method. RESULTS The present study reveals that the fish exposed to Cd showed significant reduction in Alkaline Phosphatase. While, inclination were observed in levels of SGPT & SGOT .The changes in each of the parameter are listed in table 1, while the same is depicted graphically in fig 1. SGPT SGOT Alkaline Phosphatase (IU/L) (IU/L) (IU/L) 0.0 27.13  1.85 48.23  0.12 41.89  0.57 0.1 31.20  0.69 47.67  0.75 56.24  1.76 24 Hr 0.2 30.73  1.10 47.17  0.58 67.21  1.06 0.3 35.13  0.75 46.77  0.58 81.72  2.20 0.0 31.80  1.04 47.27  1.10 53.59  1.60 0.1 38.80  0.69 45.93  0.64 74.64  1.58 7 Days 0.2 42.03  0.40 44.83  0.64 86.82  1.47 0.3 46.00  0.00 45.77  1.15 94.46  1.27 0.0 35.27  0.64 47.73  0.64 65.12  1.20 0.1 41.57  0.81 45.47  0.64 92.56  1.19 15 Days 0.2 43.70  0.87 44.00  0.17 103.99  1.11 0.3 48.77  0.46 42.03  0.40 109.17  0.96 Table 1: Biochemical observations of chronic toxicity tests DISCUSSION Different blood serum tests were carried out to estimate the toxic effect of cadmium exposure on physiology of different organs. The aminotransferases, including Alanine Amino Transferase (ALT), formerly known as Serum Glutamate Pyruvate Transaminase (SGPT), and Aspartate Amino Transferase (AST), formerly known as Serum Glutamate Oxaloacetate Transaminase (SGOT), are good indicators of damage to hepatocyte. Although the magnitude of the increase in ALT or AST is directly proportional to the number of hepatocytes affected, it is not related to the reversibility/irreversibility of the change. This is also true for increased AST with muscle damage or disease (e.g., myocardial infarction). SGOT or SGPT are measured for cardiac necrosis, liver damage, muscular dystrophy or cholecystis.

Alkaline Phosphatase Alkaline Phosphatase gives measure of liver damage and hepatic diseases. The present study reveals that the Serum Alkaline Phosphatase level decreases upon exposure of cadmium with increasing level of cadmium and duration of exposure. The decrease in activity of ALP in fish exposed to various pollutants or stressors has been reported by different researchers (Das et al. 2003, Ogueji et al., 2007, Rashatwar et al., 1983). Depletion of enzymes in the fishes exposed to cadmium can be attributed to increased cadmium levels in the tissues. Furthermore, accumulation of cadmium in liver and muscles could be the possible reason for variation of enzyme activities. The inhibition in protein level may also be due to the decrease in alkaline phosphatase activity as it plays an important role in protein synthesis.

SGPT and SGOT SGOT and SGPT are measured for cardiac necrosis, liver damage or muscular dystrophy. Our study demonstrated that the exposure of cadmium caused significant rise in the activities of SGOT and SGPT. Several researchers have found same result in the fish treated with cadmium, zinc and copper (Al-Attar, A.M., (2005); Singh et al., 1990; Hilmy et al., 1987). The increased SGOT may reflect hepatic toxicity which leads to extensive liberation of the enzymes into the blood circulation (Daabees et al., 1992). The tissue damaged by toxicants exhibit a sharp rise in activity of mitochondrial enzymes SGOT. SGOT has been strongly implicated in the production of energy in tissues (Srivastava et.al. 1999) and is considered as a stress indicator (Gould et.al. 1976). The intoxication of cadmium combine with an enzyme to form an enzyme inhibition complex, which react with various functional groups of the enzymes inhibit the enzyme activity of major metabolic site. The increase in concentration of SGPT and SGOT in blood plasma indicates impairment of liver.

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120.00 100.00

) 24 hours 80.00 ■ 7 Days 60.00 ■ 40.00 ■ 15 Days AST AST (IU/L 20.00 0.00 Control 0.1 0.2 0.3 Exposure Concentration mg/L -

50.00 48.00 ■ 24 hours 46.00 7 Days 44.00 ■

(IU/L) 42.00 ■ 15 Days 40.00 38.00 Control 0.1 0.2 0.3 ALKALINE ALKALINE PHOSPHATASE Exposure Concentration mg/L

50.00

40.00

30.00 ■ 24 hours

■ 7 Days 20.00

SGPT SGPT (IU/L) ■ 15 Days 10.00

0.00 Control 0.1 0.2 0.3

Exposure Concentration mg/L

Fig 1. Deviation in Bio-Chemical parameters of Oreochromis niloticus upon chronic exposure to CdCl2, 2H2O REFERENCES 1. Abbass, H. H., K. H. Zaghloul and M. A. A. Mousa. (2002). Effect of some heavy metal pollutants on some biochemical and histopathological changes in blue tilapia; Oreochromis niloticus. Egyptian Journal of Agricultural Research, 80(3): 1385-1411.

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2. Abdel-salam, E.B., S.E.I. Adam and G. Tartour, (1982). Mixture of dieldrin ndphosphamidon rises GOT, GPT and suppress cholinesterase in blood serum. Zentralale Veternaemed Reihe A, 29: 136- 138. 3. Al-Attar, A.M., (2005). Biochemical effects of short term cadmium exposure on the freshwater fish, Oreochromis niloticus. J. Biol. Sci., 5: 260-265. 4. Al-Attar, A.M., (2010). Hematological, biochemical and histopathological studies on marsh frog, Rana ridibunda,naturally infected with Waltonella duboisi, Int. J. Zool. Res., 6: 199-213. 5. Almeida JA, Novelli EL, Dal Pai Silva M, Junior RA (2001). Environmental cadmium exposure and metabolic responses of the Nile tilapia, Oreochromis niloticus. Environ Pollut. 114 (2): 169-175. 6. Asha Agrawal and Poonam Sharma. (1999). Effect of Sulphur Dioxide on total lipid and cholesterol level in the blood of albino rats. Journal of Environ.Biol.20 (4). 335-338. 7. Chen CY, Wooster GA, Getchell RG, Bowser PR, Timmons MB (2003).Blood chemistry of healthy, nephro-calcinosis affected and ozone-treated tilapia in a recirculation system, with application of discriminant analysis. Aquaculture, 218: 89-102. 8. Chowdhury MJ, Pane EF, Wood CM (2004). Physiological effects of dietary cadmium acclimation and waterborne cadmium challenge in rainbow trout: respiratory, ionoregulatory and stress parameters. Comp Biochem. Physiol. Toxicol. Pharmacol., 139(1-3): 163-173 9. Daabees, A. Y.; EL Domiatty, N. A.; Soliman, S. A. and El-Toweissy, M. Y. (1992). Comparative action of three synthetic pesticides on serum liver and brain enzymes of the freshwater Clarias lazera,J. Egypt. Ger. Soc. Zool., 10 (A): 105 -119. 10. Das B. K. and Mukherjee S. C. (2003). Toxicity of cyper methrinin Labeo rohita fingerlings: Biochemical, enzymatic and haematological consequences.Comp. Biochem. Physiol. C 124:109– 121 11. Early J.L., V.K. Nonavinakere and Z.Mallory, (1990). Effect of cadmium on blood glucose level in the rat. Toxicol. Lett. 54: 199-205 12. Gopal, V., S. Paravathy and P.R. Balasubramanian, (1997). Effect of heavy metals on the blood protein biochemistry of the fish Cyprinus carpio and its use as a bio-indicator of pollution stress.Environ. Monit. Assess. 48: 117-124. 13. Gould, E., R.S. Collier, J.J. Karoulus and S.A. Givenus, (1976). Heart transaminase in the rock crab, Cancer irrortus exposed to cadmium salts. Bull. Environ. Contam. Toxicol. 15: 635-643. 14. Hilmy, A.M., El-Domiaty, N.A., Weshana, K. (1987) Acute and Chronic Toxicity of Nitrite to Clarias Lazera.Comp. Biochem. Physiol. C., 86(2):247-253. 15. Hilmy A. M. and Domaity N. El (1987a). Some physiological and biochemical indices of zinc toxicity in two freshwater fishes, Clarias lazera and Tilapia zillii. Comp. Biochem. Physiol. 87: 297-301. 16. Honda. R.T, De-Castilho. F.M., and Val A.L., (2008), Cadmium-induced disruption of environmental exploration and chemical communication in matrinxa, Brycon amazonicus. 17. Aquatic Toxicolology 89 pp 204-206 18. Hontela A, Daniel C et al.(1996) Effect of acute and sub-acute exposure to cadmium on the inter renal and thyroid function in rainbow trout, Oncorhyncus mykiss. Aqut. Toxicol. 35: 171-182 19. Kirubagaran, R., and Joy, K.P. (1994). Effects of short-term exposure to methyl mercury chloride and its withdrawal on serum levels of thyroid hormones in the catfish Clarias batrachus. Bull. Environ. Contamin. Toxicol. 53:166–170 20. Levesque HM, Moon TW, Campbell PGC and Hontela A (2002). Seasonal variation in carbohydrate and lipid metabolism of yellow perch (Perca flavescens) chronically exposed to metals in the field. Aquatic Toxicology. 60(3-4): 257-267. 21. Mona S Zaki, Olfat M.F., Suzan O.M., Isis AMF. (2010). Biochemical studies on Tilapia nilotica exposed to climate change and cadmium sulphate (0.50p.p.m.) New York Science Journal 3: 90-95 22. Muazzez Oner, Guluzar Atli, Mustafa Canli, (2009). Changes in serum biochemical parameters of freshwater fish Oreochromis niloticus following prolonged metal (Ag, Cd, Cr, Cu, Zn) exposures. Environmental Toxicology and Chemistry 27(2): 360–366 23. Murray, I.K., P.A. Mayer, O.K. Granner and V. Rodwel, (1990). Cholesterol Biosynthesis, Degradation and Transport. In: Harper’s Biochemistry. 2nd Edn, Prentice Hall International Inc., pp: 240-254. 24. Ogueji EO and Auta J. (2007). Investigation of biochemical effects of acute concentrations of lambda- cyhalothrin on African catfish Clarias gariepinus- Teugels. Journal of Fisheries International 2(1): 86-90 25. Rashatwar S. S. and Ilayas R. (1983). Effect of chronic herbicide intoxication on the in vivo activities of certain enzymes in the liver of fresh water fish Nemacheilus denisonii. Toxic. Lett. 16: 249–252

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26. Ruparelia S.G., Y. Verma, S. R. Saiyed, U. M. Rawal, (1990). Effect of cadmium on Blood of tilapia, Oreochromis mosambicus (Peters), during prolonged exposure. Bull. Environment Contam. Tox. 45:305-312 27. Salah El-Deen, M. A., Sharada, H., Abu El-Ella, S. M. (1996). Some metabolic alteration in grass carp Ctenopharyngodon idella induced by exposure to cadmium. J. Egypt. Ger. Soc. Zool., 21A: 441- 457 28. Sastry, K.V. and Subhadra, K. (1985) In vivo effects of cadmium on some enzyme activities in tissues of the freshwater catfish Heteropneustes fossilis. Environ. Res., 36 : 32-45 29. Shakoori, A. R., Javed Iqbal, M., Latif Mughal, A., SyedShahid A., (1994). Bio chemical changes induced by inorganic mercury on the blood, liver and muscles of fresh Ctenopharyngodon idella. J. Ecotoxicol. Environ. Monit.,4: 81–92. 30. Singh, H.S. and T.V. Reddy, (1990). Effect of copper sulfate on hematology, blood chemistry and hepato-somatic index of an Indian catfish, Heteropneustes fossilis (Bloch) and its recovery. Ecotoxicol. Environ. Saf, 20: 30-35. 31. Srivastava, A.S., I. Oohara, T. Suzuki and S.N. Sing, (1999). Activity and expression of aspartate amino-transferase during the reproductive cycle of a fresh water fish, Clarias batrachus. Fish Physiol. Biochem., 20: 243-250 32. Verma, S. R., Bansal, S. K., Gupta, D. K. and Dalela, R. C. (1979). Pesticide induced haematological alterations in a freshwater fish, Saccobranchus fossils. Bull. Environ. Contam. Toxicol. 22: 467-474. 33. Vutukuru, S.S., (2005). Acute effects of Hexavalent chromium on survival, oxygen consumption, hematological parameters and some biochemical profiles of the Indian Major carp, Labeo rohita. Int. J. Environ. Res. Public Health. 2(3): 456-462. 34. Yang J and Chen HC (2003) Effects of gallium on common carp, Cyprinus carpio; Acute test, serum biochemistry and erythrocyte morphology. Chemosphere, 53: 877-882

