UTILIZATION OF INDUSTRIAL WASTE (CHEESE WHEY) FOR THE BIOSYNTHESIS OF β- GALACTOSIDASE
SYEDA UM-E-KALSOOM NAQVI Ph.D SCHOLAR
DEPARTMENT OF ENVIRONMENTAL SCIENCE LAHORE COLLEGE FOR WOMEN UNIVERSITY, LAHORE 2017
PhD THESIS
SYEDA UM
-
E
-
KALSOOM NAQVI
2017
UTILIZATION OF INDUSTRIAL WASTE (CHEESE WHEY) FOR THE BIOSYNTHESIS OF β– GALACTOSIDASE
A THESIS SUBMITTED TO LAHORE COLLEGE FOR WOMEN UNIVERSITY IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN ENVIRONMENTAL BIOTECHNOLOGY
By SYEDA UM-E-KALSOOM NAQVI
DEPARTMENT OF ENVIRONMENTAL SCIENCE LAHORE COLLEGE FOR WOMEN UNIVERSITY, LAHORE 2017
CERTIFICATE
This is to certify that the research work described in this thesis submitted by Ms. Syeda Um-e-Kalsoom Naqvi to Department of Environmental Science, Lahore College for Women University has been carried out under my direct supervision. I have personally gone through the raw data and certify the correctness and authenticity of all results reported herein. I further certify that thesis data have not been used in part or full, in a manuscript already submitted or in the process of submission in partial fulfillment of the award of any other degree from any other institution or home or abroad. I also certify that the enclosed manuscript has been prepared under my supervision and I endorse its evaluation for the award of PhD degree through the official procedure of University.
______Name Supervisor Date:
Verified By
______Name Chairperson Department of ______Stamp
______Controller of Examination Stamp Date: ______
DEDICATION This thesis is dedicated to my grandfather (Syed Zahur-ul-Hassan Naqvi), most beloved mother (Kaneez Sughra) and my very supportive and loving father (Syed Zain-ul-Aba Naqvi) for their unconditional love and prayers that motivates me to set highest targets in my life. I also dedicated my thesis to my husband (Syed Faisal Abbas) and my sons (Hussain Abbas and Nalain Abbas) for their love and moral support in my hard times. Also, this thesis is dedicated to my respected teachers for their unlimited kindness, scholarly guidance and moral support.
ACKNOWLEDGMENTS Up and above anything else, all gratitude and praises are due to ALMIGHTY ALLAH alone, the most Gracious, Merciful and Compassionate, the Creator of the universe, who enabled us to complete this work successfully. We offer our humble and sincerest words of praise to the Holy Prophet HAZRAT MUHAMMAD (S.A.W) and FAMILY OF HOLY PROPHET (S.A.W) who forever is a torch of knowledge and guidance for humanity. I am greatly indebted to worthy Vice Chancellor Prof. Dr. Uzma Qureshi, Lahore College for Women University, Lahore for providing me a bright chance to carry out this research work. I offer my profound thanks to Prof. Dr. Bushra Khan, Dean Faculty of Natural Sciences and Head of Chemistry Department, Lahore College for Women University, for providing me this opportunity to avail the research work. It is a great honor and pleasure for me to express my deep feelings of gratitude to my respected Supervisor Prof. Dr. Arifa Tahir, Head of Environmental Science Department, Lahore College for Women University, for her encouragement, advice and criticism throughout the course of this study. My thanks are due to Prof. Christopher, Department of Plant and Environmental Sciences University of Copenhagen Frederiksberg, Denmark, for the invitation and guidance to continue my research work in the University of Copenhagen, Denmark. I am extremely obliged to my foreign supervisor Associate Prof. Peter Stougaard, Department of Plant and Environmental Sciences University of Copenhagen Frederiksberg, Denmark, for his supervision throughout the experimental work and for his help and guidance in the interpretation of results. Furthermore, I would like to thank, in particular Ph.D Student Yanan Qin, Department of Plant and Environmental Sciences University of Copenhagen Frederiksberg, Denmark, for her help to design the experimental plan of work and for intriguing scientific discussions related to my project. I want to express my heartiest gratitude to Ulla, Dorte and Susanne for technical assistance and also thankful to Ph.D students, Mikkel, Morten, Benhosh and Raju, Department of Plant and Environmental Sciences University of Copenhagen Frederiksberg, Denmark, for helping me in scientific calculations and protocols. Highly obliged to Associate Prof. Mikkel Andreas Glaring, Department of Plant and Environmental Sciences University of Copenhagen Frederiksberg, Denmark, for
providing me the Antarctic samples and Witold for helping me in the interpretation of whole genome sequencing data. My greatest appreciation to Helle J. Martens, University of Copenhagen, Denmark for performing the transmission electron micrographs of strains NAQVI-58 and NAQVI-59T and Kjeld Pyrdol Nielsen, Process Technologist/ Process with responsibility for dairy and brewery, Institute of Food Science, IFV, University of Copenhagen, Denmark for the provision of Cheese Whey. I am extremely thankful to my father (Syed Zain-ul-Aba Naqvi) for his prayers, motivation, counseling and help to look after my kids during the course of my study, without his help it is almost impossible for me to complete my work. I would like to thank my husband (Syed Faisal Abbas) for his love, support, encouragement and motivation to complete my project at any cost. Extremely obliged to Uncle Riaz Shah and his family in Denmark for their help, support and loyalty towards my family which really helped me a lot to complete my project successfully.
KALSOOM NAQVI
CONTENTS
Title Page No.
List of Table i List of Figures ii List of Abbreviations x Abstract xi Chapter 1 : Introduction 1 Chapter 2: Review of Literature 10 Chapter 3: Materials and Methods 44 Chapter 4: Results 55 Chapter 5: Discussion 150 References 160 Annexures xiii Plagiarism Report ci List of Publications ciii
i
LIST OF TABLES
Table No Description/Title Page No 1. Showing the highest sequence similarity with other bacterial 109 strains. 2. Whole genome sequencing results showing the sequence 111 similarity with other bacterial strains. 3. Description of new Pararhizobium sp. 111 4. Morphological characteristics of selected bacterial isolates 123 (NAQVI-58 and NAQVI-59). 5. Distinctive features of strains NAQVI-58 and NAQVI-59 are 127 represented in the table. 6. Biochemical characteristics of strains NAQVI-58,NAQVI-59 136 and three reference strains. 7. Cellular fatty acid (%) composition of strains NAQVI-58 and 139 NAQVI-59.
ii
LIST OF FIGURES
Figure Description/Title Page No. No. 1 β-galactosidase producing bacterial strains from Antarctica on 56 R2 agar medium with X-gal.
2 Number of β-galactosidase producing bacterial colonies (turned 57 blue on X-gal) from Antarctica (Mc Murdo, USA) and Ikka columns (Greenland).
3 Isolation of β-galactosidase producing bacterial strains from 58 Antarctica on Marine agar without X-gal from Antarctic samples (1A, 2A, 3A, 4A and 5A).
4 Isolation of β- Isolation of β-galactosidase producing bacterial 59 strains on Marine agar without X-gal from Ikka column samples (1K, 2K, 3K, 4K, 5K, 6K, 7K, 9K and 10K).
5 Bacterial strains on Marine agar medium without X-gal 60 supplemented with cheese whey.
6 Bacterial strains on Marine agar medium supplemented with 62 cheese whey and X-gal (Plate 1A).
7 Bacterial strains on Marine agar medium supplemented with 63 cheese whey and X-gal (Plate 2A and 3A).
8 Bacterial strains on Marine agar medium supplemented with 64 cheese whey and X-gal (Plate 4A and 5A).
9 Number of β-galactosidase producing bacterial colonies from 65 Antarctica (Mc Murdo, USA) (Plate 1A).
10 Number of β-galactosidase producing bacterial colonies from 66 Antarctica (Mc Murdo, USA) (Plate 2A).
11 Number of β-galactosidase producing bacterial colonies from 67 Antarctica (Mc Murdo, USA) (Plate 3A).
12 Number of β-galactosidase producing bacterial colonies from 68 Antarctica (Mc Murdo, USA) (Plate 4A). iii
13 Number of β-galactosidase producing bacterial colonies from 69 Antarctica (Mc Murdo, USA) (Plate 5A).
14 β-galactosidase producing bacterial strains on Marine agar 70 medium with X-gal.
15 β-galactosidase producing bacterial strains from Antarctica 71 were categorized on the basis of their colour with X-gal on Marine agar medium.
16 Isolation of β-galactosidase producing bacterial strains from 73 Antarctic samples A1, A2, A3, A4 and A5 on R2 agar plates supplemented with cheese whey and X-gal.
17 β-galactosidase producing bacterial strains from Antarctica 74 were categorized on the basis of their size with X-gal on R2 agar medium.
18 Isolation of β-galactosidase producing bacterial strains from 75 Antarctic samples A1, A2, A3, A4 and A5 on R2 agar plates supplemented with cheese whey and X-gal.
19 Isolation of β-galactosidase producing bacterial strains from 76 Antarctic samples A1, A2, A3, A4 and A5 on Marine agar plates supplemented with cheese whey and X-gal.
20 β-galactosidase producing bacterial strains from Antarctica 77 were categorized on the basis of their size with X-gal on Marine agar medium.
21 Isolation of β-galactosidase producing bacterial strains on R2 80 agar medium with X-gal from Ikka samples I(1), I(2), I(3), I(4), I(5), I(6), I(7), I(8), I(9) and I(10).
22 Isolated colonies were purified again on new R2 agar medium 81 plates with X-gal.
23 β-galactosidase producing bacterial strains from Antarctica 82 were screened again on new R2 agar medium plates with X-gal.
iv
24 Isolated colonies are purified again on new R2 agar medium 84 plates with X-gal.
25 β-galactosidase producing bacterial strains from Antarctica are 85 purified again on new R2 agar medium plates with X-gal.
26 β-galactosidase producing bacterial strains from Antarctica 86 were categorized on the basis of their color on Marine agar medium with X-gal.
27 Isolated colonies are purified again on new R2 agar medium 87 plates with X-gal.
28 Isolated colonies are purified again on new Marine agar plates 88 with X-gal.
29 β-galactosidase producing bacterial strains were categorized on 90 the basis of their color on Marine agar medium with X-gal.
30 Isolated colonies are purified again on new Marine agar plates 91 with X-gal.
31 Isolated colonies are purified again on new Marine agar plates 92 with X-gal.
32 β-galactosidase producing bacterial strains were categorized on 93 the basis of their color on Marine agar medium with X-gal.
33 Isolated colonies are purified again on new Marine agar plates 94 with X-gal.
34 β-galactosidase producing bacterial strains (purified colonies) 97 obtain on R2 agar medium with X-gal.
35 Band patterns of diluted (10 times) and undiluted DNA 98 templates used for the amplification of PCR reaction. Strains 1- 12 were used as undiluted (1st column) and diluted (2nd column) form in the PCR reaction.
36 Diluted (10 times) and undiluted DNA templates were used for 99 the amplification of PCR reaction. Strains 12-24 were used as undiluted (1st column) and diluted (2nd column) form in the PCR reaction, band patterns were shown in the figure.
v
37 Diluted (10 times) and undiluted DNA templates were used for 100 the amplification of PCR reaction. Strains 24-36 were used as undiluted (1st column) and diluted (2nd column) form in the PCR reaction, band patterns were shown in the figure.
38 Diluted (10 times) and undiluted DNA templates were used for 103 the amplification of PCR reaction. Strains 37-48 were used as undiluted (1st column) and diluted (2nd column) form in the PCR reaction, band patterns were shown in the figure.
39 Diluted (10 times) and undiluted DNA templates were used for 104 the amplification of PCR reaction. Strains 49-60 were used as undiluted (1st column) and diluted (2nd column) form in the PCR reaction, band patterns were shown in figure.
40 Diluted (10 times) and undiluted DNA templates were used for 105 the amplification of PCR reaction. Strains 1-12 were used as undiluted (1st column) and diluted (2nd column) form in the PCR reaction, band patterns were shown in the figure.
41 β-galactosidase producing strains were selected for UP-PCR 106 reaction. Band patterns were used to differentiate between bacterial strains as shown in the figure.
42 β-galactosidase producing strains were selected for UP-PCR 107 reaction. Band patterns were used to differentiate between bacterial strains as shown in the figure.
43 Band patterns for different strains PCR reaction (amplification 108 of 16s rDNA) of 14 strains (1, 8, 9, 16, 19, 20, 21, 22, 38, 53, 55, 56, 58 and 59) as shown in the figure.
44 Distribution of genes on the basis of their function for strain 112 NAQVI-58 (5,309,041 bp) with contigs 162 and NAQVI-59 (5,457,546 bp) with contigs 161 were shown in the figure.
45 Figure showed the comparison of data between two organisms 115 (number of contigs, genes and length of reference organism (NAQVI-58) were compared with number of contigs and genes of test organism (NAQVI-59) respectively). vi
46 Gene cluster for strain NAQVI-58 and NAQVI-59 was the 116 same and contained (ABC transporter sugar binding protein, Glycerol-3-phosphate ABC transporter permease protein and β- galactosidase producing gene) with Ribose ABC transporter, oxidoreductase and β-galactosidase as shown in figure.
47 Maximum likelihood tree showing the phylogenetic relationship 117 between strains NAQVI 58 and NAQVI 59T and related species based on 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 500 replications) are given at the nodes. GenBank accession numbers are given in parantheses. Bar shows estimated nucleotide substitutions per site.
48 Maximum likelihood tree showing the phylogenetic relationship 118 between atpD sequences of strains NAQVI 58 and NAQVI 59 and related species. The tree is based on sequences of atpD shown in Annexure Table 16. Bootstrap values expressed as percentages of 1000 replications are shown next to the nodes. Bar indicates estimated substitutions per site.