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CAGE CULTURE TECHNIQUE OF PANGASIUS (Pangasius sutchi) FARMING IN BANSWARA, RAJASTHAN Bhagchand Chhaba 1*, Dr. Bhatt Nakul A.2, Vikash Chahar 3 1,3Research scholar, college of fisheries sciences, W.B.U.A.F.S, Kolkata,700094 2Ph.D, college of fisheries, Maharana Pratap University of agriculture and technology, Udaipur-313001 *Corresponding author: Email- [email protected] ABSTRACT: Pangasius Commonly known as Sutchi catfish or striped catfish or Thai-Pangasius. There are 28 species in the Pangasiidae family, divided into four genera, out of which the genus Pangasius is most important. Pangasius is riverine freshwater. It is omnivorous, feeding on algae, higher plants, zooplankton, and insects, while larger specimens also take crustaceans, fish etc. This species is benthopelagic, typically living within a pH and temperature ranging from 6.5-7.5 and 22- 26 °C respectively. It is a highly migratory riverine fish Pangasius sutchi is introduced in India through Bangladesh during mid-90. About 17,000 hectare area in A.P. is under culture of Pangasius sutchi. Pangasius grows to 1 – 1.5 kg during 6-7 month compare to carp grow very slow an average 1 kg during one year. The Pangasius is one of the most suitable catfishes for rearing in ponds. Pangasius culture has proved itself as a profitable enterprise due to year round production, quick growth and high productivity in south East Asian countries. In addition, pangasius can be stocked at a much higher density in ponds compared to other cultivable species (Ali et al., 2005). In recent cage culture practices in southern Rajasthan under ICAR project. Banswara district located on the southern part of the Rajasthan. Mahidam of the Banswara is recently using for the cage culture practices. There is a huge demand for pangasius in local markets.

INTRODUCTION Cage culture practices is quite peculiar in that the fish to be cultured is kept in cages of metal mesh, bamboo mesh or nylon mesh, left in the flowing water. In past few decades, it has become a major source of aquaculture production, particularly highly pangasius, murrels, sea Bass, Groupers sp. etc [5]. The cage culture originated about 200 years ago in Kampuchia from where it has spread to Indonesia, Thailand and other Asian Countries.Pangasius sutchi, the Pangas catfish, is a species of shark catfish native to fresh and brackish waters of Bangladesh, India, Myanmar, and Pakistan. It has also been introduced to Cambodia and Vietnam [6]. This species grows to a standard length of 3 metres (9.8 ft). This species is important as a food fish. In fish species are easy to kept and easy to monitor in cages. Cages are established in southern rajasthan under the ICAR project. Cages are established in Mahi Bajaj Sagar (Banswara Dist.) and pangasius are kept in this cage. Floating type’s cages are used [8].

Why Pangasius Farming in Cages? [7] • Because of their high survival rate. • Better food conversion efficiency. • Ability to survive in dissolved oxygen deficient water bodies. • Faster growth rate. • Better market demand. • Greater consumer preference. • Easy culture practice. • Preference towards supplemental feeds. • Better yield from unit area within a short period of culture. • Economically important.

Advantages of cage culture 1 It requires less investment. 2 Its installation is easy. 3. Fishes can be stocked at high density rate. 4. Enclosed fishes are protected from predators 5. Harvesting is very simple.

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Cage culture techniques use in Southern Rajasthan (Banswara): Types of cages: Floating types cage No. Of cages: 20 Cage size: 20-40 m3

Stocking density: 100-150 fish / m2 Feeding: - Artificial feed (rice bran: ground nut oil cake) Monitoring: Every week Yield: 100-120 kg/m3

Floating type’s cages: Component use of Floating cage such as HDPE Pipes, Filling Material for Pipes, Brackets, Stoppers, Nets for Fish Cages. Floating cages are basically supported by floating frame, where from net bags are kept hanging in water without touching the basin. It is generally practiced in water bodies with depth of water more than 5 meter in reservoirs, 3 m in wetland and 2 m in canal. The size of the most of the cage varies from 500 to 1000 m3. Cages commonly include living quarters, storage and feeding facilities on top of the submerged cage portion [1].

In India Different types of cages are used: Four types of cages are being used for cages aquaculture such as

1. Fixed cages 2. Floating cages 3. Submersible and 4. Submerged

1. Fixed cages: A fixed cage is essentially a net bag supported by posts which are anchored to the bottom of a river or lake. Although they are inexpensive, their use is limited to shallow, protected water with soft substrates [2].

2. Floating cages: Floating cages are made from netting supported by a buoyant collar or a stable frame. This is the most widely used method of cage aquaculture because the cages can be made any size or shape.

3. Submersible: These cages are built with a rigid frame and because they are submersible, they can be moved up and down in the water column to take advantage of water conditions. If the weather is rough, the cage is lowered to calmer water, but in calm conditions the cage remains near the surface [3].

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4. Submerged: These cages are the least common and are permanently kept under the water. They consist of a frame with slats for openings and are anchored to the substrate in flowing water [4].

CONCLUSION Carps from the mainstay of aquaculture practice in India contributing over 85% of the total aquaculture production. Now a day’s pangasius is fastest growing species, better survival rate and good market demand etc. Pangasius farming started in reservoirs, pond, cages and pen culture system without affecting indigenous species. REFERENCES 1. Datta, S. N., Dhawan, A., Kumar, S., Singh, A., & Parida, P. (2017). Standardization of stocking density for maximizing biomass production of Pangasius pangasius in pond cage aquaculture. Journal of Environmental Biology, 38(2), 237. 2. Gurung, S., Khatri, K. B., Jaisawal, S., & Pandey, S. (2017). Existing scenario and culture techniques of Pangas (Pangasius hypophthalmus) in Rupandehi and Nawalparasi districts of Nepal. 3. Islam, M. S., Huq, K. A., & Rahman, M. A. (2008). Polyculture of Thai pangus (Pangasius hypophthalmus, Sauvage 1878) with carps and prawn: a new approach in polyculture technology regarding growth performance and economic return. Aquaculture research, 39(15), 1620-1627. 4. Keshavanath, P. (2016). SCOPE FOR SOCIAL ENTREPRENEURSHIP IN AQUACULTURE. 5. Malik, A., Kalhoro, H., Shah, S. A., & Kalhoro, I. B. (2014). The Effect of Different Stocking Densities on Growth, Production and Survival rate of Pangas (Pangasius hypophthalmus) Fish in Cemented Tanks at Fish Hatchery Chilya Thatta, Sindh-Pakistan. 6. Rahman, M. M., Islam, M., Halder, G. C., & Tanaka, M. (2006). Cage culture of sutchi catfish, Pangasius sutchi (Fowler 1937): effects of stocking density on growth, survival, yield and farm profitability. Aquaculture Research, 37(1), 33-39. 7. Sayeed, M. A. B., Hossain, G. S., Mistry, S. K., & Huq, K. A. (2008). Growth performance of thai pangus (pangasius hypophthalmus) in polyculture system using different supplementary feeds. University Journal of Zoology, Rajshahi University, 27, 59-62. 8. Vaishnav, M., Sharma, S. K., Sharma, B. K., & Ojha, M. L. (2017). Growth performance of Pangasius Sp. cultured at different stocking density in floating net cages in Mahi Bajaj Sagar Dam of Banswara (Rajasthan).

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COMMERCIAL IMPORTANT FISHING GEAR OPERATED AT GUJARAT COAST *R. A. Khileri, A. Y. Desai, S. I. Yusufzai, S. R. Lende College of Fisheries, Junagadh Agricultural University, Veraval (Gujarat) *Corresponding Author: [email protected] Abstract: A fishing gear is the tool with which aquatic resources are captured, whereas the fishing method is how the gear is used. Gear also includes harvesting organisms when no particular gear (tool) is involved. Furthermore, the same fishing gear can be used in different ways. A common way to classify fishing gears and methods is based on the principles of how the fish or other preys are captured and, to a lesser extent, on the gear construction. A basic understanding of the properties, function and operation of the major fishing gears and methods is therefore fundamental for decision making in fisheries management, particularly when it comes to technical measures in fisheries regulations. The commercial important gears like Trawls and dredges, Gillnets, Dol nets, Hook and line fishing and Cast nets etc. are discussed in detail.

INTRODUCTION Gujarat with about 20% (1600 km) of the country’s coastline, 33% of the continental shelf area (1,64,000 sq. km) and over 2,00,000 sq. km of EEZ, ranks second amongst the maritime states in marine fish production in the country. The width of the Indian continental shelf is the greatest off Gujarat, offering scope for exploitation of several types of fin fish and shellfish resources by both traditional and mechanized fishing (Mohanraj et al. 2009). The estimated marine fish production from Gujarat in 2012 has been 690,396 t showing a considerable increase of 9.14% from that of the previous year. For the first time in the last 10 years, the marine fish production in Gujarat crossed 6 lakh tonnes mark. Mechanized multi-day trawlers have contributed 55% of the total fish landing in Gujarat, which was followed by mechanized dol-netters (30%) and outboard gill-netters (10%). The landings of both mechanized multi-day trawlers and mechanized dol-netters have increased by 2% each (CMFRI 2012). Some criteria for the ideal fishing gear could be: ➢ Highly selective for the target species and sizes, with negligible direct or indirect impact on nontarget species, sizes and habitats. ➢ Effective, giving high catches of target species at lowest possible cost. ➢ Quality orientated, producing catches of high quality. According to these and additional criteria that could be added to the list, it can easily be stated that the ideal fishing gear does not exist, as no fishing gear fulfils the complete list of desired criteria and properties. However, in the process of moving towards sustainable fisheries management, different fishing gears with their specific properties and potential for improvement are an important compartment in the fisheries. MATERIALS AND METHOD Collection of the data All the relevant data about the fishing gear and fishing method were collected through the field survey with the help of the local fishermen. A large number of fishermen were personally interviewed. The data were collected during the study period of January-December, 2014. Procedure of the study All the description of the gear and methods and other relevant statistics provided by the fishermen were recorded in the relevant section of the questionnaire. The gear and methods were - types of gears, seasonal variation of gears, types of indigenous fishing devices and fish composition. The fishing gears are classified into several groups for the better study of the same. Mehsana Wifd Ass... Sanctuary 0 Bhuj 0

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Navsari 0 Map~ '1:>20~5 Google Fig: A view of Gujarat state www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 144 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Zoology

RESULTS TYPES OF FISHING GEARS Fishing gears are commonly classified in two main categories: passive and active. This classification is based on the relative behavior of the target species and the fishing gear. With passive gears, the capture of fish is generally based on movement of the target species towards the gear (e.g. traps), while with active gears capture is generally based on an aimed chase of the target species (e.g. trawls, dredges). A parallel on land would be the difference between the trapping of and hunting for animals. In the following sections a short description of the major gear types is given, including their catching principle, construction, operation and common target species.

1. Prawn Trap 2. Dive 3. Bottom Longline 4. Shrimp Beam Trawl 5. Otter Trawl 6. Midwater Trawl 7. Hook and Line

8. Salmon Purse Seine 9. Midwater Salmon Gillnet 10. Pelagic Longline 11. Harpoon 12. Purse Seine

13. Otter Trawl 14. Offshore Hydraulic Clam Dredge 15. Dredge 16. Pot and Trap 17. Bottom Gillnet 18. Bottom Longline 19. Pot and Trap 20. Dive.