49 Maximum likelihood tree showing the phylogenetic relationship 119 between recA sequences of strains NAQVI 58 and NAQVI 59 and related species. The tree is based on sequences of recA shown in Annexure Table 16. Bootstrap values expressed as percentages of 1000 replications are shown next to the nodes. Bar indicates estimated substitutions per site.
50 Maximum likelihood tree showing the phylogenetic relationship 120 between rpoB sequences of strains NAQVI 58 and NAQVI 59T and related species. The tree is based on sequences of rpoB shown in Annexure Table 16. Bootstrap values expressed as percentages of 1000 replications are shown next to the nodes. Bar indicates estimated substitutions per site.
vii
51 Maximum likelihood tree showing the phylogenetic relationship 121 between strains NAQVI 58 and NAQVI 59 and related species. The tree is based on concatenated sequences of atpD, recA, and rpoB. Bootstrap values expressed as percentages of 1000 replications are shown next to the nodes. Bar indicates estimated substitutions per site.
52 Growth of bacterial strain NAQVI-58 on YMA at pH 7 and 124 incubated at 28 ˚C for 3 days. Colonies were creamy white, circular, smooth and half transparent with diameter of 3 mm for NAQVI-58 as shown in the figure.
53 Growth of bacterial strain NAQVI-59 on YMA at pH 7 and 125 incubated at 28 ˚C for 3 days. Colonies were creamy white, circular, smooth and half transparent with diameter of 2 mm for NAQVI-59 as shown in the figure.
54 Strains NAQVI-58 and NAQVI-59 were inoculated in the 128 sterile semi solid agar (YMA) at pH 7 and incubated at 20 ˚C for 3 days. Growth were spread across the semi solid medium (0.5 % agar) and showed the motility of the strains.
55 Catalase activity for the strains NAQVI-58 and NAQVI-59 was 129
determined by the production of bubbles in 3 % (v/v) (H2O2) as shown in the figure.
56 Strains NAQVI-58 and NAQVI-59 were inoculated on YMA 130 (Yeast Mannitol Agar) at pH 7 and incubated at 20 ˚ for 3 days. Absolutely no growth was observed for both strains in an anaerobic jar as shown in the figure.
57 YMA (Yeast Mannitol Agar) plates at pH 7 were used to check 131 the susceptibility of antibiotics. Discs impregnated with 2 µl of
antibiotic, Kanamycin 25 mg/ml (MQ- H2O) were used and zone of growth inhibition was measured after incubating at 20 ˚C for 3 days. Both strains (NAQVI-58 and NAQVI-59) were sensitive to antibiotic Kanamycin (Km) as shown in the figure. viii
58 YMA plates at pH 7 were used to check the susceptibility of 132 antibiotics. Discs impregnated with 2 µl of antibiotic, Penicillin 1 mg/ml were used and zone of growth inhibition was measured after incubating at 20 ˚C for 3 days. Both strains (NAQVI-58 and NAQVI-59) were resistant to antibiotic Penicillin (Pen) as shown in the figure.
59 YMA plates at pH 7 were used to check the susceptibility of 133 antibiotics. Discs impregnated with 2 µl of antibiotic, Tetracyklin 10 mg/ml (70 % Et OH) were used and zone of growth inhibition was measured after incubating at 20 ˚C for 3 days. Both strains (NAQVI-58 and NAQVI-59) were sensitive to antibiotic Tetracyklin (Tet) as shown in the figure.
60 YMA plates at pH 7 were used to check the susceptibility of 134 antibiotics. Discs impregnated with 2 µl of antibiotic, Ampicilin 1000 mg/ml (70 % Et OH) were used and zone of growth inhibition was measured after incubating at 20 ˚C for 3 days. Both strains (NAQVI-58 and NAQVI-59) were resistant to antibiotic Ampicilin (Amp) as shown in the figure.
61 Biochemical tests were performed using API 20E kits for 137 strains NAQVI-58, NAQVI-59 and three reference strains (Pararhizobium giardinii H152T, Parahizobium herbae CCBAU 83011T and P. sphaerophysae CCNWGS 0238T).
62 Strains NAQVI-58, NAQVI-59 and three reference strains 140 (Pararhizobium giardinii H152T, Parahizobium herbae CCBAU 83011T and P. sphaerophysae CCNWGS 0238T) were grown on YMA at pH 7 and incubated at 28 ˚C for 3 days. Oxidase test was performed by using strips, change in color showed that strains were positive for Oxidase test.
ix
63 Two dimensional thin layer chromatography (TLC) of polar 141 lipids of strains NAQVI-58 and NAQVI-59. L, lipid; GL, glycolipid; AL, aminolipid; PL, phospholipid; PG, phosphatidylglycerol; PE, phosphatidylethanolamine and PME, phosphatidylmethanolamine.
64 Transmission electron microscopy for strains NAQVI-58 and 142 NAQVI-59 showed the size, shape and arrangement of cells. It showed 1 µm long rod shaped cells, mostly occurred in pairs as shown in the figure.
65 Effect of temperature on the production of β-galactosidase from 145 strains NAQVI-58 and NAQVI-59 respectively.
66 Effect of pH on the production of β-galactosidase from strains 146 NAQVI-58 and NAQVI-59 respectively.
67 Effect of Incubation time on the production of β-galactosidase 147 from strains NAQVI-58 and NAQVI-59 respectively.
68 Graph showed the maximum enzyme activity for strains 148 NAQVI-58 and NAQVI-59 at 37 ˚C while maximum enzyme activity for strain NAQVI-22 was observed at 28 ˚C as shown in the figure.
69 Graph showed the maximum enzyme activity for strain 149 NAQVI-58 and NAQVI-59 at pH 7 while maximum enzyme activity for strain NAQVI-22 was observed at pH 8 as shown in the figure.
x
LIST OF ABBREVIATIONS
Abbreviation Explanation ANI Average Nucleotide Identity BOD Biological Oxygen Demand CFU Colony Forming Unit COD Chemical Oxygen Demand DNA Deoxyribonucleic Acid EPA Environmental Protection Agency FAO Food and Agricultural Organization GOS Galacto-oligosaccharide GCC Galactose containing chemicals GDP Gross Domestic Product GEBA Genomic Encyclopedia of Bacteria & Archaea GRAS Generally regarded as safe IPTG Isopropyl β-D-1- thiogalactopyranoside LAB Lactic Acid Bacteria NCBI National Centre for Biotechnology ONP O-nitro Phenol ONPG O-Nitrophenyl-β-D- galactopyranoside PCR Polymerase Chain Reaction USDEC United States Dairy Export Council WGS Whole genome shotgun X-gal 5-bromo-4-chloro-3-indoxyl-β- D-galactopyranoside
xi
ABSTARCT
The dairy industry is associated with the production of contaminated waste water. The whey disposal remains a serious pollution problem for dairy industry, particularly in developing countries. Direct disposal of whey in the environment creates serious pollution problems, it destroys the physical and chemical structure of soil which decreases the crops yield and if discarded in water bodies, it reduces the aquatic life. The best solution to this environmental problem is the enzymatic hydrolysis of whey by using β-galactosidase which catalyses the hydrolysis of lactose (main constituent of whey) into its basic monomers, glucose and galactose. β-galactosidase can be obtained from different sources like plants, animals and microorganisms whereas bacterial β-galactosidase is generally regarded as safe.
The basic aim of present research is to investigate the utilization of dairy industrial waste (cheese whey) as a substrate for the biosynthesis of β-galactosidase to convert environmental waste into useful biomaterial from a noval β-galactosidase producing bacterial isolate from Antarctica. Two hundred and thirty five isolates were obtained from five samples (ice, water and microbial mats) collected from different sites of Antarctica and screened for their ability to produce β-galactosidase by using X-gal. A total of 61 bacterial isolates which turned blue on X-gal were then cultured in R2 medium and Marine medium aseptically at 10˚C for one month. The most potent bacterial isolates were identified using a polyphasic taxonomical approach. Cells were found strictly aerobic, Gram negative, rod shaped, motile and formed creamy white, half transparent colonies. Growth occurred at 4°C to 28°C with an optimum at 20°C, with 0 – 5.0 % (w/v) NaCl (optimum at 0 - 1.0 %) and at pH 4.0 – 11.0 (optimum at pH 7.0 - 9.0). The major fatty acid was C18:1 ω7c. Respiratory quinone was ubiquinone 10 (Q-10). The DNA G+C content was 60.7 %. The polar lipids were phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmethanolamine in addition to three unidentified lipids, one unknown glycolipid, and five unidentified phospholipids. Comparative analysis of 16S rRNA gene sequences showed highest sequence similarity (98.1 %) to Pararhizobium giardinii H152T, P. herbae CCBAU 83011T, and “P. polonicum” F5.1T. In silico average nucleotide identity (ANI) and genome-to-genome distance calculator (GGDC) showed 81.1 % identity (ANI) and 22.6 % identity (GGDC) to the closest relative, “P. polonicum” F5.1T. On the basis of phenotypic, phylogenetic, genomic and chemotaxonomic data, the two strains xii represent a novel species of the genus Pararhizobium, for which the name Pararhizobium antarcticum sp. nov. is proposed. The type strain is NAQVI 59T LSRP00000000 (=DSMZ 103442T = LMG29675T). Strains NAQVI-58 and NAQVI- 59T showed the highest enzyme production (0.21 U/ml) for strain NAQVI-58 and (0.33 U/ml) for strain NAQVI-59 with cheese whey as a substrate at pH (7), 28 ˚C and after 48 hours of incubation respectively. In this study, a new Pararhizobium sp. is discovered by using dairy industrial waste cheese whey as a substrate which is further used for the production of β-galactosidase.
1
INTRODUCTION
β-galactosidases are available in a wide assortment of living organisms including plants, animals and microorganisms, and are known to catalyze both hydrolytic and transglycosylation reactions (Asraf and Gunasekaran, 2010). This enzyme hydrolyzes lactose, the basic carbohydrate in milk, into galactose and glucose, which can be retained over the intestinal epithelium (Vasiljevic and Jelen, 2001; Troelsen, 2005; Heyman, 2006). β-galactosidase has two enzymatic actions, one is in charge of the hydrolysis of lactose and furthermore severs cellobiose, cellotriose, cellotetrose and to a specific degree cellulose and alternate, parts β-glycosides (Troelsen, 2005; Heyman, 2006; Elmira et al., 2010).
Low action of β-galactosidase causes stomach related deficiency, called lactose intolerance as a rule (Vasiljevic and Jelen, 2001; Karasova et al., 2002). The side effects of lactose intolerance, for example, stomach agony and loose bowels, queasiness, tooting, or potentially bloating after the ingestion of lactose or lactose containing nourishment substances which can prompt reduction personal satisfaction, and day by day activities. Treatment is moderately straightforward by disposing of lactose from the eating regimen or by utilizing of supplemental β-galactosidase enzyme substitution (Vasiljevic and Jelen, 2001; Elmira et al., 2010).
Milk sugar lactose is commercially manufactured in sustenance and pharmaceutical evaluations. The wide uses of lactose depend on some favourable dietary and innovative properties of it. Be that as it may, lactose may cause vital issues predominantly in three regions, wellbeing (lactose intolerance), sustenance innovation (unreasonable lactose crystallization bringing about the dairy items with a sandy or abrasive surface) and environment (a waste item from cheese whey production). The enzymatic hydrolysis of lactose by β-galactosidase offers a few answers to tackle these issues (Maity et al., 2013).
The enzyme β-galactosidase is a commercially significant compound since it not just catalyzes the hydrolysis of lactose into promptly hydrolysable sugars glucose and galactose, additionally the transglycosylation response also. For the reaction of hydrolysis, the lactose-diminished fixings in the diet and dairy items are monetarily delivered for lactose intolerant people (Rajakal et al., 2006). The catalyzed transglycosylation response is helpful for the creation of probiotic 2 galactooligosaccharide enhancing auxiliary and useful adjustment of sustenance materials or pharmaceutical compounds (Novalin et al., 2005).
Amid the most recent three decades, lactase has pulled in consideration of numerous analysts as it has a wide range of uses in dairy, confectionary, preparing and sodas ventures (Panesar et al., 2010; Maity et al., 2013). Contrasted with plant and animal sources, the microbial enzyme is delivered at higher yields and is more industrially significant (Nam et al., 2011; Kumari et al., 2011; Mozumder et al., 2012).
The transglycosylation action has been utilized for the production of galacto- oligosaccharide and galactose containing chemicals (GCC) lately (Akcan et al., 2011). So it is vital to choose a microorganism with high possibility to create galactosidase (Zuzana et al., 2006; Jayashree et al., 2012). Interestingly, lactic acid bacteria (LAB) are not favoured as a processing enhancer because of the powerlessness to shape spores and can't endure the acidic condition of the stomach and inevitability (Sreekumar et al., 2010). β-galactosidases delivered from bacteria are utilized for the treatment of milk, whey and other dairy results of neutral pH in light of the fact that the enzyme is active at pH (6.5-7) (Jayashree et al., 2012).
β-Galactosidases are additionally present in plants particularly in apricots, almonds, peaches, apples and animal organs (Nagy et al., 2001; Haider and Husain, 2007; Nurullah, 2011). Fungal β galactosidases are thermostable; be that as it may, they are more delicate to item hindrance fundamentally by galactose (Boon et al., 2000).
Economically accessible β-galactosidase is gotten from microorganisms of various genera specifically from Kluyveromyces, Candida, Aspergillus, Bacillus sp. what's more, E. coli (Thigiel and Deak 1989; Pinheiro et al., 2003; Panesar et al., 2006; Gopal et al., 2015). This enzyme has numerous industrial and restorative applications like cleavage of blood group A and B glycotopes, biosensors for lactose assurance and enzymatic hydrolysis of lactose (Asraf and Gunasekaran, 2010).