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FISHING GEARS USED IN GUJARAT 1. TRAWLS AND DREDGES Trawls and dredges are often called towed gear or dragged gear. 1.1. Catching principle The catching principle and construction of trawls are shown in Figure 4. 1.2. Construction Trawls and dredges are in principle netting bags that are towed through the water to catch different target species in their path. During fishing, the trawl entrance or trawl opening must be kept open. With beam trawls and dredges this is done by mounting the trawl bag on a rigid frame or beam. With otter trawls the opening is maintained by so-called otter boards (trawl doors) in front of the trawl which keep the trawl open sideways while the vertical opening is maintained by weights on the lower part (ground-rope) and floats on the upper part (headline). With pair trawling, the vertical opening is also maintained by weights and floats, while the lateral opening is maintained by the distance between the two vessels that are towing the trawl. In otter trawling, the trawl is connected to the trawl boards by a pair of sweeps (rope or steel wire) and the trawl doors are connected to the vessel by a pair of warps (normally steel wire). In otter trawling and partially in pair trawling, the sweeps and warps are also parts of the catching system, as they will herd fish towards the centre of the trawl path and the approaching trawl, so that the trawl may catch fish over a larger area than that of the trawl opening. With beam trawl and dredges there is little or no herding of target species in front of the trawl, so the effective catching area is that of the trawl or dredge opening. 1.3. Operation Beam trawls and dredges are exclusively operated on the bottom, where they are towed for a certain length of time (towing time) and distance before being retrieved for the emptying of the catch and being set again for another tow. Otter trawls and pair trawls are most often operated on the bottom to catch different demersal target species. However, these gears are also commonly used for pelagic (or mid-water) trawling at different depths between the surface and the sea bed. This is done by attaching more floats to the head rope of the trawl opening as well as regulating the trawl depth by varying the length of warp and towing speed. In most pelagic trawling, the trawl depth is monitored by depth sensors on the trawl, so that the fishing depth can easily be adjusted to that of the fish targets.

Figure 4. Catching principle and construction of an otter trawl

1.4. Target species Beam trawls are mainly used for catching flatfishes such as plaice and sole as well as for different species of shrimp. Dredges are commonly used for harvesting scallops, clams and mussels. Demersal otter and pair trawls are used to catch a great variety of target species like cod, haddock, hake, sandeel, flatfish, weakfish, croakers as well as shrimps. Pelagic trawls are used in the fisheries for various pelagic target species, like herring, mackerel, horse-mackerel, blue whiting and Pollock. 2. GILLNETS The catching principle and construction of gillnets are shown in Figure 1. 2.1 Catching principle The gillnet is named after its catching principle, as fish are usually caught by gilling i.e. the fish is caught in one of the meshes of the gillnet, normally by the gill region (between the head and the body). Thus, fish capture by gillnets is based on fish encountering the gear during feeding or migratory movements. As fish may avoid the gillnet if they notice the gear, catches are normally best at low light levels or in areas with turbid water.

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2.2 Construction A gillnet consists basically of a wall or panel (e.g. 5 by 30 m) of meshes made from fine thread. The mesh panel is mounted with reinforcing ropes on all sides. To obtain a vertical position of the net in the sea, floats and weights are fastened at regular intervals to the top rope (float line, cork line) and bottom rope (sinker line, lead line), respectively. The size of the meshes and hanging ratio (number of meshes per length of gillnet) are chosen to fit the desired target species and size. Mesh size is commonly given as the length (in mm) either of a whole stretched mesh or the half length (also called bar-length). Today, gillnets are almost exclusively made from synthetic fibres, normally nylon (polyamide) either as multifilament thread or monofilament (gut). The latter is increasingly being used because of its low visibility and correspondingly higher catch efficiency. Multi-monofilament is also becoming more common.

Figure 5. Catching principle and construction of a gill nets 2.3 Operation Gillnets are most commonly operated as a stationary gear anchored to the bottom at either end, but may also be so-called drift-nets which float freely in the water. Stationary nets may be set on the sea bed, at different depths in the water column or with the float line at the surface. Similarly, drift-nets may be operated with the float line at the surface or suspended from surface floats and corresponding float lines to the desired fishing depth in midwater. Gillnets may be operated from vessels ranging from the smallest non-mechanized fishing boats to big, well equipped vessels capable of large-scale deep sea fishing . The gear used in small- and large- scale fishing is basically the same: the unit gillnet. However, with increased vessel size a larger number of net units can be carried and operated per day. Single gillnets are then linked into long fleets of up to several hundred nets. Gillnets can also be operated from shallow to large depths and can be used for fishing on rough bottom and at wrecks. One specific problem with gillnets is so-called ghost fishing”. This refers to gillnets that are lost (most commonly after being stuck on a rough bottom) and continue to catch and kill fish over long periods of time. The Code of Conduct requires that the incidence of ghost fishing should be minimized. 2.4 Target species Gillnets are used to catch a large variety of fish species. In general, bottom gillnets are used for catching demersal species like cod, flatfish, croakers and snapper, while pelagic gillnets are used for species like tuna, mackerel, salmon, squid and herring. 3. DOL NETS Dolnets are operated in three regions viz., Umbergaon to Kavi along the southern Gujarat, Siyalbet to Diu along the Saurashtra coast and Takkara to Modhwa in the Gulf of Kutch region. Among these, Saurashtra is the important region and the main fish landing centers are Jaffarabad, Rajpara, Navabunder and Goghla. Out of these the first three are the most important with more than 600 dol netters under operation. The 'dol' net is' one of the most important gear used in strong tidal current along the coasts of Maharashtra and Gujarat. The Bombay duck, Harpodon nehereus (Ham.) is the main constituent of the catch so much that the 'dol' net fishery has become synonymous with Bombay duck fishery. However, a detailed catch composition revealed that Bombay duck forms 68.6-77% of the total 'dol' net landings along the Saurashtra coast. 3.1 Vessel Overview Boat is not necessary to operate stow nets. Various models of shrimp stow nets are set directly from the shoreline, in the inter-tidal zone. Boats operating "dol" net, in North West coast of India, are wooden boats, ranging from 12 m to 16 m in length, powered by marine diesel engines of 40-100 HP.

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3.2 Craft, gear and area of operation Dolnets are fixed bag nets using two steel poles at a fixed depth. Each boat carries 3-5 nets, which in turn makes 2-3 hauls, with duration of 6 hr/haul. Tidal fishing is done depending on the full moon day at different depths, changing the fishing depth twice in a year. Jaffarabad is one of the major landing centers with 350 dolnet units under operation, out of which 140 are 4 netters, 95 with 3 netters and the rest with 2 netters, operating at a depth of 10-50 m, South-east, South direction. Major landings were observed during April-May and October-December. At Rajpara, around 230 dolnet units arc operated, of which 120 are 4 netters, 60 are 3 netters and the rest with 2 nets, operating at a depth of 10- 50 m, South east, South direction. Peak fishery was noticed from April to May and from September to December. Navabunder is the second largest dolnet landing centre, with 280 dolnetters and 40-50 gill netters. Cat fishes are landed in dolnets as well as gillnet operating at this centre at a depth of 30-40m in South east and South west directions.

Bochi Patiya Aor

Trijo Bungu Chothi Figure 5. Different parts of dol net from Mouth to cod end 3.3 Target species Dolnet catch consists mainly of Harpodon nehereus, Protonibea diaccmthns, penaeid prawns, non- penaeid argentens prawns, Pampus argenteus, Trichiurus sp., carangids and catfishes. Bombay duck contributes a major share to the dolnet catches. 4. HOOK AND LINE FISHING Different fishing methods are based on the use of fish hooks; long lining, trolling and various forms of hand lining such as jigging. The general catching principle of hook fishing is to attract the fish to the hook and entice the fish to bite and/or swallow the hook so that the fish becomes hooked and retained. 4.1 Hand lining and Trolling The catching principle and construction of hand lining are shown in Figure 6. 4.1.1. Catching principle The fish is attracted to the hook by visual stimuli, either natural bait or more commonly in the form of artificial imitations of prey organisms like lures, jigs, rubber worms etc. 4.1.2. Construction The gear is simple: a nylon monofilament is commonly used as line with one to several hooks at the end with bait or lures. 4.1.3. Operation In hand lining the fishing line is vertical and is operated from a drifting or anchored vessel. Hand lining is also conducted from the shore, with and without the use of a pole. From using only a single line, the operation can be scaled up by using several lines on larger vessels. In recent years jigging has become mechanized and automated by the development of jigging machines. Hook and line can also be used in trolling where the fishing line is towed behind the moving vessel. Semi-automation has also been developed in trolling where power reels are often used for hauling the lines. www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 148 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Zoology

Trolling is considered to be a separate type of fishing gear from hand lining in the International Standard Statistical Classification of Fishing Gear (Nedelec and Prado, 1990).

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Figure 6. Catching principle and construction of trolling (left) and jigging (right). Expanded view: examples of lures and jig.

4.1.4. Target species Typical target species with hand lining are demersal fishes like cod and snapper as well as squid. Trolling is mainly directed towards pelagic species like mackerel, tuna and salmon. 4.2. Long lining The catching principle and construction of long lines are shown in Figure 7. 4.2.1. Catching principle Long lining is based on attracting fish by bait attached to the hook. While hand lining and trolling generally exploit the visual sense of the fish to attract it to the hook by artificial lures, long lining exploits the chemical sense of the fish. Odour released from the bait triggers the fish to swim towards and ingest the baited hook with a high probability of being caught. 4.2.2. Construction As the name of the gear indicates, this is a long line (mainline) with baited hooks attached at intervals connected to the mainline with relatively shorter and thinner leader lines (snoods, gangions). Depending on the type of fishery, there are great variations in the gear parameters, such as thickness and material of main and leader lines, the spacing between hooks, as well as hook and bait types.

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Figure 7. Catching principle and construction of long lines. Pelagic/drift (top) and bottom set (lower). Expanded view: baited hook connected with gangion (snood, branchline) to mainline.

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Today, main and leader lines are almost exclusively made from synthetic materials like polyamide (nylon) or polyester. Multifilament (rope) is generally used for main and leader lines with demersal long lines (set on the bottom), while monofilament (gut) is commonly used in pelagic long lining. Hook type (size and shape) varies greatly with target species. Naturally, larger hooks and correspondingly stronger main and leader lines are used for larger fish. There is also a great variation in baits used in different long line fisheries, but the major types of bait are either different pelagic fish (e.g. herring, mackerel, sardine) or different species of squid. 4.2.3. Operation The long line fishing cycle includes the following main operations: baiting (threading a piece of bait on each hook), setting, fishing (soaking the line for some hours), retrieval, removal of fish and old bait, gear maintenance, baiting, etc. As with gillnets, the gear is basically the same in small and large-scale operations with the length of the line and number of hooks increasing with vessel size. Small, open vessels normally fish a few hundred hooks, while the largest long line vessels (LOA 50-60 m) may operate 50-60 km of long line and as many as 40-50000 hooks per day. With increased vessel size there is normally an increased degree of mechanized gear handling. Most long line vessels are equipped with power haulers. In so called auto-lining the laborious baiting process is also mechanized with machines that can bait up to four hooks per second as the line is set into the sea. 4.2.4. Target species Pelagic (drifting) long lines are typically used for catching species like tuna, swordfish and salmon, while bottom set long lines are used for demersal species like snapper, cod, haddock, halibut, ling, tusk, hake and tooth fish. 5. CAST NETS A cast net, also called a throw net, is a net used for fishing. It is a circular net with small weights distributed around its edge. The net is cast or thrown by hand in such a manner that it spreads out on the water and sinks. This technique is called net casting or net throwing. Fish are caught as the net is hauled back in. This simple device is particularly effective for catching small bait or forage fish, and has been in use, with various modifications, for thousands of years. On the US Gulf Coast, it is used specially to catch mullet, which will not bite a baited hook. 5.1 Construction and technique Contemporary cast nets have a radius which ranges from 4 to 12 feet (1.2 to 3.6 meters). Only strong people can lift the larger nets once they are filled with fish. Standard nets for recreational fishing have a four- foot hoop. Weights are usually distributed around the edge at about one pound per foot (1.5 kilograms per meter). Attached to the net is a hand line, one end of which is held in the hand as the net is thrown. When the net is full, a retrieval clamp, which works like a wringer on a mop, closes the net around the fish. The net is then retrieved by pulling on this hand line. The net is lifted into a bucket and the clamp is released, dumping the caught fish into the bucket. Cast nets work best in water no deeper than their radius. Casting is best done in waters free of obstructions. Reeds cause tangles and branches can rip nets. The net caster may choose to stand with one hand holding the hand line, and with the net draped over the other arm so that the weights dangle, or, with most of the net being held in one hand and only a part of the lead line held in the other hand so the weights dangle in a staggered fashion (approximately half of the weights in the throwing hand being held higher than the rest of the weights). The line is then thrown out to the water, using both hands, in a circular motion rather as in hammer throwing. The net can be cast from a boat, or from the shore, or by wading. CONCLUSION ➢ Unsustainable /destructive fishing practices are highly prevalent in many areas. These need to be totally prohibited. ➢ It becomes highly relevant to comply with the provisions of the CCRF with regard to responsible fishing practices. ➢ Within the broad areas mentioned in the code, there are priority activities that need to be addressed. ➢ A common issue considered fundamental to progress towards implementing responsible fisheries practices is that of awareness, education and training. ➢ Turtle excluder device should be used in shrimp trawl net ➢ All fishing gear should have square mesh size at cod end. ➢ Fishing ban period should strictly follow. REFERENCES Alverson, D.L., Freeberg, M.H., Murawski, S.A. and Pope, J. 1994. A global assessment of fisheries bycatch and discards. FAO Fisheries Technical Paper 339. FAO, Rome. pp. 233. Bjordal, A. and Lokkeborg, S. 1996. Longlining. Fishing News Books, Blackwell Science Ltd.,Oxford. pp. 156.

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Brandt, A. 1984. Fish Catching Methods of the World. Fishing News Books, Farnham. Cowx, I.G. and Lamarque, P. 1990. Fishing with Electricity (Applications in freshwater fisheries management). Blackwell Science Ltd., Oxford. pp. 272. Central Marine Fisheries Research Institute. 2012. In: Annual Report of CMFRI; 2012-13. Published by Cen. Mar. Fish. Res. Inst.. Cochin, pp. 32. Dunbar, Jeffery A (2001) Casting net NC Coastal fishing. Retrieved 25 August 2008. Ferno, A. and Olsen, S. 1994. Marine Fish Behaviour in Capture and Abundance Estimation. Fishing News Books, Blackwell Science Ltd., Oxford. pp. 221. Gabriel, O. 1992. Fish Catching Methods of the World. (Fourth Edition). Blackwell Science Ltd., Oxford. pp. 448. Hall, S. 1999. The Effects of Fishing on Marine Ecosystems and Communities. Fishing News Books, Blackwell Science Ltd., Oxford. pp. 296. Kaiser, M.J. and Groot, S.J. 2000. The Effects of Fishing on Non-target Species and Habitats. Blackwell Science Ltd., Oxford. pp. 399. Moore, G. and Jennings, S. 2000. Commercial fishing (the wider ecological impacts). Blackwell Science Ltd., Oxford. pp. 72. Mohanraj, G.; Nair, K. V. S.; Asokan, P. K. and Ghosh, S. 2009. Status of marine fisheries in Gujarat with strategies for sustainable and responsible fisheries. Asian Fish. Sci.. 22: 285-296. Nedelec, C. and Prado, J. 1989. FAO Catalogue of Small Scale Fishing Gear. Blackwell Science Ltd., Oxford. pp. 224. Nedelec, C. and Prado, J. 1990. Definition and classification of fishing gear categories. FAO Fisheries Technical Paper, 222 (1). FAO, Rome. pp. 92. Sainsbury, J.C. 1996. Commercial Fishing Methods (an introduction to vessel and gear) Blackwell Science Ltd., Oxford. pp. 368. Scharfe, J. 1989. FAO Catalogue of Fishing Gear Designs. Blackwell Science Ltd., Oxford. pp. 160.

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ANTIMICROBIAL PROPERTIES OF SKIN MUCUS FROM FRESHWATER FISHES H.D. Kaher1, S.S. Bhatt2, A.V.R.L. Narasimhacharya3, A.N. Upadhyaya4 1.Department of Biosciences, Sardar Patel University, Vallabh Vidhyanagar. 2.Department of Biology, Shree D.K.V. Arts & Science College, Jamnagar. Email id:[email protected] ABSTRACT: The fishes are living in the medium rich in pathogenic microbes. The mucus secreted by the skin of fish showed more antimicrobial properties. The mucus secreated by the skin of fish showed more antimicrobial properties. The mucus collected from the six freshwater fishes were tested against the four pathogenic bacteria (E.coli, P. aeruginosa, B.subtilus and S.aureus ) and two pathogenic fungi namely (Candida albicans and Aspergillus niger) by well diffusion method. The activity was measured in terms of zone of inhibition in mm. The findings of the current investigation showed that the mucus of snake head fish (Channa punctatus) may be a potential source antimicrobial agent than the walking catfish (Clarius batrachus), grass carp (Ctenopharyngodon idella), mrigal (Cirrhinus mrigala), bighead carp (Aristicthys nobilis), and tilapia (Oreochromis niloticus). Keywords: Mucus, Antimicrobial properties, Pathogenic bacteria, well diffusion method, fish

1. INTRODUCTION Modern chemotherapeutic techniques have become highly reliable due to the advanced improvements and newer formulations (Ongyeongwei etal.,2013).Extractsand preparation made from the animal origin have been a great healing tool in folk and modern medicine (Kuppulakshmi et al., 2008).Now-a-days the development of resistance by a pathogen to many of the commonly used antibiotics provides an impetus for further attempts to search for new antimicrobial agents which combat infections and overcome the problems of resistance with no side effects.The global trade in animal based medicinal products accounts for billions of dollars per year (kunin and Lawton, 1996). According to WHO, out of 252 traditional medicines, 8.7 % come from animals (Marques, 1997). Indeed, animals are therapeutic arsenals that have been playing significant roles in the healing processes, magic rituals, and religious practices of peoples (Costa and Marques, 2000). All living organisms including fish coexist with a wide range of pathogenic and non- pathogenic microorganisms and therefore, posses complex defense mechanisms which contribute to their survival. One mechanism is the innate immune system that combats pathogens from the moment of their first contact (kimbrell and Beutler, 2001). The specific immunity including antibody and specific cell-mediated responses are significantly less diverse than those of higher (Ellis, 1974; Manning, 1998). The development of resistance by a pathogen to many of the commonly used antibiotics provides an impetus for further attempts to search for new antimicrobial agents, which overcome the problems of resistance and side effects. Action must be taken to reduce this problem such as controlling the use of antibiotics, carrying out research to investigate drugs from natural sources. Drugs that can either inhibit the growth of pathogen or kill them and have no or least toxicity to the host cell are considered for developing new antimicrobial drugs. It is well known that the global trade in animal based medicinal products accounts for billions of dollars per year (Kunin and Lawton, 1996). 2. MATERIAL AND METHODS Fish collection: Hypophthalmicthys nobilis, cirrhina mrigala, ctenopharyngodon idella and tilapia were obtaind from juhu- krupa farm, pij village, taluka nadiad (dist. Kheda). From there we are transport the fish in polythene bag with aerated water and immediately collected the mucus. Mucus collection: Mucus collection was carried out by using the method of Ross et al., (2000). the fishes were transfered in to polythene bags conaining 50mM Nacl solution at the rate 10ml for 100g fish. The bags were shaken by hand for 5 minutes to collect mucus and following this treatment fish were retuurned to recover in the beaker. The mucus was immediately transffered into 15ml sterile centrifuge tubes and placed on dry ice.the sample was centrifuged at 1500 rpm for 10 min at 4C and the supernatant obtained was alliqouted into 2ml centrifuge tubes, freeze dried and stored at -20c for further analysis. Antimicrobial activity by Agar Well Diffusion assay: In vitro antimicrobial assay was carried out by disc diffusion technique (Bauer et al., 1996). Whatman No.1 filter paper discs with 4 mm diameter were impregnated with known amount (10 μl) of test sample of fish mucus and a standard antibiotic disc. At room temperature (37ºC) the bacterial plates were incubated for 24 h. The fungal plates were incubated at 30ºC for 3 to 5 days for antifungal activity. 0.8% soft agar was seeded with overnight grown culture of 100µl of test organism which www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 152 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Zoology was then overlaid on the 2% Luria agar base. With the help of sterile cork borer wells of 6mm diameter was made and filled with 100µL sample under study. Appropriate control was also added in to wells. Plates were incubated at 37 C for 24 h. The diameter of zone of inhibition was observed and measured in millimeter. The spectrum of antimicrobial activity was studied using four different strains of human pathogenic bacteria and two species of fungal pathogens. 3. RESULT In the present experiment, the antibacterial activity has been analyzed in six diffrent selected fishes against two gram positive bacteria ( B.subtilus, S.aureus ) and two gram negative bacteria bacteria ( E.coli, P.areuginosa ). The activity was measured in terms of zone of inhibition in mm. Mucus secreted by fish play a major role in protection against major infectious agents such as bacteria and fungi haniffa et al., (2014). The mucus of Channa Punctata showed more effect in controlling the growth of gram-negative bacteria E.Coli with an inhibition zone of 37 mm in diameter. Next to Ecoli, the mucus of Channa Punctata showed a better effect on B.subtilus having an inhibition zone of 28 mm in diameter. That was followed by the P.areuginosa with an inhibition zone of 27 mm in diameter. Among the four bacteria tested S.aureus showed very less sensitivity to the mucus of Channa Punctata with an inhibition zone of 24 mm in diameter. The mucus of Clarius batrachus showed more effect in controlling the growth of gram-negative bacteria E.Coli with an inhibition zone of 33 mm in diameter. Next to Ecoli, the mucus of Clarius batrachus showed a better effect on B.subtilus having an inhibition zone of 25 mm in diameter. That was followed by the P.areuginosa with an inhibition zone of 22 mm in diameter. Among the four bacteria tested S.aureus showed very less sensitivity to the mucus of Clarius batrachus with an inhibition zone of 19 mm in diameter.

Table No.1 Antibacterial effect of mucus of selected diffrent fishes analysed by well diffusion assay method. (Zone of inhibition in mm ) Bacteria E.Coli B.subtilus S.aureus P.areuginosa Fish Channa Punctata 37 28 24 27 Clarius batrachus 33 25 19 22 Cirrhina mrigala 25 12 7 6 Ctenopharyngodon idella 29 28 14 11 Aristhichthys nobilis - - 4 7 Oreochromis niloticus 19 14 9 11

40 35 □ E.Coli 30 - B.subtilus 25 ~ ■ 20 ~ i;;; ~ □ S.aureus 15 ~ ~ □ P.areuginosa 10 ~ ~ - 5 - - I ■ 0 n- I rn- 11

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Table No.2 Antifungalal effect of mucus of selected diffrent fishes analysed by well diffusion assay method. (Zone of inhibition in mm)