The bacterial species at present utilized by the dairy business which delivered β- galactosidase enzyme have a place with genera of Lactobacillus and Bifidobacterium (Fernandez et al., 1999; Xanthopoulos et al., 1999; He et al., 2008). These microscopic organisms are Generally Regarded As Safe (GRAS) so the β- galactosidase enzyme gotten from them may be utilized without broad refinement (Vasiljevic and Jelen, 2001). A few strains have probiotic activity, for example, 3 enhanced assimilation of lactose and an appropriate strain selection must be done to fabricate probiotic dairy items (Vinderola and Reinheimer, 2003; Elmira et al., 2010).
It is normal that the bacterial isolates from dairy emanating are more suitable for the production of β-galactosidase utilizing whey (Kumar et al., 2012; Natarajan et al., 2012; Princely et al., 2013; Arijit et al., 2016).
Properties, structure and specificity of β-galactosidase altogether depends on the microbial source of the enzyme, e. g. diverse atomic weight, amino acids chain length, position of the active site (Zhou et al., 2001). In spite of the fact that the most examined β-galactosidase is the one created by Escherichia coli, conceivable lethal components related with coliforms make it impossible that rough segregates of this enzyme will be allowed in sustenance forms (Santos et al., 1998; Zuzana et al., 2006).
In Aspergillus sp. β-galactosidase is emitted to the extracellular medium. These fungal enzymes have a pH ideal in the acidic range (2.5–5.4) and a high temperature maximum that permits their utilization at temperatures up to 50 ˚C (Zadow, 1984; Panesar et al., 2006). Their fundamental application is in the hydrolysis of acidic whey, which gets from the generation of new or delicate cheeses (Yang and Silva, 1995; Carla et al., 2011). Then again, in Kluyveromyces sp. the β-galactosidase is intracellular; lactose is first transported to the inside of the yeast cell by a permease and afterward hydrolyzed intracellularly to galactose and glucose, which take after the glycolytic pathway or the Leloir pathway, individually (Domingues et al., 2010).
The yeast enzyme has a close neutral maximum pH (6.0–7.0) and along these lines has a more extensive scope of utilizations, especially in the hydrolysis of sweet whey and milk (gotten from hard cheese fabricating) (Zadow, 1984; Yang and Silva, 1995; Panesar et al., 2006). In light of its intracellular nature, the enzyme should be separated from the yeast cells by disturbing or permeabilizing the cells utilizing substance or potentially mechanical medicines (Panesar et al., 2006). Expanded interest for β-galactosidase requires great financially savvy generation techniques to guarantee the monetary practicality of lactose hydrolysis at business scale (Nor et al., 2001; Manera et al., 2008).
The general cost of β-galactosidase production and downstream handling is the significant snag against the fruitful utilization of any innovation in the enzyme industry (Gupta et al., 2002; Nurullah, 2011). To meet the developing requests in the 4 industry it is important to enhance the execution of the framework and consequently increment the yield without expanding the cost of production (Gangadharan et al., 2008).
The dairy business is partitioned into a few areas, which are related to the generation of debased wastewaters. These effluents have diverse attributes, as per the item got (yogurt, cheese, spread, milk, frozen yogurt, and so forth.). In addition, the wastewater administration, atmosphere, working conditions and sorts of cleaning set up additionally impact the dairy effluents portrayal (Pattnaik et al., 2007). Cheese whey is the most tainted waste created in the generation of cheese (Rajeshwari et al., 2000; Ana et al., 2012).
The cheese generation is an exceptionally regular process the world over and it brings a lot of cheese whey, which speaks to a genuine ecological issue for its transfer. The maturation of cheese whey utilizing yeasts can be a practical procedure for the generation of proteins, as β-galactosidase (Manera et al., 2011; Brayam et al., 2013) and bio ethanol (Joshi et al., 2011).
Cheese whey portrayal relies upon the milk quality utilized (goat, bovine, sheep and wild ox), which may differ contingent upon animal breed, nourishment, wellbeing and lactation stage (Wit, 2001). Cheese whey can cause an abundance of oxygen utilization, impermeabilization, eutrophication, poisonous quality, and so forth in the accepting environments. The volume of effluents created in the cheese fabricating industry has expanded with the expansion in cheese generation. Around the world, 40.7-106 tons for every time of cheese whey are delivered, half of which is created in USA (Tejayadi and Cheryan, 1995; Ana et al., 2012). Cheese whey is a green- yellowish fluid coming about because of the precipitation and expulsion of milk casein in cheese making forms (Siso, 1996; Ana et al., 2012).
The yellowish shade of whey is caused by riboflavin (vitamin B2) (Wit, 2001; Ana et al., 2012). Most of the milk lactose, around 39-60 kg, stays in the cheese whey, constituting the primary portion (90 %) of the natural load (Kisaalita et al., 1990; Ghaly and Kamal, 2004). Other than milk, the principle resource of lactose is cheese whey, which is an essential by product of cheese manufacturing. Around 9 L of whey stream are created amid the creation of 1 kg of cheese, adding up to more than 160 million tons of whey delivered worldwide every year (Guimarães et al., 2010). 5
Whey's natural load is high (Biological Oxygen Demand of 30–50 g/L and Chemical Oxygen Demand of 60–80 g/L), basically on account of the lactose content, which together with the high volumes to which it is produced makes cheese whey a very concerning ecological issue, and answers for its valorisation are unequivocally required (Carla et al., 2011).
The transfer of whey remains a significant issue for dairy industry, particularly in creating nations where a moderately unimportant part of whey is utilized for generation of whey protein focuses or penetrates. A noteworthy piece of whey is arranged into the water streams, causing genuine water contamination issues emerging from high Biological Oxygen Demand (BOD) mostly in light of the presence of 5 % broke down solids, of which lactose is the fundamental constituent (Marwaha and Kennedy 1988; Gopal et al., 2015). One option is the utilization of whey and other agro-modern squanders as the essential medium for different maturation forms including the creation of commercially important enzymes, ethanol, methane, yeast protein, xanthan gum and different natural acids (Siso 1996; Pandey et al., 1999; Gopal et al., 2015).
The greater part of the world's cheese whey is arranged off as a waste. Direct transfer of whey causes genuine contamination issues, it demolishes the synthetic and physical structure of soil which diminishes the yield of harvests and when disposed of into water it diminishes the oceanic life (Gonzalez, 2007).
Previously, a large portion of the cheese plants arranged their effluents via land application or direct release to accepting waters (streams, lakes, sea, and so on.) with no pre-treatment. Different less sensational arrangements examined the development of capacity tanks/tidal ponds, the release into the civil sewage framework or even animal feeding. By and by, the utilization of concentrated cheese whey may include some critical downsides. Utilization of dairy effluents in cultivating hones has continually been lessened (Malaspina et al., 1996; Ana et al., 2012). The weakening of cheese effluents is an option that considers the blending of cheese whey with less contaminated waste waters like household wastewater (Minhalma et al., 2007; Gannoun et al., 2008).
Be that as it may, even weakened effluents may disable the proficiency and solidness of microorganisms in organic procedures done in metropolitan wastewater treatment 6 plants. Whatever the case, these choices are not adequately alluring, particularly for little medium processing plants, the cheese emanating administration turning into a vital test because of strict lawful necessities (Mawson, 1994; Farizoglu et al., 2007). Three unique choices in cheese profluent administration can be considered. The first depends on the utilization of valorization techniques. These advances are acquainted with recoup significant compounds, for example, proteins and lactose. Cheese whey contains around 50 g/L of lactose and 10 g of proteins with a high wholesome and useful esteem (Domingues et al., 1999; Ana et al., 2012).
Lactose substance of whey achieves 4.8 %, and it incorporates moderately abnormal amounts of different supplements that make it reasonable as a microbial culture medium. Microorganisms equipped for utilizing lactose as the sole carbon and vitality source are makers of β-D-galactosidase (Maria et al., 1985; Swati et al., 2012).
Lactases were first proposed for dairy applications in 1950 (Warffemius and Schermerhorn, 1950). Lactose hydrolysed milk and dairy items have been a work in progress since the 1970s, when the first β-galactosidases turned out to be commercially accessible. These days lactase is a standout amongst the most critical enzymes utilized as a part of sustenance preparing (Panesar et al., 2006). The procedure of lactose hydrolysis is straightforward and does not require uncommon gear in dairy plants (Zadow, 1986; Harju et al., 2012).
The streamlining of fermentation conditions, especially physical and synthetic parameters, are critical in the improvement of fermentation procedures because of their effect on the economy and practicability of the procedure (Francis et al., 2003). The development and enzyme production of the organism are emphatically affected by medium structure consequently streamlining of media components and cultural parameters is the essential errand in a natural procedure (Dakhmouche et al., 2006). Choice of suitable carbon and nitrogen sources or different supplements is a standout amongst the most basic stages in the improvement of a productive and monetary process (Konsoula and Kyriakides, 2007).
Two sorts of β-galactosidases are of expanding centrality in modern handling, thermostable and cold active (Pivarnik et al., 1995; Zuzana et al., 2006). Their utilization gives various points of interest. Utilization of thermostable enzymes at high temperatures is associated with diminished viscosities of the substrate arrangement 7 and with a lessening of undesired microbial defilement (Wolosowska et al., 2004). Cold active enzymes give treatment of milk and dairy products under mellow conditions so that taste and dietary esteems stay unaltered (Fernandes et al., 2004).
Marine conditions can have tremendous microbial biodiversity and thusly potential for the disclosure of exploitable biotechnological assets. Truth be told, an extensive variety of enzymatic exercises have as of now been gotten from refined marine microorganisms (Kennedy et al., 2008; Hector et al., 2015). Moreover, marine microorganisms can be found as intracellular or extracellular symbionts in marine creatures, for example, vertebrates or spineless creatures, where must have a wide gamma of enzymes for to the prerequisites of the host living beings (Trincone, 2011).
The enthusiasm for the differences of marine microorganisms and the request of biocatalysts adjusted to extraordinary conditions (low or high temperatures, acidic or basic arrangements, and high salt content) have expanded in the business (Alvarenga et al., 2011). These days, many research organizations and organizations have built up accumulations of living beings from an assortment of normal and outrageous situations (e.g., soils, seawater, hot springs, Antarctic ice, and antacid lakes), yielding an assortment of enzymes that catalyze responses under typical or extraordinary conditions (Lee et al., 2010; Hector et al., 2015).
Bacterial diversity is sorted out into discrete phenotypic and hereditary bunches, which are isolated by huge phenotypic and hereditary crevices, and these groups are perceived as species (Fred, 2002; William et al., 2006). The 16S rRNA sequences have been generally utilized as a part of environmental microbiology and have been significant for revealing the tremendous differing qualities of microbial life (De Long and Pace, 2001; William et al., 2006), and for relegating unculturable organisms as new species (Hugenholtz et al., 1998; Garrity et al., 2002; William et al., 2006).
Phylogenetic deliberate analysis in light of 16S rDNA sequences have been broadly used to describe groups of microorganisms found in an assortment of marine environments (Hagström et al., 2002) and have explained novel connections between obscure microorganisms and existing bacterial taxa, e.g., Proteobacteria and Cytophaga-Flexibacter-Bacteroides (Junge et al., 2002; Hector et al., 2015).
Two strains with 16S rRNA sequences that are under 97 % indistinguishable are along these lines doled out with high certainty to various species, however DNA– 8
DNA hybridization is as yet required to set up whether strains that have 97 % or more 16S rRNA likeness ought to or ought not be put in similar species (Vandamme et al., 1996; William et al., 2006).
The innovation of PCR and mechanized DNA sequencing, and following work on 16S rDNA sequencing of bacteria, and additionally 18S rDNA sequencing of eukaryotes, has prompted the collection of a huge measure of sequence data on the rDNA gene sequencing of the littler subunit of the ribosomes in countless life forms. Study of these sequences has demonstrated that the rDNA gene sequences are very rationed inside organisms of similar genera and species, yet that they contrast between life forms of other genera and species.
Utilizing these rDNA gene sequences for phylogenetic investigations, three spaces of life, Archaea, Bacteria and Eukarya, rather than the conventional characterization of living beings into prokaryotes and eukaryotes just, were depicted (Woese et al., 1990; Woo et al., 2008). Two decades have gone since the first bacterial genome was totally sequenced (Fleischmann et al., 1995; Fraser et al., 1995; Miriam et al., 2015).
As of late, the examination of different protein-encoding housekeeping genes has turned into a widely applied tool for the examination of taxonomic relationships (Wertz et al., 2003; Adekambi and Drancourt, 2004; Christensen et al., 2004; Holmes et al., 2004; Naser et al., 2005; Thompson et al., 2005; Miet et al., 2008). The house- keeping genes are utilized for this reason as they comparatively evolve slowly (however more quickly than 16S rRNA sequencing) and the majority of the variety that aggregates in these genes is thought to be specifically nonpartisan. Moreover, house-keeping genes encode items that are probably going to be fundamental to the bacteria and thus are relied upon to be available in all strains of a genus (William et al., 2006).
For a long time, ribosomal RNA operons, particularly the 16S rRNA genes, were utilized as the essential instrument for taxonomic assignment and phylogenetic trees (Mizrahi et al., 2013; Miriam et al., 2015). Full genome sequencing alongside extra instruments can thoroughly dissect and group hundreds or thousands of genomes. These new instruments have prompted new understandings of hereditary connections that the 16S rRNA gene just approximates. A striking improvement in the second 9 decade of bacterial genome sequencing was the era of metagenomic information, which covers all DNA display in a given specimen (Mende et al., 2012).