Fungi A.niger C.albicans

Fish Channa Punctata 28 23

Clarius batrachus 21 16

Cirrhina mrigala 10 7

Ctenopharyngodon idella 15 19

Aristhichthys nobilis - 7 Oreochromis niloticus 12 14

30 25 20 A.niger 15 C.albicans 10 5 0

The mucus of Cirrhina mrigala showed more effect in controlling the growth of gram-negative bacteria E.Coli with an inhibition zone of 25 mm in diameter. Next to Ecoli, the mucus of Cirrhina mrigala showed a better effect on B.subtilus having an inhibition zone of 12 mm in diameter. That was followed by the S.aureus with an inhibition zone of 6 mm in diameter. Among the four bacteria tested P.areuginosa showed very less sensitivity to the mucus of Cirrhina mrigala with an inhibition zone of 7 mm in diameter.The mucus of Ctenopharyngodon idella showed more effect in controlling the growth of gram-negative bacteria E.Coli with an inhibition zone of 29 mm in diameter. Next to Ecoli, the mucus of Ctenopharyngodon idella showed a better effect on B.subtilus having an inhibition zone of 28 mm in diameter. That was followed by the S.aureus with an inhibition zone of 14 mm in diameter. Among the four bacteria tested P.areuginosa showed very less sensitivity to the mucus of Ctenopharyngodon idella with an inhibition zone of 11 mm in diameter. The mucus of Aristhichthys nobilis showed No effect in controlling the growth of gram-negative bacteria E.Coli and B.subtilus. That was followed by the P.areuginosa with an inhibition zone of 4 mm in diameter. Among the four bacteria tested S.aureus showed very less sensitivity to the mucus of Aristhichthys nobilis with an inhibition zone of 7 mm in diameter.The mucus of Oreochromis niloticus showed more effect in controlling the growth of gram-negative bacteria E.Coli with an inhibition zone of 19 mm in diameter. Next to Ecoli, the mucus of Oreochromis niloticus showed a better effect on B.subtilus having an inhibition zone of 14 mm in diameter. That was followed by the P.areuginosa with an inhibition zone of 11 mm in diameter. Among the four bacteria tested S.aureus showed very less sensitivity to the mucus of Oreochromis niloticus with an inhibition zone of 9 mm in diameter. The mucus of Channa Punctata showed more effect in controlling the growth of pathogenic fungai A.niger with an inhibition zone of 28 mm in diameter. Next to A.niger, the mucus of Channa Punctata showed a better effect on C.albicans having an inhibition zone of 23 mm in diameter. The mucus of Clarius batrachus showed more effect in controlling the growth of pathogenic fungai A.niger with an inhibition zone of 21 mm in diameter. Next to A.niger, the mucus of Clarius batrachus showed a better effect on C.albicans having an inhibition zone of 16 mm in diameter. The mucus of Cirrhina mrigala showed more effect in controlling the growth of pathogenic fungai A.niger with an inhibition zone of 10 mm in diameter. Next to A.niger, the mucus of Cirrhina mrigala showed a better effect on C.albicans having an inhibition zone of 07 mm in diameter. The

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 154 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Zoology mucus of Ctenopharyngodon idella showed more effect in controlling the growth of pathogenic fungai A.niger with an inhibition zone of 15 mm in diameter. Next to A.niger, the mucus of Ctenopharyngodon idella showed a better effect on C.albicans having an inhibition zone of 14 mm in diameter. The mucus of Aristhichthys nobilis showed No effect in controlling the growth of pathogenic fungai A.niger with an inhibition zone of mm in diameter. Next to A.niger, the mucus of Aristhichthys nobilis showed a better effect on C.albicans having an inhibition zone of 7 mm in diameter.The mucus of Oreochromis niloticus showed more effect in controlling the growth of pathogenic fungai A.niger with an inhibition zone of 12 mm in diameter. Next to A.niger, the mucus of Oreochromis niloticus showed a better effect on C.albicans having an inhibition zone of 14 mm in diameter. 4. DISCUSSION The epithelial surfaces of fish, such as the skin, gills and the alimentary tract provide first contact with potential pathogens. The biological interface between fish and their aqueous environment consists of a mucus layer composing of biochemically-diverse secretions from epidermal and epithelial cells (Ellis, 1999). This layer is thought to act as a lubricant to have a mechanical protective function, to be involved in osmoregulation and play a possible role in immune system of fish. Fish tissue and body fluids contain naturally occurring proteins or glycoproteins of non-immunoglobulin nature that react with a diverse array of environmental antigens and may confer an undefined degree of natural immunity to fish. Antimicrobial peptides are among the earliest developed molecular effectors of innate immunity and are significant in the first line of host defense response of diverse species. Most antimicrobial peptides found through out the animal and plant kingdom are small, functionally specialized peptides (Boman, 1995). Several endogenous peptides with antimicrobial activity from fish, especially from the skin and skin mucus are reported (Park et al., 1997). Endogenous peptides play an important role in fish defense, possess broad spectrum of antimicrobial activity against bacteria, yeast and fungi. The epidermic and the epithelial mucus secretions act as biological barriers between fish and the potential pathogens of their environment (Shephard, 1993). In the present study, variation in their antimicrobial activity was observed among the fish mucus. This may be due to the variation in the relative levels of lysozyme, alkaline phosphatase, cathepsin B and proteases of the epidermal mucus of all fish species (Subramanian et al., 2007). 5. CONCLUSION Falling in line with the above observation, the indigenous fish species such as Channa Punctata and Clarius batrachus show higher antimicrobial activity than that of the exotic fish species such as C. idella. This is the first report on the antimicrobial activity of skin mucus of cultivable indigenous fishes of India. Moreover, the mucus of fish possesses antimicrobial agents which could be used to formulate new drugs for the therapy of infectious diseases caused by pathogenic and opportunistic microorganisms. These properties of mucus suggest that it may be beneficial in aquaculture and human health-related applications. Further studies are needed to isolate the bioactive compounds (antimicrobial substances) from the mucus of these cultivable fish species and the mechanism of antimicrobial action. 6. ACKNOWLEDGEMENT The authors are thankful to department of Biosciences Sardar Patel University and D.K.V Arts and Science College for providing administrative and infrastructural facilities for doing this work. 7.REFERENCE Boman Hg (1995). peptide antibiotics and their role in innate immunity. ann. rev. immunol., 13: 61-92.Costa Em, Marques Jgw (2000). faunistic resources used as medicines by artisanal fishermen from siribinha beach, state of bahia, brazil. j. ethnobiol., 20: 93-109. Dhanaraj M, Haniffa M, Arun A, Singh Sv, Muthu Rc, Manikandaraja D, Milton Jm (2009). antibacterial activity of skin and intestinal mucus of five different freshwater fish species viz., channa striatus, c. micropeltes, c. marulius, c. punctatus and c. gachua. malay. j. sci., 28(3): 257-262. Ellis A (1999). immunity to bacteria in fish. fish shellfish immunol., 9: 291-308. Ellis Ae (1974). non-specific defense mechanisms in fish and their role in disease processes. dev. biol. stand., 49: 337-352. Ellis Ae (2001). the immunology of teleosts, in: roberts rj (ed), fish pathology, 3rd edition. elsevier, new york. pp. 133-150. Kunin Wk, Lawton Jh (1996). does biodiversity matter? evaluating the case for conserving species. in:gaston kj (ed), biodiversity: a biology of numbersand differences, oxford:

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blackwell science. 283-308.Marques Jgw (1997). fauna medicinal: recurso do ambienteou ameaca a biodiversidade? mutum.1-4 Kuppulakshmi C, Prakash M, Gunasekaran G, Manimegalai G, Sarojini S (2008). antibacterial properties of fish mucus from channa punctatus and cirrhinus mrigala. european review for medical and pharmacological sciences. 12:149-153. Manning Mj (1998). immune defense systems. in: blank kd, pickering ad (eds), biology of farmed fish. academic press, sheffield. 180-221. Ong Yeong Wei R, Xavier K Marimuthu (2013). screening of antibacterial activity of mucus extract of snakehead fish, channa striatus (bloch). european review for medical and pharmacological sciences. 14:675-681. Park Cb, Lee Jh, Park Iy, Kim Ms, Kim Sc (1997). a novel antimicrobial peptide from the loach misgumus anguillicaudatus. febs lett., 411: 173-178. Shephard Kl (1993). mucus on the epidermis of fish and its influence on drug delivery. adv. drug. deliv. rev., 11: 403-417. Subramanian S, Mackinnon Sl, Ross Nw (2007). a comparative study on innate immune parameters in the epidermal mucus of various fish species. comp. biochem. physiol., 148b: 256-263. Subramanian S, Ross Nw, Mackinnon Sl (2008). comparison of antimicrobial activity in the epidermal mucus extracts of fish. comp. biochem. physiol., 150 (b): 85-92. Yang Jy, Shin Sy, Lim Ss, Hahm Ks, Kim Y (2000). structure and bacterial cell selectivity of a fish derived antimicrobial peptide, pleurocidin. j. microbiol. biotech., 16: 880-888. Zasloff M (2002). antimicrobial peptides of multicultural organisms. nature, 415: 389-395. Zuchelkowski Em, Lantz Rc, Hinton De (1981). effects of acid-stress on epidermal mucus cells of the brown bullhead-ictalurus nebulosus (leseur): a morphometric study. anat. rec., 200: 33-39

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SALIVARY COMPONENTS OF THERAPEUTIC LEECH AND ITS USES K.K.Koriya, A.N. Upadhyay, H.D.Kaher Department of Biology, D.K.V Arts & Science College, Jamnagar (Gujarat) [email protected] ABSTRACT-Medicinal leech (Hirudinaria Granulosa) used up in leech therapy in conventional treatment of pain, skin diseases and inflammatory diseases. The majority of these treatment benefits are not due to blood sucked during the biting, rather than the various bioactive ingredients of saliva has particulate effect on human body. It contains more than 100 bioactive substance including Hyaluronidase, Hirudin, Calin, Destabilase, Acetycholine, Collagenase ect. These secretion components are responsible for suppression of protecting mechanism of patient organisms at wound, their functioning allows long term blood sucking. When saliva injested into the wounds acts on various system of the human body directly linked to the homeostatic system. Key words: Leech saliva, diseases, therapy, salivary components

1.INTRODUCTION Amongst haematophagous animals, Leeches have been familiar therapeutic animal (Abdualkader et al,2013). Traditionally, Leech application was used in many treatments like skin problems, Nervous system abnormalities, urinary and reproductive system problems and in ocular inflammation, dental problems and hemorrhoids etc( Bhrigu kumar das 2013). Doctors had used Leeches since antiquity for a variety of therapeutic purposes. Saliva of leech contains many biological active compounds that are mainly proteins and peptides. These proteins and peptides have particular effects during the therapy on the skin of patients some clinicians use leech therapy to improve blood circulation (Mohamed alaama et al ,2011). During the application by using their proboscis to puncture through the skin and release anesthetic to eliminate pain of bite and inficted. The salivary components of hirudinaria granulosa are prevent the blood from clotting and allow maintenance of anticoagulation of blood. Salivary components of leech has hyluronidase, hirudin, calin, destabilase, and many other component which play imp role in hirudo therapy (I.P.Baskova, 2004). 2. AIMS AND OBJECTIVES The present study is aimed at understanding the benefits of salivary components of Leech during the Therapy. The major aims include the following: To study bioactive ingredient present in saliva of Hirudinaria Granulosa. To analyze the function of Leech saliva in Hirudo therapy. To Study the function of salivary component of Leech. to analyze the effect of Leech saliva on skin of Patients. To study the effect of salivary components on blood flow.

3. MATERIALS AND METHOD Study area: shri gulab kunvarba ayurved mhavidhyalay, Jamnagar. which is also known as dhanvantri mandir. Its providing bachelor of ayurvedic medicine and surgery course. Data collection: the study was carried out from January 2015 to May 2015. I was visited regularly during these periods of time. Methodology: First a fall clean the part of leech bite and then the mouth of leech brought to the site of infected skin. When once starts sucking blood elevates its neck and fix its head to supporting site of infection. When the neck and body of the leech are covered with a large of wet cotton which is to be moistened throughout process. After some time, the detachment of leech from the skin of patient. I observed the difference between skin of eczema patient before the 1st sitting and after every sitting till 5th sitting of leech therapy (Bhrigu kumar das 2013). 4.RESULT AND DISCUSSION After every sitting of Leech, the skin of patient become dry and the flow of blood from the site of leech bite is increased. During therapy Acetylcholine and Histamine like substance are responsible for the dilation of blood vessels that’s help prevent the blood from clotting and allow maintenance of anticoagulation of blood. (I.P.Baskova, 2004). www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 157 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Zoology

That indicates that there is substance like Hyaluronidase, Hirudin, Calin, Destabilase, Bdellins, Acetylcholine, Histamine like substance, Hirustasin,Eglins, Factor Xa inhibitor, Carboxy peptidase-A inhibitors, Collagenase, Complement inhibitors, present in the leech saliva which prevents blood clotting and increased flow of blood ( Bhrigu kumar das 2013).

Salivary Bioactive substance of hirudinaria Granulosa their functions Bioactive substance Their functions Hirudin It binds with thrombin and prevent blood clotting for some time Destabilase It has thromboiytic effects and dissolves fibrin. Hyaluronidase For penetration and diffusion of pharmacologically active substances into tissues and specially in joint pain. It also has antibiotic properties. Calin It binds to Von Willebrand factor to collagen and inhibits blood coagulation last upto 12 hours. Hirustasin It inhibits neutropholic cathepsin G, trypsin, kallikrein,and chymotrypsin. Bdellins Anti-inflammatory effect and inhibits acrocin, plasmin and trypsin Acetylcholine A vasodilator Histamine like substances A vasodilator increases the inflow of blood at the bite site.