The investigation of metagenomes was so new in the last survey that the term should have been characterized, as around then there were just two metagenomic projects available. Now a days, greater than twenty thousands metagenomic projects are publically accessible. It was seen that ten years back, the assorted qualities of bacteria keeps on growing and astonishment (Lagesen et al., 2010).
AIMS AND OBJECTS
Isolation and screening of β-galactosidase producing bacterial strains.
Utilization of cheese whey as a substrate for the biosynthesis of β- galactosidase.
To inspect the growth of organism and production of enzyme, fermentation parameters like pH, temperature and incubation time will be optimized.
To change over natural waste into helpful biomaterial.
To diminish natural issues of dairy industrial waste.
10
LITERATURE REVIEW
β-galactosidase is a commercially essential catalyst that catalyzes the hydrolysis of lactose into its constituent monosaccharides glucose and galactose.
Presentation of hydrolysis of lactose by β-galactosidase. (Prescott, et al., 1990). β-galactosidase has a potential significance in the dairy business (Voget et al., 1994; Montanari et al., 2000; Domingues et al., 2005). This compound is broadly distributed in nature, found in various plants, creatures and microorganisms including yeast, parasites and microscopic organisms. The protein from a few sources have been all around portrayed, particularly the enzyme from Escherichia coli serves a model for the comprehension of the activity of the catalyst (Jacobson, 1994).
β-galactosidase compound is utilized as a part of dairy industry and is delivered by most lactobacilli (Karasova et al., 2002; Corral et al., 2006; Nguyen et al., 2007). The thermostable β-galactosidase from Aspergillus niger, Bacillus stearothermophilus, Pyrococcus woesei, Thermus sp. are generally steady from 35–80 ˚C (Asraf and Gunasekaran, 2010).
Numerous life forms have been known as high β-galactosidase maker for business utilize. In spite of the fact that yeast (intracellular), parasites or molds (extracellular) enzymes are known to deliver β-galactosidase (Gekas and Lopez, 1985), bacterial sources have increased more significance and they are ideal because of simplicity of maturation, high exercises of catalyst and great solidness. The monetarily misused wellsprings of β-galactosidase have been of microbial birthplace. Bacterial β- galactosidases are described by impartial pH optima also.
They are differing in their ideal temperature with variety amongst microscopic organisms and even between strains of same microbes. Various microbes have been considered as potential β-galactosidase sources, for example, L. lactis, L. acidophilus, L. bulgaricus (Cover and Jelen, 2000; Vasiljevic and Jelen, 2001; Gueimonde et al., 2002; Akolkar et al., 2005). 11
Thermophilic sources have been found to create thermo stable β-galactosidase. S. thermophilus β-galactosidase has an ideal temperature of 55 °C (Greenberg and Mahoney, 1982). Lactobacilli strains are usually utilized as a part of the business as probiotic. It is notable that β-galactosidase from lactic acid bacteria is an intracellular catalyst, and it is not discharged to the outside of cells under ordinary fermentation conditions (Cover et al., 2001).
Lactobacillus delbrueckii sub sp. bulgaricus 11842, utilized as a part of the creation of yogurt is fit for delivering moderately elevated amounts of intracellular β- galactosidase in contrast with other dairy societies (Cover and Jelen, 2000). There is a positive requirement for β-galactosidase that is steady at high and low temperatures and could be affirmed as GRAS for hydrolysis of lactose in drain and other dairy items (Kim and Rajogopal, 2000).
An examination was directed by Ariosvana et al. (2013) on the generation of β- galactosidase by various strains of Kluyveromyces, utilizing lactose as a carbon source. The most extreme enzymatic action of 3.8 ± 0.2 U/mL was accomplished by utilizing Kluyveromyces lactis strain NRRL Y1564 after 28 h of maturation at 180 rpm and 30 ˚C. β-galactosidase was then immobilized onto chitosan and described in view of its ideal operation pH and temperature, its thermal stability and kinetic parameters, utilizing o-nitrophenyl β-d-galactopyranoside as substrate. The ideal pH for solvent β-galactosidase action was observed to be 6.5 while the ideal pH for immobilized β-galactosidase action was observed to be 7.0, while the ideal working temperatures were 50 ˚C and 37 ˚C, separately.
Rech et al. (1999) examined the usage of protein-hydrolyzed sweet cheese whey as a medium for the generation of β-galactosidase by the yeasts Kluyveromyces marxianus CBS 712 and CBS 6556. The conditions for development were resolved in shaking cultures. The best development happened at pH 5.5 and 37 ˚C. Strain CBS 6556 developed in cheese whey in nature, while strain CBS 712 required cheese whey supplemented with yeast remove. Every yeast was developed in a bioreactor under these conditions. The strains created comparable measures of beta-galactosidase. To streamline the procedure, strain CBS 6556 was developed in concentrated cheese whey, bringing about a higher beta-galactosidase generation. The β-galactosidase delivered by strain CBS 6556 created greatest action at 37 ˚C, and had low stability at 12 room temperature (30 ˚C) and additionally at a capacity temperature of 4 ˚C. At - 4 ˚C and - 18 ˚C, the enzyme kept up its action for more than 9 weeks (Rech et al., 1999).
Ayesha et al. (2016) taken a shot at different bacterial strains were confined and screened for β-galactosidase generation. The most extreme catalyst creating strain was recognized as Bacillus strain B-2 on the premise of morphological and biochemical attributes. In the wake of choosing the fitting development medium, distinctive maturation parameters were enhanced utilizing submerged fermentation methodology. It was watched that disconnected bacterial strain delivered most extreme β- galactosidase with 1 % lactose as a substrate after 48 h of incubation. The present discoveries show that β-galactosidase from Bacillus strain B-2 can be an important contender for various industrial applications.
Fungal enzymes have pH optima in the scope of 3 to 5. In this manner fungal enzymes are appropriate for preparing acidic whey and pervade. They have additionally generally high ideal temperature which is between 55-60 °C. At it is known, mix of low pH and high temperature debilitates microbial development.
Be that as it may, fungal β-galactosidase is not as unadulterated as a yeast source and it might contain different compounds, for example, protease, lipase or amylase. As an outcome of these confinements, fungal applications of β-galactosidase have been restricted to high acidic items and pharmaceutical arrangements (Mahoney, 1985).
Be that as it may, yeast β-galactosidase is portrayed by their impartial pH optima. Therefore, they are generally utilized as a part of the hydrolysis of lactose in milk (pH 6.6), sweet whey (pH 6.2). Milk additionally supplies the potassium and magnesium particles required for movement. Yeast β-galactosidase can be created in exceptional returns at moderately low costs and are seen as safe for use in sustenances. Be that as it may, the most essential normal for this compound is its low heat stability.
In the event that the temperature increments over 55 °C, the enzyme is inactivated quickly. To accomplish high transformations at these temperatures, with negligible oligosaccharide generation, high amounts of enzymes are required. This would expand the handling costs. Keeping in mind the end goal to maintain a strategic distance from these issues, hydrolysis is regularly done at 4-6 °C for 16 to 24 hours where microbial waste is limited (Mahoney, 1985). 13
The enzymes gotten from different microbial sources have distinctive properties, for example, protein chain length, and the position of the dynamic site. Be that as it may, it has been discovered as of late that β-galactosidase from various sources have a similar amino acid residue, glutamic acid, as their synergist site. Molecular weight of β-galactosidase shifts between life forms too. E. coli β-galactosidase has a sub-atomic weight of 116,353 kDa per monomer (around 540 kDa per particle). The impacts of mono and divalent cations have been very much archived (Greenberg and Mahoney, 1982; Garman et al., 1996; Kreft and Jelen, 2000; Vasiljevic and Jelen, 2002).
Divalent cations, for example, magnesium and manganese may improve the β- galactosidase action, though monovalent cations may have a positive or negative impact (Garman et al., 1996; Kreft and Jelen, 2000). In an examination performed by Garman et al., six types of lactic acid bacteria were utilized (Garman et al., 1996).
The rate of lactose hydrolysis by β-galactosidase from every species was improved by Mg+2 however the impact of K+ and Na+ varied from strain to strain. In another investigation, manganese was observed to be the best cation taken after by magnesium for the greatest β-galactosidase movement of Streptococcus thermophilus (Greenberg and Mahoney, 1982). Ca+2 was known as an inhibitor of β-galactosidase (Greenberg and Mahoney, 1982). Be that as it may, the greater part of the calcium in milk is bound to casein. As it is not free in arrangement, it doesn't repress β- galactosidase movement (Garman et al., 1996).
The wholesome estimation of lactose is constrained because of the way that a substantial segment, for example, 50 % of world's occupants does not have this compound and can't use lactose in this way creating lactose maldigestion or narrow mindedness (Furlan et al., 2000; Vasiljevic and Jelen, 2002). This however makes a potential market for the utilization of β-galactosidase. The offer of nourishment catalysts is 37 % of aggregate enzyme deals comparing to 720 million dollar. This esteem is required to increment 863 million dollar, expanding the interest for the revelation of new species, delivering enzymes, for example, β-galactosidase with novel attributes, which will be of incredible incentive to the compound business for various applications (Cortes et al., 2005).
Enzymatic hydrolysis of lactose affects absorption of sustenances containing lactose for lactose bigoted populace, and also conceivable mechanical and ecological points 14 of interest for modern applications (Linko et al., 1998; Jurado et al., 2002). These can be compressed, for example, enhancing the innovative and sensorial attributes of nourishments by expanding the dissolvability, giving more noteworthy sweetening force and arrangement of monosaccharides (Jurado et al., 2002).
Besides, the use of β-galactosidase is imperative in the change of cheese whey, a waste from dairy industry into various esteem included items (Linko et al., 1998). Despite the fact that β-galactosidase (lactase) has been found in various natural frameworks, microorganisms, for example, yeasts, bacteria and mold still remain the main hotspots for business purposes (Vasiljevic and Jelen, 2001).
Lactose prejudice, as it were powerlessness to hydrolyze lactose (a kind of sugar found in milk and other dairy items), is an issue pervasive in the greater part of total populace (Vasiljevic and Jelen, 2001). It is caused by the insufficiency of the β- galactosidase enzyme.
The nonappearance of β-galactosidase can be depicted as intrinsic, essential or auxiliary inadequacy. Inherent insufficiency is a greatly uncommon condition in which perceivable levels of β-galactosidase are missing during childbirth (Pray, 2000). Essential β-galactosidase inadequacy is hereditarily acquired, age related abatement in β-galactosidase action. It happens in early youth and advance through the life (Miller et al., 1995).
Be that as it may, auxiliary β-galactosidase insufficiency can happen at any age. It is a transient condition of β-galactosidase inadequacy because of the harm to the intestinal mucosa where β-galactosidase is created. This harm can be caused by a serious episode of gastroenteritis, hunger, uncontrolled coeliac illness, inflammatory bowel disease (IBS), malignancy or poisons (Savaiano and Levitt, 1987).
The fundamental side effects of lactose bigotry incorporate fart, bloating, loose bowels and stomach torment. The manifestations are caused by undigested lactose going from the small digestive system into the colon. In the colon, the microbes ordinarily show mature unabsorbed lactose creating short chain unsaturated fats and gasses (carbon dioxide and hydrogen). Gas creation may bring about fart, bloating and distension torment. Unabsorbed lactose additionally has an osmotic impact in the gastrointestinal tract, drawing liquid into the lumen and causing looseness of the bowels (Vresa et al., 2001). 15
As lactose intolerant individuals don't have capacity to produce β-galactosidase enzyme, the treatment of milk and its subsidiaries with β-galactosidase is required. Along these lines, items free of lactose or low lactose substance can be devoured with no issues by lactose bigoted individuals (Furlan et al., 2000). Yogurt is normally preferred endured over fresh milk items by lactose maldigestors.
This is on the grounds that the β-galactosidase in the live yogurt microscopic organisms can help lactose assimilation in the colon. Marteau et al. (2001) likewise abridged that lactose prejudiced individuals have better assimilation and resilience of the lactose contained in yogurt. Among β-galactosidase sources, bacterial sources are best because of simplicity of maturation, high catalyst exercises and great solidness (Vasiljevic and Jelen, 2001).
Lactose hydrolysis can be performed in two ways including acidic hydrolysis and enzymatic hydrolysis. To begin with method for lactose hydrolysis is acid hydrolysis. It is completed by a homogenous response in acidic arrangement or in a heterogeneous stage with ion exchange resin. Acid hydrolysis can be performed under brutal conditions. For instance; 80 % hydrolysis might be accomplished in three minutes at pH 1.2 and 150 °C (Gekas and Lopez, 1985).
In spite of the fact that this methodology is by all accounts straightforward, it has a few hindrances. Most imperative one is protein denaturation because of low pH and high temperature. It causes decrease in the capacity of the proteins. Hence, it keeps their utilizations in numerous items (Bury et al., 2001).
Also, the nearness of salts in whey causes deactivation of acid and requires a demineralization step. Different hindrances are offensive and off flavor arrangement. Because of numerous disadvantages of acid hydrolysis, enzymatic hydrolysis by β- galactosidase is the favored technique for lactose hydrolysis (Gekas and Lopez, 1985). Enzymatic hydrolysis of lactose is a standout amongst the most vital biotechnological forms in the nourishment business. Lactose hydrolysis instrument was clarified by utilizing the catalyst β-galactosidase gotten from Escherichia coli (Jacobson, 1994).
A twofold removal response instrument was proposed in which β-galactosidase framed and hydrolyzed a glycosyl compound middle of the road by means of carbonium particle galactosyl transition state (Wallfels and Malhrotra, 1961). In 16 writing, it was recommended that the dynamic site of β-galactosidase contains cysteine and histidine amino acids which work as proton contributor and proton acceptor, individually. Cysteine amino acid contains the sulphydryl amass gone about as proton benefactor and histidine deposits contains imidazole bunch gone about as nucleophile site to encourage cleavage of the glycosidic bond, separately, amid the enzymatic hydrolysis methodology (Mahoney, 1998; Zhou and Chen, 2001).