Factor Xa inhibitor It prevents the activity of coagulation factor Xa.

Carboxypeptidase-A Increase the flow of blood. inhibitors Collagenase Reduces collagen.

Complement inhibitors Replace natural complement inhibitors if they are deficient.

List of Eczema patients (from January 2015- may 2015)salivary effect on blood flow Name of Volume of Volume of Volume of Volume of Volume of patient blood from nd blood from blood from blood from st blood from 2 rd th th 1 sitting sitting 3 sitting 4 sitting 5 sitting

Patient 01 15cc 21cc 25cc 29cc 32cc

Patient 02 12cc 18cc 23cc 28cc 30cc

Patient 03 10cc 13cc 18cc 20cc 23cc

Patient 04 15cc 18cc 22cc 26cc 30cc

Patient 05 10cc 13cc 18cc 22cc 25cc

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Blood volu me (cc) after every sitting

patie nt 01 patient02 patie nt 03 p atie nt 04 patient OS 11 5th sitting 32 30 23 30 25 a 4t h sitting 29 28 20 25 22 • 3 rd sitting 25 23 18 22 18 • 2nd sitting 21 18 13 18 13 • 1st sitting 0 12 10 15 10

List of Eczema patients (from January 2015- may 2015) salivary effect on skin Name of Effect on Effect on skin Effect on skin after Effect on skin Effect on skin patient skin after after 2nd sitting 3rd sitting after 4th sitting after 5th 1st sitting sitting

Patient 01 No more Slightly dry Become hard and Completely dry Completely change moderately dry and hard skin dry and hard skin Patient 02 No more Moderately dry Become hard and Completely dry Completely change highly dry and hard skin dry and hard skin

Patient 03 No more Slightly dry Moderately dry Become hard Leech not able change and highly dry to bite with out puncture

Patient 04 No more Moderately dry Become hard and Completely dry Completely change completely dry and hard skin dry and hard skin Patient 05 No more Skin becom hard Become hard and Completely dry Leech not able change and moderately completely dry and hard skin to bite with out dry puncture

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Skin change after every sitting of leech therapy 70

40 teatment

Dry skin after after skin Dry 20

0 patient 01 patient 02 patient 03 patient 04 patient 05 Number of patient

5. CONCLUSION On the basis of this study, there are more than 100 bioactive substance presents in the saliva of Leech out of these 13 bioactive substances are highly effect during the leech therapy. Acetylcholine and Histamine like substance are responsible for the dilation of blood vessels the main substance like Hirudin, Calin, Destabilase, Factor Xa inhibitor, carboxypeptidase-A inhibitors, Collagenase, which are responsible for the prevent the blood coagulation by binding the blood components of patients. Other substance has their other particular effect on the Hirudo therapy. And repeatedly large volume of blood flow without coagulation there for the skin becomes dry and hard. 6. ACKNOWLEDGEMENT The authors are thankful D.K.V Arts and science collage and shri gulab kunvarba ayurved mhavidhyalay for providing lab facility and their valuable support. 7. REFERENCES Abdualkader, bdualrahman Mohammed, Ghawi, Abbas Mohammad, Alaama, Mohamed,Awang, Mohamed, Merzouk, Ahmed; (2013); “ In vivo anti-hyperglycemic activity of saliva extract from the tropical leech Hirudinaria manillensis”; Chinese Journal of natural Medicines; vol.11(5); P-488- 493.;doi:10.3724/SP.J.1009.2013.00488 Bhrigu Kumar das (2014); “An overview on Hirudotherapy/leech therapy”;Indian research journal of pharmacy and science Ghos, kuntala: tripathi pareshc (2012). Clinical effect of virechana and shamona chikitsa in tamaka shwasa Panchkarma therapy in school roga pbumed central canada (2013). I.P.Baskova ,L.L.Zavalova, A.V.Basanova ,S.A.Moshkovskii ,,and V.G.Zgoda ,(2004); “protein profiling of the Medicinal Leech Salivary Gland Secretion by Proteomic Analytical Methods.”;Biochemistry (Moscow); vol.69; No.7; P-770-775. Mohamed Alaama, manar alnajjar, abdualraman M. abdualkader, abbas Mohammad and ahmed merzouk;(2011); “Isolation and Analytical characterization of local malasian leech saliva extract”;IIUM Engineering Journal; vol.12; No.4; P-51-59.

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 160 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Zoology DIVERSITY AND DENSITY OF MIGRATORY BIRDS AND ITS CORRELATION WITH PHYSICO-CHEMICAL PARAMETERS OF LAKHOTA LAKE, JAMNAGAR, GUJARAT A.N. Upadhyaya1, P. S. Oza2 and B. A. Jadeja3, H.D. Kaher4, K.K.Koria5 1Department of Biology, Shree D.K.V. Arts & Science College, Jamnagar. 2Department of Chemistry, Shree D. K. V. Arts & Science College, Jamnagar. 3Department of Botany, M.D. Science College, Porbandar. 4Department of Biology, Shree D.K.V. Arts & Science College, Jamnagar. 5Department of Biology, Shree D.K.V. Arts & Science College, Jamnagar. [email protected]

ABSTRACT: The key physical and chemical parameters are one of the major habitats for birds. The present study deals with the interactions between these physico-chemical parameters and bird diversity of lakhota lake in Jamnagar of Gujarat, India. The study was carried out monthly but was tabu- lated seasonally. Result showed different species of birds have been cited belonging to 9 orders and 18 families during the year in year 2010. The correlation made between birds’ density and physico- chemical parameters to analyses the relative features of the both variable. The aggregation of birds in the area is mainly related to the increase in migratory population of birds during winter. The variations in bird aggregations as well as physico-chemical parameters are discussed. From the results it has been observed that the lake water is suitable for the growth of different biological parameters like algae, planktons, insect larva, fishes, molluscans etc. which ultimately attract birds towards lake. Key Words: Diversity, density, migratory, physico-chemical parameter, LakhotaLake

1. INTRODUCTION Water is a basic and primary need of all vital processes and it is now well established that the life first arose in aquatic environment. Ever since the pre-historic times man has been intimately associated with water and it has been continuously proved by the evidences of past civilization that all historic human settlements were around inland freshwater resources. These wetlands are traditional zones that occupy intermediate position between dry land and open water. These wetlands are rich in flora and fauna and birds are one of the important biotic factors which prefer to live near these wetlands (M. C. Vachant 2012).To study any ecosystem the birds serve as important component as they have the ability to fly away and avoid any obnoxious condition. Hence, they are considered as important health indicators of the ecological conditions and productivity of an ecosystem (Newton, 1995; Desai and Shanbhag, 2007). Distribution and abundance of water birds was affected by several factors. Little change in physical, chemical or biological properties put forth intense effects on bird’s habitats (Murphy et al., 1984). Any change in the physical, chemical and biological factors in the catchment exerts severe impact on the wetland as habitat for aquatic communities. These in turn affect the wetland dependent communities as well as the ecosystem attributes such as species richness, its distribution and density (Burkert et al., 2004). Wetland supports congregation of large number of migratory and resident bird species as it has high nutritional value as well as productivity (Paracuellos 2006). Freshwater wetlands hold more than 40% bird species of the entire world and 12% of all animal species (Patra et al. 2010). The physical and chemical characteristics of water bodies affect the species composition, abundance, productivity and physiological conditions of aquatic organisms (Bhat et al. 2009). Trivedi (1981) has emphasized the importance of species diversity in assessing the water quality and reported that polluted water supports low organism diversity while the clean water supports high diversity. the physical and chemical properties of freshwater body are characteristics of the climatic, geochemical, geomorphological as well as pollution conditions prevailing in the drainage basin and the underlying aquifer (Ramachandra et al. 2002). These characteristics with natural or manmade changes determine the quality of water (Anonymous 2003). Thus, wetlands are highly complex ecosystems due to various interactions between the components like water, soil, biosphere and atmosphere (Bodegom et al. 2004). Also, the study of interactions between biotic and abiotic factors becomes essential to understand the community structure of an ecosystem (Dunson and Travis 1991). No systematic work has been done in Jamnagar on the distribution of birds (biotic) in relation to physico-chemical parameters (abiotic) of a water body. This study intended to assess water quality parameters to examine its relationship with bird numbers and species richness. Knowledge on the composition of the bird community in Lakhota lake facilitates to manage programs aiming at protection and conservation of bird species and their habitats.

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 161 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Zoology 2. MATERIAL AND METHODS Study Area:

2.1 Lakhota Lake 2.2 View of Lakhota Lake (satellite View)

Bird surveys Bird surveys were carried out at seasonal intervals (winter, spring and summer) at Lakhota lake in the year in year 2010. Direct count of the birds was carried out by using the binoculars. The counts were conducted between (07.30 & 11.30hrs) in the morning. Vantage points (open-water areas, mudflats and short-grass flats) were identified that covered large sections of the lake where birds were less disturbed and the chances of visibility was high. Birds were identified with a popular field guide and the book of Indian birds by salim ali. Scientific nomenclature and systematic order of birds follows salim ali. Physico-chemical analysis: The analysis of filtered water samples was carried out for the parameters, as pH Electrical Conductivity (EC), Total Dissolved Solids (TDS), Total Hardness (TH), Major Constituents Calcium (Ca) and Chloride (Cl), Total Alkalinity (TA), Phosphate (PO4), indicator parameter Dissolved Oxygen (DO), Biological Oxygen Demand (BOD) of the samples were done according to standard methods APHA, (1999). The results of analyses were averaged out seasonally and statistical variables computed using WindowTM/Excel/2007. Analysis of bird community For the analysis of bird community, Shannon diversity index (Shannon & Wiener 1949). Statistical analysis The significant difference if any in the mean values of water quality parameters with species richness and bird number was performed by WindowTM/Excel/2007. Pearson’s correlation coefficients were calculated to evaluate the parametric relationships between the abiotic (i.e., physico-chemical parameters) and biotic factors (i.e., bird) supposedly in interaction. The tests were all two tailed and the correlations were tested at 5% and 1% level of significance. Correlation: The degree or the intensity of relationship between two variables was ascertained by computing the value of coeffient of correlation. Karl Pearson’s has given a formula for measuring correlation. The result of this formula varies between +1 and –1. The following chart shows degrees of correlation according to Karl Pearson’s formula, which is used time to time in different chapters.

I Degree of correlation I Positive Negative -,7 Perfect correlation +1 -1 - Very high degree of correlation +0.9 or more -0.9 or more - Fairly high degree of Correlation From +0.75 to +0.9 From -0.75 to -0.9 - Moderate degree of correlation From +0.50 to +0.75 From -0.50 to -0.75 - Low degree of correlation From +0.25 to +0.50 From -0.25 to -0.50 - Very low degree of correlation Less than +0.25 Less than -0.25 ' - I Absence of correlation I 0 l 0 _J Diversity Index: A diversity index is a mathematical measure of species diversity in a community. Diversity indices provide important information about rarity and commonness of species in a community. The ability to quantify diversity in this way is an important tool for biologists trying to understand community structure.