The second reaction happened is called galactosyl (transgalactosyl) response. In this reaction, β-galactosidase exchanged the galactosyl moiety from the middle to an acceptor containing a hydroxyl gathering (Mahoney, 1998). At the point when this acceptor is water, free galactose is produced by hydrolysis. However, under specific conditions, different sugars can go about as acceptors and offer ascent to oligosaccharide development (Mahoney, 1998).
Enzymatic hydrolysis of lactose is joined by galactosyl exchange to different sugars, there by delivering oligosaccharides. The sum and nature of the oligosaccharide development by transgalactosyl response depends principally on the compound source and the nature and concentration of the substrate. The yield of oligosaccharides can be expanded by utilizing higher substrate as well as by diminishing the water content (Mahoney, 1998).
As of late, examines recommended that oligosaccharide generation is valuable to human wellbeing. They have been added to newborn child recipe as potential "bifidus elements" to advance the development and the foundation of bifidobacteria in the digestive tract (Hsu et al., 2005). Additionally, other announced remedial advantages of oligosaccharide utilization incorporate diminished serum cholesterol levels, upgraded retention of dietary calcium and improved combination of B complex vitamins (Onishi et al., 1995).
Proposed mechanism of β-galactosidase for galactosyl transfer reaction (Richmond et al., 1981). 17
The level of polymerization of GOS can shift uniquely, going from 2 to 8 monomeric units. GOS are consequently mind boggling blends of various oligosaccharides, and the range of the oligosaccharides making up these blends emphatically relies upon the wellspring of the β-galactosidase utilized for the biocatalytic responses and also on the transformation conditions utilized as a part of their production. GOS can fill in as fermentable substrates for specific individuals from the gut microbiota, and have been found to tweak the colonic greenery by incitement of valuable microscopic organisms, for example, bifido microorganisms and lactobacilli, and restraint of less alluring microorganisms (Holzapfel et al., 2002, Rastall et al., 2005, Macfarlane et al., 2008; Barbara et al., 2016).
Barbara et al. (2016) detailed that β-Galactosidase from Streptococcus thermophilus was over communicated in a sustenance review living being, Lactobacillus plantarum WCFS1. Research center developments yielded 11,000 U of β-galactosidase action per liter of culture comparing to around 170 mg of compound. Rough cell free compound concentrates gotten by cell interruption and consequent evacuation of cell garbage demonstrated high soundness and were utilized for change of lactose in whey saturate.
The catalyst demonstrated high transgalactosylation action. When utilizing an underlying grouping of whey penetrate comparing to 205 g/L lactose, the most extreme yield of galacto oligosaccharides (GOS) gotten at 50 ˚C came to around 50 % of aggregate sugar at 90 % lactose change, implying that effective valorization of the whey lactose was acquired. GOS are of awesome enthusiasm for both human and animal nourishment; in this way, proficient change of lactose in whey into GOS utilizing an enzymatic approach won't just diminishing the ecological effect of whey transfer, additionally make extra esteem.
The milk sector alongside the meat preparing industry is the biggest segment of modern sustenance production. The preparing of raw milk into sustenance items, for example, cheese, yogurt, drain and whey powder, exceptionally particular dietary items like without lactose milk items and unique child nourishment and lactose and extraordinary proteins for pharmaceutical purposes, creates noteworthy amounts of fluid squanders (e.g. whey), which postures genuine contamination issues for the encompassing condition, since it's Chemical Oxygen Demand (COD) of 50 kg O2/ton (Jelen, 2003; Grazina et al., 2016). 18
Whey postures noteworthy difficulties to the dairy business' natural insurance systems. High generation of cheese whey and whey saturate and additionally their high ecological effect and dietary substance make them an essential subject for cautious valorization examines. Distinctive methods for whey valorization have been examined, both diminishing natural effect and investigating the potential outcomes of reusing supplements (Banaszewska et al., 2014; Barbara et al., 2016).
Archana et al. (2015) portrayed the world exchange of whey and whey items for 2014 was evaluated at 5.6 million tons, of which the significant item was whey powder (Sossna, 2014a). Cheese fabricating is anticipated to achieve 25.3 million metric tons by 2023 (OECD-FAO, 2014), which would bring about gigantic measures of accessible whey pervade. In 2013, the worldwide market for whey powder and proteins was assessed at $9.8 billion US and was determined to be worth $11.7 billion US (Sossna, 2014b). In this manner, discovering novel approaches to utilize whey saturate is critical. The utilization of whey saturate as an immediate lactose source has been executed in some dairy facilities; however, this requires broad handling, including demineralization and dewatering (Jelen, 2009; Archana et al., 2015).
Whey has been used for the creation of ethanol, exopolysaccharide and single cell protein by utilizing β-galactosidase from microorganisms like Aspergillus oryzae, Kluveromyces lactis, Kluveromyces marxianus, Lactobacillus delbrueckii sub sp. bulgaricus, Saccharomyces cerevisiae. Transglycosylation and transgalactosylation properties of β-galactosidase from A. niger, Bacillus megaterium, Beijerinckia indica, Bifidobacterium infantis, Bifidobacterium longum, Enterobacter cloacae, Geobacillus stearothermophilus, K. marxianus, Lactobacillus sp, Lactobacillus reuteri, Penicillium expansum have been used for creation of glucose, galactose, heteropolysaccharide, galacto-oligosaccharides (Hussain, 2010).
β-galactosidase based medicinal and modern applications incorporate treatment of lactose malsorption, generation of lactose hydrolysed milk. Microbial β- galactosidases have an unmistakable position as far as their part underway of different modernly significant items like biosensor, lactose hydrolyzed milk, ethanol (Asraf and Gunasekaran, 2010). Marrakchi et al. (2008) have built up a biosensor partner two particular enzymatic exercises, that of the β-galactosidase and that of the glucose oxidase, keeping in mind the end goal to apply it for the quantitative location of 19 lactose in coomercial samples of milk (Marrakchi et al., 2008; Asraf and Gunasekaran, 2010).
Cheng et al. (2006) have utilized Bacillus sp. for the production of low-content GOS from lactose that brought about the most noteworthy yield of trisaccharides and tetrasaccharides. GOS creation was enhanced by blending β-galactosidase with glucose oxidase. The low substance GOS syrups, created by β-galactosidase was subjected to the maturation by K. marxianus, where by glucose, galactose, lactose and different disaccharides were at a low level, bringing about up to 97 % and 98 % on a dry weight premise of high content GOS with the yields of 31 % and 32 %, separately (Cheng et al., 2006; Asraf and Gunasekaran, 2010).
Other than the hydrolysis of lactose β-galactosidases because of their transgalactosylation property are exceptionally helpful to deliver esteem included inferred results of lactose (Gosling et al., 2010). Particularly GOS are generally utilized as a part of the planning of soda pops, bread, sweeteners (low calorie) and confectioner's items. To deliver GOS by utilizing β-galactosidase is likewise looked into as of late (Park and Oh, 2010b)
Iqbal et al. (2010) have watched that a recombinant β-galactosidase from Lactobacillus plantarum has a high transgalactosylation action and was utilized for the blend of prebiotic GOS. Hence, β-galactosidase assumes a huge part underway of galacto-oligosaccharides that can be utilized as sustenance and nourish for people and animals individually (Iqbal et al., 2010; Asraf and Gunasekaran, 2010).
Lactose (disaccharide comprised of glucose and galactose, connected by β-(1-4)-O- glycosidic bound can be hydrolysed synthetically (acid hydrolysis) or by the utilization of enzymes. Contrasted with chemical hydrolysis, the enzymatic have a few advantages, no side-effects, no debasement of mixes in dairy items, no extra frightful flavours, scents and hues. Moreover, milk treated by the protein holds its unique nourishment esteem, particularly since glucose and galactose are not evacuated (Ladero et al., 2003; Zuzana and Michal, 2006).
Lactose is normally found in exceedingly amassed just in milk and milk items. Dairy animals' milk contains 4.5–5 % lactose, which is more than 33 % of the solid phase in milk, roughly 20 % in frozen yogurt and around 72 % in whey solids. The disaccharide performs critical natural capacities, for example, invigorating the 20 development of bifidobacteria and providing galactose, a fundamental supplement for the arrangement of galacto-oligosaccharides and cerebral galactolipids (Maldonado et al.,1998; Zuzana and Michal, 2006).
Appearance of lactase lack relies upon ethnic and racial gathering of populace. The vast majority of the Asians (over 90 %), the majority of Africans (80–100 %), Native Americans (over 90 %), Southern Europeans (over 80 %) are accounted for to be lactose intolerant. On the opposite side, genuine lactose prejudice is kept mostly to individuals whose inceptions lie in Northern Europe (Sweden, Great Britain, Holland, Germany, and so on under 5 %) or the Indian subcontinent and is because of "lactase industriousness" (Alm, 2003; Zuzana and Michal, 2006).
An assortment of lactose free dairy items and milk are accessible in the market that are created by utilizing the β-galactosidases during the time spent enzymatic hydrolysis (Zadow, 1984). The sweetness and dissolvability of lactose is less when contrasted with different sugars, for example, sucrose, fructose, glucose and galactose (Ganzle et al., 2008).
As indicated by the strategy utilized for the precipitation of casein, whey was isolated into two principle sorts. Whey which was delivered from delicate or new cheese like curds and cream was called acid whey having (pH-5) while whey which was acquired from matured/hard cheeses was called sweet whey (pH 6-7) (Yang and Silva, 1995; Siso, 1996).
There are a wide range of wellsprings of whey as there are different sorts of cheeses. Sweet whey (pH ≥ 5.8) that is acquired either from the fabricate of regular catalyst created cheeses (Cheddar, Edam) i.e., cheese whey or from the generation of (rennet) caseinates, i.e., rennet casein whey. Acid whey (pH < 5.0) acquired from the production of fresh acid cheeses and acid casein whey, gotten from the making of acid casein by fermentation of skimmed milk. Medium acid whey (pH 5.0 to 5.8), that is gotten from the generation of some new acid cheeses (Akbache et al., 2009; Zadow, 1994; Muhammad et al., 2013).
Sweet whey and acid whey are two primary sorts of fluid whey. Both begin from the assembling procedure of rennet casein or natural cheese. Fresh whey is dried to plan both sorts of sweet whey powder and acid whey powder. pH of acidic whey by definition has 5.1 or lower. Acid whey is a by product of the assembling procedure of 21 fermented cheeses, for example, cottage and cream cheese (Welderufael and Jauregui, 2010).
It contains all the first constituents of acid whey with the exception of the water. Milk was fermented to a pH of 4.6, and soon thereafter the casein coagulated and accelerated so acid whey was created. It is higher in mineral substance than sweet whey particularly calcium phosphate. It might be utilized as a part of nibble sustenances, solidified dishes and serving of mixed greens dressings. The protein substance of acid whey and sweet whey powder are like the scope of 11-14.5 % (USDEC, 2003; Muhammad et al., 2013).
Sweet whey is made by evacuating a significant bit of water from fresh sweet whey which is the whey isolated from the creation of renneted cheeses have a grayish to cream hued item (Muhammad et al., 2013). Acid whey powder has a fat substance of 0.5-1 % which is lesser than sweet whey powder (1-1.5 %). Both sweet whey and corrosive whey have an equivalent measure of protein, 11-13.5 %, however the lactose substance are more in sweet whey (63-75 %) (USDEC, 2003; Muhammad et al., 2013).
In the sustenance business sweet whey is most ordinarily utilized and its pH is 5.8-6.3 and titratable sharpness of 0.1 (Pouliot, 2008). There are some different sorts of fluid whey as demineralized whey and lessened lactose whey. Demineralized whey means lessened mineral whey which is acquired by the expulsion of some bit of minerals from the purified whey. There are different strategies to make demineralized whey, generally it was set up by division systems, for example, particle trade and should not surpass 7 % cinder content (USDEC, 2003).
The essential utilization of demineralised whey is its utilization in sustenance frameworks where mineral fixation and substance are significant. A few items in which it is being utilized are slim down nourishment details, baby edibles and arranged dried blends. Mineral concentrated whey additionally marked as lessened lactose whey, is a cream to dull cream hued product and is fabricated by drying the whey that has as of now been dealt with to evacuate a little part of the lactose content (Szajewska and Horvath, 2010).
The procedure of lactose hydrolysis is basic and does not require exceptional gear in dairy plants (Zadow, 1986). When utilizing a solitary utilize chemical for lactose 22 hydrolysis, a few variables must be considered. These incorporate substrate focus, pH of operation, greatest temperature and contact time admissible, enzyme activity and cost. A broad contact time at 35-45 ˚C might be required to decrease costs, yet with milk this as a rule brings about broad microbial development. On the other hand, overnight holding at refrigeration temperature might be utilized (Zadow, 1986).
In whey just about 85 to 95 % of the volume of milk is available which contains 55 % of the supplements of milk. The most over the top supplements which are available in the whey are lactose (4.5 to 0.5 % w/v), lipids (0.4 to 0.5 % w/v), mineral salts (8 to 10 % dried concentrate) and solvent proteins (0.6 to 0.85 w/v). Impressive measures of different parts are likewise present in the whey like citrus extract, lactic (0.05 % w/v), B aggregate vitamins and non-protein nitrogenous compounds, for example, urea and uric acid (Siso, 1996).
In days of yore there were diverse strategies for the transfer of whey, for example, it is arranged off into lakes, waterways and sea, toss them in the fields, pipe it in the caverns or use as creature bolster. Whey has an incredible COD and BOD esteems else it can likewise be arranged off in the tidal ponds for the procedure of oxidation or in the civil waste yet it will defile the framework and cause contamination issue (Smithers, 2008).