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 162 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Zoology Shannon diversity index: The Shannon diversity index (H) is commonly used to characterize species diversity in a community. Shannon's index accounts for both abundance and evenness of the species present. The proportion of species i relative to the total number of species (pi) is calculated, and then multiplied by the natural logarithm of this proportion (lnpi). The resulting product is summed across species, and multiplied by -1: ' JI' - Lt>~ln p: l:1

Where: H = the Shannon diversity index,Pi = fraction of the entire population made up of species i, S = numbers of species encountered, ∑ = sum from species 1 to species S. Interpretation: Typical values are generally between 1.5 and 3.5 in most ecological studies, and the index is rarely greater than 4. The Shannon index increases as both the richness and the evenness of the community increase. The fact that the index incorporates both components of biodiversity can be seen as both a strength and a weakness. It is a strength because it provides a simple, synthetic summary, but it is a weakness because it makes it difficult to compare communities that differ greatly in richness. Simpson index Species richness as a measure on its own takes no account of the number of individuals of each species present. It gives equal weight to those species with very few individuals & those with many individuals. Thus, one daisy has much influence on the richness of the area as 1000 buttercups. A community dominated by one or two species is considered to be less diverse than one in which several different species have a similar different species have a similar abudance.Simpson’s index (D) is a measure of diversity, which takes into account both species richness & an evenness of abudance among the species present. In essence it measures the probability that two individuals randomly selected from an area will belong to the same species. The formula for calculating D is Presented as : D = 휀푛푖(푛푖 − 1) N(N-1) Where, ni = total number of organisms of each individual species N = total number of organisms of all species. The value of D ranged from 0 to 1. With this index, 0 represents infinite diversity & 1 represents no diversity. That is, the bigger the value the lower the diversity.This does not seem intuitive or logical, so some texts use derivations of the index, Such as the inverse (1/D) or the difference from 1 (1-D). The equation used here is the original equation as derived by Edward H. Simpson in 1949. To calculate Simpson’s index for a particular area, the area must be sampled. The number of individuals of each species must be noted. For example, the diversity of the ground flora in a woodland might be determined by sampling with random quadrats. The number of plant species in each quadrat, as well as the number of individuals of each species should be noted. There is no necessity to be able to identify all the species provided that they can be distinguished from each other, further, Percentage cover can be used to determine plant abundance but there must be consistency, either all by “number of individuals” or all by “Percentage cover”. Low species diversity suggests: Relatively few successful species in the habitat. The environment is quite stressful with relatively few ecological niches & only a few organisms really well adapted to that environment. Food webs which are relatively simple. Changes in the environment would probably have quite serious effects. High species diversity suggests: A greater number of successful species & a more stable ecosystem. More ecological niches are available & the environment is less likely to be hostile. Complex food webs Environmental change is less likely to be damaging to the ecosystem as a whole. Species biodiversity may be used to indicate the biological health of a particular habitat. However, care should be used in interpreting biodiversity measures. Some habitats are stressful & so few organisms are adapted for life there but those that do may well be unique or indeed rare. Such habitats are important even if there are little biodiversity. Nevertheless, if a habitat suddenly begins to loss its animal & plant type, ecologists become worried & search for causes. Alternatively, an increase in the biodiversity of an area may mean that corrective measures have been effective.

3. RESULTS AND DISCUSSION During present study, 30 species (table no.2) of migratory birds were observed in and around Lakhota Lake in year 2010. The variations in the bird density and the species richness are noted according to the seasonal changes. Density of Birds Maximum density of total birds (Table: 3) was observed in February and minimum in September with highly significant seasonal variations of the total birds. Registered birds were belong different order like as Pelecani- formes (6 Species), Charadriiformes (8 species), Gruiformes ( 2 species), Passeriformes (2 species ), www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 163 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Zoology Coracciiformes (2 species ), Suliformes (2 species ), Podicipediformes (2 species ), Anseriformes ( 5 species ) Apodiformes (2 species ) showed in diagram no.1. In this study period order Charadriiformes was dominated over rest of the bird’s population data give in diagram no.1.Population of birds gradually raised from September onwards and touched peak reached in February. Correlation An inverse relationship was found between physicochemical parameters and birds biomass. Birds showed the negative correlation with temperature (-0.632), Phosphate, (0.895) and DO (-0.621). However, a positive relationship was found with and pH (0.020), EC (0.741), TDS (0.742), TH (0.793), ALK (0.896), BOD (0.30) and CL (0.718) which indicated that total alkalinity and BOD were found very low. From the results it has been observed that the pond water is suitable for the growth of different biological parameters like algae, planktons, insect larva, fishes, molluscans etc. which ultimately attract birds towards pond and adjoining area. Represents in table no 6. Diversity indices The value of Simpson’s index varies between 0 and 1. Simpson index of recorded Migratory birds in the Lakhota Lake during year in year 2010 is 0.26 (Table No.4). High dominance value during winter reflects diversified resources in the habitat available for components of the community. High species diversity suggests A greater number of successful species & a more stable ecosystem. More ecological niches are available & the environment is less likely to be hostile. Complex food webs Environmental change is less likely to be damaging to the ecosystem as a whole. Species biodiversity may be used to indicate the biological health of a particular habitat. However, care should be used in interpreting biodiversity measures. An inverse relationship was found between dominance and species diversity of birds. The seasonal distribution pattern showed two peaks of Shannon diversity index, one in winter (1.66) and the other in autumn (1.54 ) (Table 5). A number of reasons including north-south migration, breeding, food availability and vegetation changes could be attributed to this pattern.

Table no.1 The mean, standard deviation and range of Physico–Chemical Parameter of Water of Lakhota Lake in year 2010)

PARAMETERS MEAN SD MEAN ± SD Temp 27.3 3.72 27.3 ± 3.72 pH 7.57 0.23 7.57 + 0.23 EC 583.72 340.51 583.72 + 340.51 TDS 379.42 221.33 379.42 + 221.33 TH 180 98.07 180 + 98.07 Cl 140.83 42.95 140.83 + 42.95 ALK 132.5 24.17 132.5 + 24.17

PO4 - P mg/l 0.18 0.19 0.18 + 0.19 DO 6.82 0.46 6.82 + 0.46 BOD Mg/1 5.67 1.48 5.67 ± 1.48

Table No.2 List of migratory birds in the Lakhota Lake (Year in year 2010 )

Order Family Common name Scientific name Ardeidae Indian pond heron Ardeola grayii Ardeidae Great egrat Ardea alba Pelecaniformes Ardeidae Little egrat Egretta garzetta Pelecanidae Great white pelican Pelecanus onocrotalus Pelecanidae Dalmatian pelican Pelecanus crispus Threskiornithidae Spoon bill duck Platalea Scolopacidae Black tailed godwait Limosa limosa Laridae Heuglin’s gull Larus fuscus heuglini Recurvirostridae Avocet Recurvirostra Laridae Slender billed gull Chroicocephalus genei Charadriiformes Laridae Brown headed gull Chroicocephalus brunni- cephalus Laridae Yellow legged gull Larus michahellis www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 164 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Zoology Laridae Caspian tern Hydroprogne caspia Charadriidae Red watted lapwing Vanellus indicus Gruiformes Rallidae Common coot Fulica atra Rallidae Purple moorhen Porphyrio porphyrio Passeriformes Pycnonotidae Red vented bulbul Pycnonotus cafer Sturnidae Rosy starling Pastor roseus Coracciiformes Meropidae Green bee eater Merops orientalis Alcedinidae White throated kingfisher Halcyon smyrnensis Suliformes Anhingidae Darter Anhinga Phalacrocoracidae Indian cormorant Phalacrocorax fuscicollis Podicipediformes Podicipedidae Little grebes Tachybaptus ruficollis Podicipedidae Great crusted grebes Podiceps cristatus Anatidae Tufted duck Aythya fuligula Anatidae Common pochard Aythya ferina Anseriformes Anatidae Mallard Anas platyrhynchos Anatidae Knob billed duck Sarkidiornis melanotos Anatidae Spot billed duck Anas poecilorhyncha Apodiformes Apopidae Common swift Apus apus

Diagram no.1 Order wise species composition of water birds in Lakhota lake

Species 10 8 8 6 6 5 4 2 2 2 2 2 2 1 0 ~.... .~O~/ e" o<; e" e" e" e" e" e" ~ ~ ~ ~ ~ ~ ~ ~ .~o<; -~ .~O<; .~o<; .~0<; e; .~o<; .~O<; ~ ~ -rl f.." "&(}~ f.." ~ c,'f> f..'f> (:)'-:> ~e; 'l>c;~ ',~ ~e; q_O 'I?-~ 'l?-~o q_e-::.."' q_'l>' cl

■ Species

Table No.3 Average value of seasonal density of recorded Migratory birds in the Lakhota Lake in year 2010 )

Order Season O1 O2 O3 O4 O5 O6 O7 O8 O9 Total Sep 133 610 23 169 19 5 56 237 12 1264 Autumn Oct 142 634 29 188 22 7 64 249 19 1354 Nov 146 688 31 196 28 7 69 258 26 1449 Total 421 1932 83 553 69 19 189 744 57 4067 Dec 176 715 45 255 37 9 87 276 36 1636 Winter Jan 192 792 51 286 58 10 99 301 39 1828 Feb 196 802 57 294 60 10 112 326 45 1902 Total 556 2309 153 835 155 29 298 903 120 5366 Grand Total 977 4241 236 1388 224 48 487 1647 177 9433 Percentage % 10.35 44.95 2.50 14.71 2.58 0.50 5.16 17.45 1.87

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Contribution

■ O1 ■ O2 ■ O3 ■ O4 ■ O5 ■ O6 ■ O7 ■ O8 ■ O9

10% 17%

5% 3% 1%

15% 45%

Diagram No.3 Monthly variation in Migratory birds density in the Lakhota Lake (Year in year 2010 )

Density 1828 1902 1636 1449 1264 1354

Sep Oct Nov Dec Jan Feb

Table No. 4 Simpson index of recorded Migratory birds in the Lakhota Lake (Year in year 2010 ) Order N (ni-1) N(ni-1) En(ni-1) Pelecaniformes 977 976 977*976 953552 4241 4240 4241*4240 17981840 Charadriiformes Gruiformes 236 235 236*235 55460 Passeriformes 1388 1387 1388*1387 1925156 Coracciiformes 244 243 244*243 59292 Suliformes 48 47 48*47 2256 Podicipediformes 487 486 487*486 236682 Anseriformes 1647 1646 1647*1646 2710962 Apodiformes 177 176 177*176 31152 9445 23956352

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푒푛 (푛푖 − 1) 퐷 = 푁(푛 − 1) 23956352 퐷 = 9445(9445 − 1) 23956352 퐷 = 89198580

퐷 = 0.26

Table No.5 Shannon Diversity index of Migratory birds in the Lakhota Lake (Year in year 2010)

Season Diversity index Autumn 1.54 Winter 1.66

Table No.6 Correlation matrix between migratory birds and physico-chemical parameters of Lakhota Lake (Year in year 2010)

Parame- Den- ter sity Temp PH EC TDS TH CL ALK P DO BOD Density 1 Temp -0.632 1 PH 0.020 -0.356 1 EC 0.741 -0.251 -0.562 1 TDS 0.742 -0.243 -0.567 0.999 1 TH 0.793 -0.711 0.511 0.415 0.409 1 CL 0.718 -0.281 -0.518 0.991 0.989 0.456 1 ALK 0.896 -0.540 0.152 0.457 0.462 0.647 0.39 1 P -0.895 0.901 -0.179 -0.549 -0.545 -0.809 -0.54 -0.80 1 DO -0.621 0.169 0.656 -0.710 -0.717 -0.025 -0.62 -0.62 0.46 1 BOD 0.300 0.108 0.457 -0.192 -0.182 0.261 -0.26 0.61 -0.10 -0.08 1 -Correlation is significant at the 0.01 level (2-tailed). -Correlation is significant at the 0.05 level (2-tailed).

4. CONCLUSION Lakhota Lake was rich in diversity of birds. Registered migratory birds were belong to 30 species of different order like as as Pelecaniformes (6 Species), Charadriiformes (8 species), Gruiformes (2 species), Passeriformes (2 species), Coracciiformes (2 species), Suliformes (2 species), Podicipediformes (2 species), Anseriformes (5 species) Apodiformes (2 species). In this study period order Charadriiformes was dominated over rest of the bird’s population. Population of birds gradually raised from September onwards and touched peak reached in february. It was noticed that density of migratory birds was maximum in winter, minimum in autumn. Overall it is concluded that, the diversity and density of birds depends upon the nutrient condition of water body, biotic factor, abiotic factors and BOD.

5. ACKNOWLEDGEMENT The authors are thankful D.K.V Arts and Science College for providing administrative and infrastructural facilities for doing this work.