A large portion of the world's cheese whey is arranged off as a waste however now a few conceivable outcomes for the usage of cheese whey have been found. Coordinate transfer of it causes genuine natural contamination issues, it influences the concoction and physical structure of soil which diminishes the yield of products and when disposed of into water bodies diminishes amphibian life by expending the broke down oxygen (Marwaha and Kennedy, 1988; Gonzalez-Siso, 1996).
Dumping specifically to nature causes genuine contamination issues on account of its high natural substance. A few works has been done on the bioconversion of whey into helpful metabolites, for example, ethanol, single cell protein or catalysts as an answer for natural issues (Mawson 1994; Gonzales Siso 1996; Eliwa and El-Hofi, 2010).
It is evaluated that just about 50 % of the aggregate cheese whey which is delivered on the planet is prepared and changed over into many esteem included results of nourishment. Proportion of this appears to build as a result of the nonstop research for the use of whey alongside weight which is applied on casein and cheese producers 23 through extraordinary enactment identified with the transfer of whey as a contamination. Huge measure of whey is dried and changed over into cheese whey powders in the wake of preparing (Yang and Silva, 1995).
It is an exceptionally helpful item and littler sums can likewise be utilized as a part of nourishment items like wieners, pastry shop things, desserts and other inferred results of drain and so forth however the greater part of it fundamentally used to encourage animals (Siso, 1996).
In 2009, fitting conditions for the creation of β-galactosidase from whey saturate has been assessed (Jokar et al., 2009; Asraf and Gunasekaran, 2010). Whey usage by β- galactosidase diminishes the weight of water contamination and gives advantageous items like ethanol and protein concentrates. Innovative work in the β-galactosidase will address the issues confronted in the nourishment and united ventures that search for catalysts with novel properties like cool soundness and thermo dynamic.
Immobilization of beta-galactosidase will decrease the cost of production of nourishment items and consider reusage of the compound. Novel galactooligosaccharides creation by β-galactosidase will prepare for improvement of prebiotics that can be utilized as nourishment supplement (Asraf and Gunasekaran, 2010).
The decision of reasonable β-galactosidase source relies upon response states of lactose hydrolysis. For instance, dairy yeasts with a pH ideal 6.5–7 are periodically utilized for the hydrolysis of lactose in milk or sweet whey. Then again, the contagious β-galactosidases with ideal pH 3–5 are more suited for acidic whey hydrolysis (Harju, 1987; Help et al., 2000; Zuzana and Michal, 2006). The movement of various β-galactosidases additionally relies upon nearness of particles. The contagious β-galactosidases are dynamic without particles as cofactors, the yeast β- galactosidase segregated from Kluyveromyces lactis requires particles, for example, Mn2+, Na+, and β-galactosidase from Kluyveromyces fragilis particles, for example, Mn2+, Mg2+, K+ (Jurado et al., 2002). Despite what might be expected, Ca2+ and substantial metals hinder the enzyme activity of all β-galactosidases (Čurda et al., 2001).
The changing idea of dairy effluents makes the treatment a troublesome assignment. Without a proper treatment, these effluents posture genuine ecological perils (Rivas et 24 al., 2011). Organic and physicochemical procedures are typically proposed to manage dairy effluents (Kushwaha et al., 2010).
Frozen yogurt, spread, whey, and cheese generation effluents are the most critical wellsprings of natural tainting in the dairy business. Cheese fabricating is dependable of three fundamental sorts of effluents; Cheese whey (coming about because of cheese generation), Second cheese whey (coming about because of curds creation) and cheese whey wastewater (washing water that contains diverse parts of cheese whey as well as second cheese whey). Cheese effluents speak to a critical natural effect in the dairy business due to their physicochemical attributes, specifically, minerals, add up to suspended solids, pH, phosphorus, add up to Total Kjeldahl Nitrogen, natural load and so on.
The high estimation of natural issue is caused by the lactose, protein and fats substance. This natural issue is around 99 % biodegradable (Ergüder et al., 2001). As needs be, regular medications depend on natural procedures. However, when organic procedures are not completely controlled, lactose and the casein decay produces solid scents, pulls in creepy crawlies, and so on. (Rivas et al., 2010).
The Environmental Protection Act has set a few constraints ashore spreading as a way for whey transfer, which is a support to run over supplementary uses for whey and whey items (Outinen, 2010; Muhammad et al., 2013).
The dairy business is one of the principle wellsprings of mechanical profluent era in Europe (Demirel et al., 2005). This industry depends on the handling and assembling of raw milk into items, for example, yogurt, frozen yogurt, spread, cheese and different sorts of pastries by methods for various procedures, for example, sanitization, coagulation, filtration, centrifugation, chilling, and so on (Rivas et al., 2010; Fatima et al., 2013).
As per FAO, cheese is one of the fundamental agrarian items around the world. The European Union overwhelms its generation and utilization, trailed by the United States. Whatever kind of cheese (Mozzarella, Danish blue, Camembert, Brie, Feta, Gouda, Serpa, and so forth.), the making processing plants create effluents that speak to a critical ecological effect (Ghaly and Singh, 1989; Comeau et al., 1996; Siso, 1996; Berruga et al., 1997; Lee et al., 2003; Fatima et al., 2013). 25
Cheese whey can cause an overabundance of oxygen utilization, impermeabilization, eutrophication, poisonous quality, and so forth in the getting conditions. The volume of effluents delivered in the cheese manufacturing industry has expanded with the expansion in cheese generation. Around the world, 40.7-106 tons for every time of cheese whey are delivered, half of which is created in USA (Tejayadi and Cheryan, 1995). Most of the milk lactose, around 39-60 kg, stays in the cheese whey, constituting the primary part (90 %) of the natural load (Ghaly and Kamal, 2004).
Around 50 % of aggregate world cheese whey generation is dealt with and changed into different sustenance items, of which around 45 % is utilized straightforwardly in fluid frame, 30 % as powdered cheese whey, 15 % as lactose and de lactosed by items, and the rest as cheese whey protein concentrates. Overall yearly whey generation is assessed in the vicinity of 150 and 200 million tons, with an expansion rate of 2 % every year (Smithers, 2008). The world whey generation is more than 160 million tons for each year (assessed as 9-overlap the cheese creation), demonstrating a 1–2 % yearly development rate (OECD-FAO, 2008).
Customarily, living beings were grouped, as indicated by likenesses and contrasts in their phenotypic attributes. The examination of rDNA gene sequences is a vital point of interest in the investigation of the advancement and characterization of living beings. Be that as it may, objective ordered grouping by these strategies can be troublesome in view of varieties in phenotypic attributes. Three decades back, Carl Woese and others begun to dissect and grouping the 16S rDNA genes of different microscopic organisms, utilizing DNA sequencing, a cutting edge innovation around then, and utilized the arrangements for phylogenetic examinations (Woese et al., 1990; Charm et al., 2008).
The innovation of PCR and computerized DNA sequencing, and ensuing work on 16S rDNA sequencing of microbes, and 18S rDNA sequencing of eukaryotes, has prompted the gathering of a tremendous measure of succession information on the rDNA sequences of the littler subunit of the ribosomes in an extensive number of living beings. Correlation of these groupings has demonstrated that the rDNA gene arrangements are exceptionally rationed inside living life forms of similar class and species, yet that they contrast between life forms of other genera and species. 26
Utilizing these rDNA gene arrangements for phylogenetic examinations, three areas of life, Archaea, Microscopic organisms and Eukarya, instead of the conventional grouping of living beings into prokaryotes and eukaryotes just, were depicted. Two decades have gone since the main bacterial genome was totally sequenced (Fleischmann et al., 1995; Fraser et al., 1995; Miriam et al., 2015).
In the most recent decade, because of the far reaching utilization of PCR and DNA sequencing, 16S rDNA sequencing has assumed a significant part in the exact distinguishing proof of bacterial confines and the revelation of novel microscopic organisms. For bacterial identity, 16S rDNA sequencing is especially imperative on account of microorganisms with irregular phenotypic profiles, uncommon microscopic organisms, moderate developing microbes and uncultivable microorganisms.
These days, bacterial characterization includes systems to decide both phenotypic and genotypic qualities. Of the genotypic techniques, 16S rRNA gene sequencing and genomic DNA–DNA reassociation fill in as 'highest quality levels' for bacterial species assurance (Stackebrandt and Goebel, 1994). It has been watched that life forms with add up to genomic relatedness over 70 % (evaluated by DNA–DNA hybridization) share more than 97 % 16S rRNA quality grouping similitude (Stackebrandt and Goebel, 1994).
For a long time, ribosomal RNA (rRNA) operons, particularly the 16S rRNA genes, were utilized as the essential device for ordered task and phylogenetic trees (Mizrahi et al., 2013). Full genome sequencing alongside extra instruments can exhaustively break down and arrange hundreds or thousands of genomes. These new instruments have prompted new understandings of hereditary connections that the 16S rRNA gene just approximates.
An eminent improvement in the second decade of bacterial genome sequencing was the era of metagenomic information, which covers all DNA introduce in a given example (Mende et al., 2012). The investigation of metagenomes was so new in the last audit that the term should have been characterized, as around then there were just two metagenomic ventures distributed. Today, there are more than 20,000 metagenomic projects publically accessible (Lagesen et al., 2010). 27
Two strains with 16S rRNA arrangements that are under 97 % indistinguishable are along these lines appointed with high certainty to various species, yet DNA–DNA hybridization is as yet required to build up whether strains that have 97 % or more 16S rRNA likeness might to or might not be put in similar species (Vandamme et al., 1996).
The house-keeping genes are utilized for this reason as they develop generally gradually (however more quickly than 16S rRNA genes) and the greater part of the variety that collects in these genes is thought to be specifically impartial. Moreover, house-keeping genes encode items that are probably going to be fundamental to the microbes and subsequently are relied upon to be available in all strains of a sort (Miet et al., 2008).
Coenye et al. (2005) portrayed a few novel methodologies, e.g. examination of quality request, quality substance, nucleotide piece and codon use, to evaluate bacterial connections in view of entire genome groupings (Coenye et al., 2005).
Konstantinidis and Tiedje (2005) characterized the Average Nucleotide Identity (ANI) as the rate of the aggregate genomic sequence shared between two strains. The ANI was turned out to be a strong and delicate instrument for estimation of the hereditary relatedness between partnered bacterial strains (from strain to class level and potentially family level) (Konstantinidis and Tiedje, 2005; Konstantinidis et al., 2006). Cho and Tiedje (2001) built up a technique in view of arbitrary genome pieces and DNA microarray innovation that can be connected to the recognizable proof of microbes and in addition the assurance of the hereditary separation between microorganisms (Cho and Tiedje, 2001).
Species outline is by and by in light of hereditary relatedness utilizing DNA–DNA hybridization as an intermediary measure; strains that show around 70 % or more prominent DNA–DNA relatedness are considered to have a place with same species and those that have not as much as this esteem are distinctive species (Wayne et al., 1987; Miet et al., 2008).
Strains displaying more than 70 % DNA–DNA hybridization (or which have more than 94 % average nucleotide identity over all shared genes) have been appeared to be greatly comparative in their 16S rRNA gene sequences (Konstantinidis and Tiedje 2005). A few papers have as of late tended to whether the arrangements of different 28 genes can be utilized to recognize comparative species, to advise the division of a genus into species, or to ask whether bacterial species exist (Godoy et al., 2003; Minister et al., 2004; Baldwin et al., 2005; Hanage et al., 2005a, b; Thompson et al., 2005b).
Regardless of late advances in molecular biology and in the improvement of monetarily accessible phenotype-based identification kits, recognizing bacterial strains remains a troublesome errand for some routine microbiological labs. Among the few thousand genes inside a bacterial genome, the 16S rRNA gene has filled in as the essential key for phylogeny-based identification when thought about against all around curated 16S rRNA gene sequence databases (Rossello' and Amann, 2001; Tindall et al., 2010; Ok et al., 2012).
In spite of the fact that the EzTaxon database has been generally utilized for routine identification of prokaryotic isolates, sequence from uncultured prokaryotes have not been considered. Here, the cutting edge database, named EzTaxon-e, is formally presented. This new database covers species inside the formal nomenclatural framework as well as phylotypes that may speak to species in nature. Notwithstanding with Basic Local Alignment Search Tool (BLAST) searches and pairwise worldwide gene sequences, another target strategy for evaluating the level of culmination in sequencing is proposed.
All sequences that are held in the EzTaxon-e database have been subjected to phylogenetic examination and this has brought about a total various leveled characterization framework. It is inferred that the EzTaxon-e database gives a helpful ordered spine to the distinguishing proof of refined and uncultured prokaryotes and offers a significant methods for correspondence among microbiologists who routinely experience systematically novel strains (Ok et al., 2012)
Ten years prior, we looked into the primary decade of bacterial genome sequencing (Binnewies et al., 2006). Around then, there were around 300 sequenced bacterial genomes and just two distributed metagenomic ventures; this spoke to a development of more than 100-fold from the negligible two genomes sequenced in 1995. The quantity of sequenced genomes has kept on expanding drastically over the most recent 10 years becoming another hundredfold that is, there are more than 30,000 sequenced 29 bacterial genomes presently publically accessible in 2014 (NCBI 2014) and thousands of metagenome ventures (Miriam et al., 2015).
Ventures, for example, the Genomic Encyclopedia of Bacteria and Archaea (GEBA) (Kyrpides et al., 2014) guarantee to include more genomes as well as grow the hereditary assorted genes and add to the rundown of accessible sorts of strains. For a long time, ribosomal RNA (rRNA) operons, particularly the 16S rRNA qualities, were utilized as the essential device for ordered task and phylogenetic trees (Mizrahi et al., 2013).
The 16S rRNA gene is still generally utilized on the grounds that it is available in no less than one duplicate in each bacterial genome, its moderated locales empower straightforward specimen recognizable proof utilizing PCR, and its grouping gives dependable data on bacterial family, genus, or species as a rule. This single quality correlation is currently being supplanted by more far reaching approaches. Full genome sequencing alongside extra apparatuses can completely examine and order hundreds or thousands of genomes. These new apparatuses have prompted new understandings of hereditary connections that the 16S rRNA gene just approximates (Miriam et al., 2015).
A striking improvement in the second decade of bacterial genome sequencing was the era of metagenomic information, which covers all DNA display in a given specimen (Mende et al., 2012). As watched 10 years prior, the assorted qualities of microorganisms keeps on extending and amazement (Lagesen et al., 2010). Rather than 20 Escherichia coli genomes, we now have thousands that can be looked at (Cook and Ussery (2013), despite everything they give us new bits of knowledge into the differing qualities and pliancy of bacterial genomes.
The idea of information to be broke down is evolving. For instance, microarray examination of transcriptomes is being supplanted by RNA sequencing (Westermann et al., 2012; Zhao et al., 2014; Wang et al., 2014). In concurrence with past perceptions from investigations of a littler arrangement of living beings (Bentley and Parkhill, 2004; Bohlin et al., 2010; Karpinets et al., 2012), genomes of microbes from complex ecological natural surroundings tend to be bigger in estimate and have more noteworthy GC content than those of the host related microorganisms. The GC substance of the completed bacterial genomes ranges from somewhat under 15 % to 30 around 85 %. Albeit numerous microbes are mesophiles, there are a developing number of sequenced extremophiles, for example, thermotolerant, psychrotolerant, and psychrotrophic microscopic organisms (Miriam et al., 2015).
Life forms developing great at temperatures near the point of solidification of water yet having the quickest development rate over 20°C, have been depicted as psychrotolerants. Psychrophilic living beings can't become over 20°C and have a development ideal at 15°C or lower (Cavicchioli et al., 2002; Mikkel, 2012).
Low temperature is thought to have an impact on translation and transcription. At low temperatures there appear to be a more grounded collaboration between DNA strands which subsequently disable the loosening up of the helix and in this manner the binding of RNA polymerase. Likewise, an expansion in the arrangement of troublesome RNA auxiliary structures is seen when the temperature is brought down, which is probably going to affect translation (Feller and Gerday, 2003).
In mesophilic microscopic organisms such difficulties are overwhelmed by the combination of cold shock proteins. For instance a cold shock protein from E. coli, CsdA, was found to have helix-destabilizing capacities and proposed to encourage translation at low temperature by loosening up auxiliary structures in mRNAs (Jones et al., 1996; Mikkel, 2012).
As psychrotolerant and psychrophilic microscopic organisms inhabit continually low temperatures, they don't really encounter cold shock. In any case, it has been watched, that psychrotolerant microorganisms deliver an expanded measure of a few proteins for all time amid development at low temperature, and not at higher temperatures. These proteins called cold acclimation proteins (CAPs) are believed to be a general component of cold adopted living organisms (Berger et al., 1997; Feller and Gerday 2003).
Temperature is a component that has impact on most biochemical responses. Regularly low temperatures will back off the rate of responses catalyzed by enzymes. To keep the chemical action high at low temperatures psychrophiles are thought to have an upgraded adaptability of their tertiary protein structure (Feller and Gerday 1997; D'Amico et al., 2002).
Cold active enzymes are wanted for some industrial and biotechnological forms from heat labile enzymatic absorptions in genetic engineering, to cost-sparing clothing 31 cleansers, to sustenance handling at low temperature. The desire to enhance both cost- decreasing and sterile procedures has prompted a constantly developing enthusiasm for applying the enzymes from psychrophilic microorganisms, which can be found all through the planet, e.g. in the Cold locales, on high height mountains, and in the profound seas, where temperatures are only a couple of degrees over zero (Mariane and Dwindle, 2010).
The manufacturing of cold stable β-D-galactosidases and microorganisms that ingeniously ferment lactose is of high biotechnological intrigue, especially for expulsion of lactose in milk and dairy items at low temperatures, cheese whey bioremediation and bio-ethanol generation. As of late, a quality encoding β-D- galactosidase was segregated from the genomic library of Antarctic bacterium Arthrobacter sp. 32c. Despite the fact that, the most noteworthy action of this purified enzyme was found at 50 °C, 60 % of the most elevated activity of this enzyme was resolved at 25 °C and 15 % of the most noteworthy action was recognized at 0 °C (Hildebrandt et al., 2009; Asraf and Gunasekaran, 2010).
Ram et al. (2005) portrayed Antarctica is the coldest landmass on earth and harbors an assortment of microorganisms. One of the bacterial secludes from cynobacterial mats of Schirmacher Desert garden, portrayed as Bacillus sp. developed at 5-35 ˚C with an ideal at 25 ˚C, and created intracellular cold active β-galactosidase. The most extreme activity was recorded at pH 6.8 and 40 ˚C amid late stationary stage. At 5 ˚C, the enzyme held 39.7 % activity and at 60 ˚C turned out to be totally latent inside 15 minutes. The enzyme activity was invigorated by metal particles yet was restrained by ethylene diamine tetra acidic acid. Non denaturing polyacylamide partition taken after by in situ hydrolysis of 5-bromo-4-chloro-3-indolyl-β-galactopyanoside, recommended the presence of isozymes.
The cruel natural states of mainland Antarctica have molded soil ecosystems of low assorted qualities and basic trophic structure. In many ranges of the mainland, soil creatures confront serious conditions, including low water and supplement accessibility, to a great degree frosty temperatures, visit freeze–thaw cycles, times of delayed murkiness in winter, and presentation to large amounts of UV radiation in summer (Cary et al., 2010). 32
Ineffectively investigated territories like the Antarctic present critical potential for the disclosure of novel microorganisms like microscopic organisms and natural dynamic metabolites (Moncheva et al., 2002; Marinelli et al., 2004; Tindall, 2004; Taton et al., 2006; Bull and Stach, 2007; Lee et al., 2012).
Cold adopted and cold active β-galactosidase from psychrophilic microorganisms like Arthrobacter psychrolactophilus, Pseudoalteromonas haloplanktis are when all is said in done very proficient in remunerating the lessening of response rates by incited low temperatures through change of the turnover number (kcat) or of the physiological productivity (kcat/Km) and are moderately steady from 0 - 25 °C (Asraf and Gunasekaran, 2010).
Van et al. (2014) broke down the cold active β-galactosidase from the psychrophile Pseudoalteromonas haloplanktis. Ideal parameters for proficient lactose hydrolysis in whey penetrate have been reasoned, viz. ideal brooding temperature, pH and lactose fixation. Hydrolysis efficiencies over 96.0 % were acknowledged inside 24 h at 23 °C and pH 7.0 in whey saturate with a greatest dry issue substance of 10.0 % (w/w). Likewise, the operational soundness of the cold active β-galactosidase was examined. Hydrolysis efficiencies over 90.0 % were kept up amid 7 ensuing hydrolysis cycles.
Ghosh et al. (2012) revealed that the cold active β-galactosidase from psychrophilic microscopic organisms quicken the likelihood of outflanking the present business β- galactosidase production from mesophilic sources. The present examination was done to screen and disconnect a cold active β-galactosidase delivering bacterium from significant marine waters of Bay-of-Bengal and to improve the variables for lactose hydrolysis in milk. Isolated bacterium 3SC-21 was portrayed as marine psychrotolerant, halophile, gram negative, rod shaped strain delivering an intracellular cold active β-galactosidase. Further, in view of the 16S rRNA gene sequence, bacterium 3SC-21 was distinguished as Thalassospira sp. The secluded strain Thalassospira sp. 3SC-21 had demonstrated the enzyme activity in the vicinity of 4 and 20 °C at pH of 6.5 and the enzyme was totally inactivated at 45 °C.
The factual strategy, central composite rotatable design of response surface methdology was utilized to upgrade the hydrolysis of lactose and to uncover the communications between different elements behind this hydrolysis. It was discovered that greatest of 80.18 % of lactose in 8 ml of raw milk was hydrolysed at pH of 6.5 at 33
20 °C in contrast with 40 % of lactose hydrolysis at 40 °C, proposing that the cold active β-galactosidase from Thalassospira sp. 3SC-21 would be most appropriate for assembling the lactose free dairy items at low temperature.
Hoyoux et al. (2001) considered the β-galactosidase from the Antarctic gram-negative bacterium Pseudoalteromonas haloplanktis TAE 79 was sanitized to homogeneity.
The nucleotide sequence and the NH2-terminal amino acid sequence of purified enzyme demonstrate that the β-galactosidase subunit is made out of 1,038 amino acids. This β-galactosidase shares auxiliary properties with Escherichia coli β- galactosidase (tantamount subunit mass, 51 % amino sequence identity, protection of amino acid buildups required in catalysis, comparative ideal pH esteem, and prerequisite for divalent metal particles) however is portrayed by a higher synergist effectiveness on manufactured and common substrates and by a move of evident ideal action toward low temperatures and lower thermal stability.
P. haloplanktis β-galactosidase was expressed in E. coli, and the recombinant enzyme shows properties indistinguishable to those of the wild-type enzyme. Heat induced unfurling checked by inborn fluorescence spectroscopy indicated bring down melting point esteems for both P. haloplanktis wild type and recombinant β-galactosidase contrasted with the mesophilic enzyme. Measures of lactose hydrolysis in milk show that P. haloplanktis β-galactosidase can outflank the present commercial β- galactosidase from Kluyveromyces marxianus, recommending that the cool adjusted β-galactosidase could be utilized to hydrolyze lactose in dairy items prepared in refrigerated plants (Qing et al., 2014).
In this investigation, a quality that encoded a to a great degree thermostable β- galactosidase from Pyrococcus furiosus was cloned and expressed in Escherichia coli BL21. The recombinant enzyme was purged by heat treatment and Ni-NTA proclivity chromatography. The enzyme showed ideal action at 90 °C and pH 7.0 in phosphate buffer. The specific activity of the recombinant enzyme on o-nitrophenyl-β-D- galactopyranoside was 10.2 U/mg at 0°C and 130.0 U/mg at 90 °C. The half-lives of the enzyme were 31423.4, 8168.3, 4017.7, 547.4, 309.6, and 203.5 min at 70 °C, 80 °C, 85 °C, 90 °C, 95 °C, and 100 °C, accordingly.
The recombinant enzyme showed both β-galactosidase and β-glucosidase activity. The active inclusion collections of β-galactosidase were effortlessly separated by 34 nonionic cleanser treatment and specifically utilized for lactose change in a monotonous group mode. More than 54 % (90 °C) or 88 % (10 °C) of the first catalyst action was held after 10 transformation cycles under ideal conditions. These outcomes propose that the recombinant thermostable β-galactosidase might be reasonable for the hydrolysis of lactose in milk processing (Qing et al., 2014).
Reyhan et al. (2007) portrayed an intracellular β-galactosidase from a thermoacidophilic Alicyclobacillus acidocaldarius sub sp. rittmannii was cleaned by methods incorporating precipitation with ammonium sulfate, gel saturation, ion exchange and affinity chromatography lastly by preparative electrophoresis and a few properties of the purged chemical were resolved. The homogenous enzyme had a particular action of 113 U/mg protein, with an overlap refinement of 163 and a yield of 8 %.
The Km and kcat esteems for ONPG were resolved as 8.9 mM and 1074/min, separately in the filtered β-galactosidase from A. acidocaldarius sub sp. rittmannii. The microbes deliver thermostable β-galactosidase activity, which displays its ideal at the nonpartisan pH area. The pH and temperature optima for the purified enzyme are 6.0 and 65 ˚C, individually (10 min measure). β-galactosidase particular activities of crude extracts gotten from bacterial cells developed in the nearness and nonattendance of lactose over some stretch of time (6–40 h) demonstrated that β-galactosidase synthesis is by all accounts constitutive and increments by expanding time up to 40 h of development.
β-Galactosidase activity in bacteria developing on the medium without lactose was 0.4 U/mg protein and expanded up to 0.6 U/mg protein in the cells developing on the medium with lactose at 24 h (an expansion by around 33% of its constitutive esteem), while it was 0.072 and 0.48 U/mg protein, separately at 12 h (an expansion by around 85 % of its constitutive esteem). IPTG was additionally found to increment β- galactosidase activity over a brief timeframe.
Tomoyuki et al. (2006) portrayed in the present investigation, psychrophilic yeasts, which develop on lactose as a sole carbon source at low temperature and under acidic conditions, were secluded from soil from Hokkaido, Japan. The phenotypes and sequences of 28S rDNA of the isolated strains showed a taxonomic affiliation to Guehomyces pullulans. The secluded strains could develop on lactose at beneath 5 ˚C, 35 and demonstrated cold active acidic β-galactosidase action even at 0 ˚C and pH 4.0 in the extracellular parts. Additionally, Km of β-galactosidase action for lactose in the extracellular part from strain R1 was observed to be 50.5 mM at 10 ˚C, and activity could hydrolyze lactose in milk at 10 ˚C. The discoveries in this examination demonstrate the likelihood that the confined strains create novel acidic β- galactosidases that can hydrolyze lactose at low temperature.
Frosty situations, which are the most copious conditions on the surface of our planet, have been effectively colonized by various living beings, specifically microscopic organisms, yeasts, unicellular green growth and parasites. Since these living beings don't have any temperature control, their inside temperature is close, if not indistinguishable, to that of the encompassing condition. In spite of the solid negative impact of low temperatures on biochemical responses, these life forms breed, develop and move at rates like those accomplished by firmly related species living in calm conditions.