6. REFERENCES Anonymous (2003). A Manual on Water and Wastewater Analysis. One-Day Training Programme conducted by Gujarat Pollution Control Board (GPCB), Gandhinagar, Gujarat, India. APHA (1996). Standard methods for the examination of water and wastewater. 20th Edition. American Public Health Association, American water works Association Water Environment Federation. Bhat, M.M., T. Yazdani, K. Narain, M. Yunus and R.N. Shukla. (2009). Water quality status of some urban ponds of Lucknow, Uttar Pradesh. Journal of Wetlands Ecolog 2: 67-73. Bodegom, V., P. C. Bakker and H. D. van der Gon. (2004). Identifying key issues in environmental wetland research using scaling and uncertainty analysis. Regional Environmental Change 4: 100–06.

www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788192952130, Page No. 167 Proceedings of 10th National Science Symposium (February 11, 2018) Organized by Christ College, Rajkot & Sponsored by Gujarat Council on Science and Technology (GUJCOST), Govt. of Gujarat. Zoology Burkert U, Ginzel G, Babenzien HD, Koschel R (2004). The hydrogeology of a catchment area and an artificially divided dystrophic lake - consequences for the limnology of Lake Fuchskuhle. Biogeochemistry 71:225-246. Desai M, Shanbhag A (2007). Birds breeding in unmanaged Monoculture plantations in Goa, India. Indian Forester 133:1367-1372. Dunson, W. and J. Travis. (1991). The Role of Abiotic Factors in Community Organization. The American Naturalist 138: 1067-1091. M. C. Vachant, N. Karanthi and G. Sridharan, J. Ecobiol., 30 (1), 75(2012). Murphy S, Kessel MB, Vining LJ (1984). Waterfowl population and limnological characteristics of Taiga ponds. J. Wildl. Manage. 48(4):1156-1163. Newton I (1995). The contribution of some recent research on birds to ecological understanding. J. Anim. Ecol. 64:675-696. Patra A, Santra KB, Manna CK (2010). Relationship among the abundance of waterbird species diversity, macrophytes, macroinvertebrates and physico-chemical characteristics in Santragachi Jheel, howrah, W. B., India. Acta Zool. Bulg. 62(3):277-300. Paracuellos, M. (2006). How can habitat selection affect the use of a wetland complex by waterbirds? Biodi- versity and Conservation 15: 4569-4582. Ramchandra, T., A. Kiran, N. Ahylaya and R. S. Deepa. (2002). Status of wetlands of Bangalore. Trivedi, R. C. (1981). Use of diversity Index in evaluation of water quality. In: Proceedings of the WHO work- shop on biological indicators and indices of environmental pollution. Central Pollution Control Board, New Delhi. Pp. 175 – 188.

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A DIFFERENT CULTURE TECHNIQUES FOR MUD CRAB IN GUJARAT Dr. Bhatt Nakul A. 1*, Dr. B. K. Sharma2, Dr. D. T. Vaghela3 1Research scholar, college of fisheries, Maharana Pratap University of agriculture and technology, Udaipur-313001 2Profressor, College of fisheries, Maharana Pratap University of agriculture and technology, Udaipur-313001 3Profressor, College of fisheries, Junagadh Agriculture University, Veraval-365266 *Corresponding author: Email- [email protected] ABSTRACT: Mud crab may refer to crab that lives in or near mud. There are two species of mud crab Such as Scylla serrata and Scylla tranquebarica that are the focus of both commercial fisheries and aquaculture production throughout their distribution. Mud crabs occurring in the estuarine and mangrove areas along the coast of Gujarat. The size at first sexual maturity is 120 mm (carapace width) for larger species (Scylla tranquebarica) and 83 mm (carapace width) for Scylla Serrata. For culture purpose, crab seed are mainly collected from natural resources. Juvenile crabs can be collected from estuaries, lakes, backwaters, creeks, mangroves and saltwater lagoons by using bamboo traps, lift nets or scissor nets. A Hatchery has been set up at the Central Marine Fisheries Research Institute, Kochi for the commercial production of crab seed Seeds are available in commercial scale in Rajiv Gandhi Centre for Aquaculture (RGCA). Crab fattening is widely practiced in Thailand, Taiwan, Malaysia, Singapore and Indonesia.In Pond culture, the pond size depends on the production type. Pond size of 0.5 to 2 hectares is most suitable for crab culture. The maximum stocking density should be 1 crab/m2. Young crabs are raised and grown for a certain period of 5 to 6 months till they reach marketing size and weight. In Pond culture in a mangrove area, the ponds could be constructed as described above around the mangrove plants. Maximum pond area of 100 m2 is suitable for this type of culture. In Pen culture in pond Pens size of 4 X 4 X 2.5 m could be made inside the ponds using bamboo strips. Bamboo strips are driven 1-1.5 m deep into the soil to prevent the escape of the crabs by burrowing. In Pen culture in mangrove area, the pens could be constructed using the locally available bamboo splits or are canut logs or cane. Mangrove trees in the Centre of the pen provide shade for the crabs. The crabs could be harvested after 4–7 months. Cage culture Crab fattening can be carried out in Cell-type Cane Cages of 1 m (L) X 1 m (W) X 20 cm (H) size. Cages can be partitioned into nine equal compartments. One crab should be placed in each compartment of the cages. Generally, the mud crabs are sold in live condition.

INTRODUCTION Among the marine edible crustaceans, crabs occupy third position by virtue of its delicacy, demand and price. The first two being shrimps and lobsters. Scylla serrata and Scylla tranquebarica are the common mud crabs occurring in the estuarine and mangrove areas along the coast of India. Scylla serrata is commonly called as “red crab” and it prefers to live in low saline waters, whereas S. tranquebarica, the “green crab” lives in high saline waters [1]. Mud crabs stand first in the context of both capture and culture fisheries due to their larger size, great demand and higher price. Crab fattening is widely practiced in Thailand, Taiwan, Malaysia,

Scientific Name: Scylla tranquebarica Scientific Name: Scylla Serruta Common Name: Green Mud Crab Common Name: Red Mud Crab

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Singapore and Indonesia. Mud Crab is also known as Green Crab and Mangrove Crab A total of 3,500 tonnes (2,500 tonnes from brackish water and 1000 tonnes from marine region) of mud crabs are caught annually from India. Monoculture of mud crabs is being practiced in most of the Southeast Asian countries [2]. MATERIAL AND METHODS Different Cultivation Techniques of Mud Crab Site selection for Mud crab Culture: - Site selection is an important process in farming as it decides the success or failure of the mud crab farming. It greatly influences economic viability by determining capital outlay and affecting running costs, production and mortality [1]. Seed collection & Hatchery production of Mud crab: - Crab seed are available in the nature at all sizes. Juvenile crabs can be collected from estuaries, lakes, backwaters, creeks, mangroves and salt water lagoons by using bamboo traps, lift nets or scissor nets. A hatchery is being set up at the Central Marine Fisheries Research Institute, Kochi for the commercial production of crab seed [3,4,2]. Culture Method: - a) Pond Culture b) Pond culture in mangrove area c) Pen culture in pond d) Pen culture in mangrove area e) Cage culture (Suspended or Fixed type)

RESULTS a) Pond Culture: - Pond size of 0.5 to 1 ha.is most suitable for crab culture. However, large size ponds of more than one acre can also be used for this purpose. Sandy soils with a mixture of 50% clay are ideal for culture of these crabs. A water inlet system and an outlet system to drain out water during water exchange should be constructed as in the case of shrimps [7]. A fencing of nylon net used for fishing can be placed on the dike to prevent the escape of the crabs during night time. In addition, about 1000 numbers of stone ware, pipes of 6 inch diameter and 1.5 feet length, worn-out tyres, etc., should be kept at the bottom of the pond throughout the dike. b) Pond culture in mangrove area: - The ponds could be constructed as described above around the mangrove plants. But a maximum pond area of 100 m2 is suitable for this type of culture. A canal of 1m wide and 0.5m deep, in which water will be available even during low tide, should be dug around the edge of the pond [5]. Feeding depends on the availability of organisms namely low-value fishes, mangrove snails, clams, mussels etc. c) Pen culture in pond: - Several units of pens of 4 X 4 X 2.5 m could be made inside the ponds using bamboo strips which are driven 1-1.5 m deep into the soil to prevent the escape of the crabs by burrowing. The pens could be made nearer to the dykes for easy stocking and monitoring.

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d) Pen culture in mangrove area: - The pens could be constructed using the locally available bamboo splits or arecanut logs or cane. These strips should be driven 1-1.5 m deep into the soil to keep the crabs inside and the potential predators outside. The manageable area of the pen could be 100 to 150 m2. Within the pen, a ditch of about 0.3 to 0.9 m wide and 0.3 m deep should be dug. Mangrove trees in the Centre of the pen provide shade for the crabs [8].

e) Cage culture (Suspended or Fixed type): - Crab fattening can be carried out in Cell-type Cane Cages of 1 m (L) X 1 m (W) X 20 cm (H) size, which can be partitioned into nine equal compartments. Each of these cages should be provided with a lid to prevent the escape of crabs [6]. One crab should be placed in each compartment of the cages. In this method of fattening, higher number of crabs can be fattened in a square meter area, i.e. 9 crabs/m2.

Economic of Mud crab farming

Crab culture Crab Fattening

Scylla Serrata Scylla Serrata Species Scylla tranquebarica Scylla tranquebarica

Culture periods(days) 120 15-30

Pond Size 0.5-2 ha 0.1 ha

1 crab /sq.m (400 Nos of 550-600 1 to 3 crab/sq.m(8 cm carapace Stocking density gm. each) length)

Expenditure(Rs/-) (seed,feed,pond preparation,labour) 43,860 56,200

Production(tonnes) 0.78 0.56

Income(Rs/-) 1,57,200 1,22,850

Net profit/crop (Rs/-) 1,13,340 66,650

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CONCLUSION The ability of mud crab to grow fast and its suitability for culture in brackish water and estuarine areas make it attractive to develop mud crab culture/fattening programmes in Gujarat to meet the increasing demand. The major constraint faced by the crab farmers in Gujarat is the non-availability of stocking materials in sufficient quantity at the appropriate time. Seeds are mainly obtained from backwater fishing. Due to this reason, farmers are mostly concentrated in crab fattening than grow-out culture. Crab farming is mostly performed by the farmers having direct or indirect relation with fishing activities and thereby having accessibility to wild seeds. Until and unless hatchery production is established in the country for ensuring enough supply of seeds, mud crab farming will not reach sustainable development. Aquatic pollution and environmental problems that arise due to crab culture are negligible under optimum stocking density, when compared to penaeid shrimp culture and hence it is considered as environment friendly venture. More area can be brought under aquaculture without disturbing the environmental equilibrium of the ecosystem for enhancing foreign exchange earnings of the country. REFERENCE 1. Akpaniteaku, R. C. (2014). Assessment of the approach and potential of mud crab aquacultur. Global J. of Fisheries and Aquaculture, 2(3), 148-151. 2. Azra, M. N., & Ikhwanuddin, M. (2015). Larval culture and rearing techniques of commercially important crab, Portunus pelagicus (Linnaeus, 1758): Present status and future prospects. Songklanakarin Journal of Science & Technology, 37(2). 3. David, M. H. (2009). Mud crab (Scylla serrata) culture: understanding the technology in a silvofisheries perspective. Western Indian Ocean Journal of Marine Science, 8(1). 4. Mirera, D. O. (2014). Capture-based mud crab (Scylla serrata) aquaculture and artisanal fishery in East Africa-Practical and ecological perspectives: Mud crab ecology and aquaculturen (Doctoral dissertation, Linnaeus University Press). 5. Mirera, D. O., & Moksnes, P. O. (2015). Comparative performance of wild juvenile mud crab (Scylla serrata) in different culture systems in East Africa: effect of shelter, crab size and stocking density. Aquaculture international, 23(1), 155-173. 6. Mirera, D., & Mtile, A. (2009). A preliminary study on the response of mangrove mud crab (Scylla serrata) to different feed types under drive-in cage culture system. Journal of Ecology and The Natural Environment, 1(1), 007-014. 7. Sathiadhas, R., & Najmudeen, T. M. (2004). Economic evaluation of mud crab farming under different production systems in India. Aquaculture Economics & Management, 8(1-2), 99-110. 8. Waiho, K., Mustaqim, M., Fazhan, H., & Ikhwanuddin, M. (2013). Mud crab culture system and practice in Malaysia.