They have in this way created different adjustments as finely tuned basic changes at the level of, for instance, their films, constitutive proteins and catalysts, empowering them to make up for the malicious impacts of low temperature. Catalysts, which are proteins fit for catalyzing all the biochemical responses happening inside a living being that render them good with life, are a basic focus for the adjustment of a living being to an icy situation (Tomoyuki et al., 2006).
As of late, a deliberate examination has been done to comprehend the guidelines administering their sub-atomic adjustment to low temperatures. These key viewpoints are nearly connected with a solid biotechnological enthusiasm for the interesting properties of these catalysts, which are delivered by psychrophiles (all microorganisms ready to develop at temperatures near 0 ˚C).
A large portion of the cold active enzymes that have so far been described start from the Antarctic and living space additionally apply a high particular weight on endemic microorganisms on the grounds that the temperature of their environment is unvarying. Not with standing this, densities of bacterial cells as high as 107 ml21 have been found in the Antarctic sea, like the most noteworthy densities announced in mild waters. In labs, the Antarctic strains are usually developed at ~5 ˚C, a temperature thought to be near the ideal, securing the nonattendance of cell push. This 36 enables biochemical hardware to work ideally, offering ascend to high cell densities and most-proficient generation of extracellular enzymes (Tomoyuki et al., 2006).
At low temperatures, the era time (the time required to twofold the cell's populace) ranges from two to ten hours, contingent upon the bacterial species. Higher temperatures, despite the fact that shortening the era time, prompt cell push, regularly prompting low densities of cells and poor extracellular protein creation. Psychrophiles developing at ~5˚C can likewise be found in other for all time permanently cold environments, for example, the remote ocean, icy masses and mountain areas (Tomoyuki et al., 2006).
The expression "psychrotroph" is likewise regularly used to assign psychrotolerant living beings that are not completely adjusted to the cool and have an upper development restrain at temperatures >20˚C. The particular movement of wild-type cold enzymes and of some of their recombinant structures have been resolved for many enzymes created by Antarctic and Arctic microorganisms. Normally, the particular activity of these cold active enzymes is higher than that of their mesophilic partners at temperatures of around 0-30 ˚C (Charles et al., 2000).
Cold adopted enzymes offer financial advantages through vitality investment funds, they invalidate the prerequisite for costly warming strides, work in cold situations and amid the winter season, give expanded response yields, suit an abnormal state of stereospecificity, limit undesirable concoction responses that can happen at higher temperatures and display heat lability for quickly and effortlessly inactivating the enzyme when needed (Charles et al., 2000).
The couple of species secluded from cold environments significantly speak to the assorted qualities of cold adjusted microorganisms. Endeavours are unmistakably required to create culture accumulations utilizing an extensive variety of refined strategies with tests taken from various cool natural surroundings. This will give roads to high-throughput screening of new cold active enzymes and microbial procedures with biotechnological applications. Notwithstanding thinks about with microbial disconnects, a 'genomic mining' approach can possibly quicken the distinguishing proof of genes encoding cold adopted proteins without the requirement for unadulterated cultures (Charles et al., 2000). 37
At higher temperatures, denaturation of the cold enzyme happens. It is additionally important the aspartate aminotransferase from the Antarctic Moraxella sp. (Tutino et al., 1999; Charles et al., 2000) the action of the recombinant compound acquired in Escherichia coli is bring down at 0–30 ˚C than that of the mesophilic enzyme from E. coli. Phylogenetically, the reference enzyme is excessively remote from the cold active enzyme, appearing, for instance, in citrate synthase, a low amino acid sequence identity. The absence of clear temperature adjustment at the level of enzyme particular activity may reflect definitely unique cell needs, that is, a high particular movement at low temperature may not be required for the bacterial enzyme (Charles et al., 2000).
Microorganisms are found in territories of huge assorted qualities all around the world (Whitman et al., 1998; Anna et al., 2013). They speak to the most seasoned tenants on Earth, and their high flexibility empowers them to colonize even the most outrageous situations. Ecological microbiology is an exploration region of expansive intrigue, initially determined by strain detachments from natural examples and their portrayal.
Until the middle of the twentieth century, the general conviction was that the main strategies for microbial portrayal were development and microscopy (Handelsman, 2004) yet in the end, it ended up noticeably evident that most by far of all microorganisms can't be developed utilizing set up lab techniques, requiring elective methodologies. Afterward, the 16S rRNA quality was characterized as a marker for ordered investigation (Woese, 1987; Anna et al., 2013) and utilized as an apparatus for microbial diversity investigation (Schmidt et al., 1991; Anna et al., 2013).
Encourage technical improvements have brought about the metagenomic time, (Woese, 1987; Chistoserdova, 2010) where add up to DNA (the metagenome) from a natural specimen is disconnected and broke down. The direct analysis of sequence of metagenomic DNA is directly viewed as the most precise strategy for evaluating the structure of a natural microbial group, since it doesn't include any choice (e.g. by development/enhancement) and limits specialized inclinations (as presented by PCR intensification of the 16S rRNA gene).
Strategies for ecological testing differ contingent upon the motivation behind the examination, the natural surroundings inspected and the coveted downstream investigation. Pollutions of test material have diverse effects, that is, in bioprospecting 38
(Ferrer et al., 2007), tainting contemplations are less imperative than, for instance, for group structure portrayals. In metagenomics, detached natural DNA is either cloned as libraries (e.g. for useful screening), improved sequences (regularly by PCR) or specifically sequenced utilizing a high through put stage. From numerous ecological specimens, confinement of adequate measures of DNA is testing, requesting irregular DNA intensification, as by utilizing f 29 Multiple Displacement Amplification, as connected by, for instance (Biddle et al., 2008).
In any case, mind should be adopted in such strategies since MDA is inclined to real inclinations which may bargain the centrality of results (Yergeau et al., 2010). Whole genome shotgun sequencing was the primary approach utilized for sequencing natural DNA tests, exemplified by the investigation of marine waters (Venter et al., 2004) and an acid mine drainage (Tyson et al., 2004). Inferable from the monstrous advance in sequencing innovations, natural specimens would now be able to be sequenced without earlier cloning, accordingly restricting predisposition and loss of representativeness. With expanding measures of information produced, bioinformatics are tested to continually create and enhance reasonable devices for data analysis (Scholz et al., 2012).
Numerous situations give outrageous conditions to life, for instance, identified with temperature, pH, radiation, pressure or high concentrations of salt or overwhelming metals, and microorganisms adjusted to colonize such situations harbor unique properties of expansive intrigue both from an organic perspective and for bioprospecting purposes. Metagenomics seems, by all accounts, to be an exceptionally powerful method for the investigation of these extraordinary and frequently low diversity living spaces containing living beings that are regularly troublesome or so far difficult to develop (Anna et al., 2013).
Distinctive outrageous situations give shifting difficulties to their tenants, some requiring adjustment of the whole cellular machinary (with respect to thermophiles, psychrophiles and some halophilic taxa), while different extremophiles defeat such difficulties utilizing instruments which keep up physiological intracellular conditions, for instance, by H+ directing components for taxa living at extraordinary pH and additionally some salt-tolerant taxa (Litchfield, 2011). 39
Lee et al., (2012) were effectively disconnected the fifty-seven proteobacterium species from soils of Barrientos Island of the Antarctic utilizing 11 diverse seclusion media. Examination of 16S rDNA sequencing of these segregates demonstrated that they had a place with eight unique genera, to be specific Bradyrhizobium, Sphingomonas, Methylobacterium, Caulobacter, Paracoccus, Ralstonia, Rhizobium, and Staphylococcus. All segregates were examined for ability of creating antimicrobial and antifungal optional metabolites utilizing high-throughput screening models. These outcomes demonstrated that proteobacterium species disconnects from Antarctic could fill in as potential wellspring of valuable bioactive metabolites. Proteobacteria are a noteworthy phylum of microorganisms, which are all Gram- negative microscopic organisms. This bacterial scientific classification has been much of the time found in Antarctic soils (Aislabie et al., 2008).
Before Soon et al. (2005) said by Bergey's Manual of Systematic Bacteriology, the rhizobia and agrobacteria were characterized into four genera (Rhizobium, Bradyrhizobium, Agrobacterium and Phyllobacterium) inside the family Rhizobiaceae (Jordan, 1984). Rhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium and the previous Agrobacterium were customarily arranged on the premise of phenotypic qualities, for example, nodulation, and pathogenic and physiological properties. Be that as it may, nodulation and pathogenic properties have turned out to be less essential in the taxonomic evaluation of these genera. As of late, high sequence variety in the Internally Transcribed Spacer (ITS) district has been appeared to be more useful for taxonomic assessment of Bradyrhizobium strains (Berkum and Fuhrmann, 2000; Willems et al., 2001b; Soon et al., 2005).
Seyed et al. (2015) detailed the family Rhizobiaceae suits the seven genera Rhizobium, Neorhizobium, Allorhizobium, Agrobacterium, Ensifer (syn. Sinorhizobium), Shinella and Ciceribacter. Anyhow, a few purported Rhizobium species don't show powerful phylogenetic positions. Rhizobium is to a great degree heterogeneous and need significant modification. Along these lines, a phylogenetic examination of the family Rhizobiaceae by multilocus sequence analysis of four housekeeping genes among 100 strains of the family was attempted. In view of the outcomes we propose the depiction of the new genus Pararhizobium in the Rhizobiaceae family, and 13 new species blends. 40
Pararhizobium gen. nov. (Pa.ra.rhi.zo'bi.um. Gr. prep. para, adjacent to, close by of; N.L. neut. n. Rhizobium a generic name; N.L. neut. n. Pararhizobium, a genus neighboring to Rhizobium). Oxygen consuming, Gram-negative, non-spore-forming rods that are 0.3–0.9 µm wide by 1.2–2.5 µm long. Colonies are round, raised, white or cream, translucent to misty, with a measurement of 1–4 mm in 2–4 days on YMA at 28 ◦C. The highest growth temperature is 35–40 ◦C. Articulated turbidity creates following 2–3 days in broth media. The type species of genus Pararhizobium is P. Giardinii (Amarger et al., 1997).
The genus Pararhizobium incorporates four approved species, P. giardinii, P. capsulatum, P. Herbae and P. sphaerophysae, the G+C content of which shifts from 57.6 mol % to 63.5 mol %. The individuals from the family are scattered around the world, and were secluded from, Phaseolus vulgaris, Astragalus membranaceus, Oxytropis cashmiriana, Caragana sinica, Albizia kalkora, Kummerowia stipulacea, Astragalus danicus, Sphaerophysa salsula, and new water. The unsaturated fat profiles of the novel family demonstrated that Pararhizobium species have C14:0, C16:0, C16:0 3OH, C18:0, summed include 3 (C16:1 iso I/C14:0 3OH), summed highlight 4 (C15:0 iso 2OH/C16:1 w7c), and summed highlight 7 (C18:1 w7c/C18:1 w9t/C18:1 w12t) unsaturated fats in common manner. Unmistakable components of the species now exchanged to Pararhizobium, and discriminative elements between P. giardinii and other related genera were tried completely in previous examinations (Hirsch and Müller, 1985; Sittig and Hirsch, 1992; Lajudie et al., 1994; Amarger et al., 1997; Lajudie et al., 1998; Tighe et al., 2000; Ren et al., 2011; Xu et al., 2011; Qin et al., 2012; Seyed et al., 2015).
Depiction of Pararhizobium giardinii was given by (Amarger et al., 1997; Seyed et al., 2015). Furthermore the species can be separated from other Pararhizobium species at sub-atomic level by the sequences of 16S rRNA, atpD, glnA, glnII, recA, rpoB, and thrC genes. The whole genome shotgun sequence is accessible for Pararhizobium giardinii H152T in GenBank (ARBG00000000.1) (Amarger et al., 1997; Seyed et al., 2015).
Interpretation of Pararhizobium herbae was given by (Ren et al., 2011; Seyed et al., 2015). What's more the species can be separated from other Pararhizobium species at molecular level by G+C content (57.6–59.1 mol %), and the sequences of 16S rRNA, atpD, glnII, recA, rpoB, and thrA genes (Ren et al., 2011; Seyed et al., 2015). 41
Representation of Pararhizobium sphaerophysae was given by (Xu et al., 2012; Seyed et al., 2015). What's more the species can be separated from other Pararhizobium species at sub-atomic level by G+C content (62.9–63.5 mol %), and the arrangements of 16S rRNA, atpD, recA and rpoB genes (Xu et al., 2012; Seyed et al., 2015).
Portrayal of Pararhizobium capsulatum given by (Hirch and Müller, 1985; Seyed et al., 2015). Likewise the species can be separated from other Pararhizobium species at molecular level by G+C content (58.9 - 60.2 mol %), and the sequences of 16S rRNA, atpD, recA and rpoB genes (Hirch and Müller, 1985; Seyed et al., 2015).
Bioprospecting of cold temperature conditions for novel biocatalysts, as of late explored by Martinez-Rosales and collaborators (Martinez et al., 2012) for Antarctic DNA, conveys catalysts from psychrophilic microorganisms which frequently indicate low thermal stability and higher reactant efficiencies than their mesophilic homologues ( Anna et al., 2013).
On the off chance that today the yearly market for thermostable enzymes speaks to ~US$250 million, it is likely that the potential estimation of cold active enzymes is more prominent, in perspective of the differing capacities of these compounds (Charles et al., 2000).
Using microorganisms to lessen natural tainting, for example, in soils and waste waters, is not new but rather has all the earmarks of being an attainable contrasting option to physicochemical strategies (Timmis and Pieper, 1999; Charles et al., 2000)
In mild areas, expansive regular varieties in temperature decrease the viability of microorganisms in corrupting natural poisons, for example, oils and lipids. In any case, bioaugmentation and immunization of defiled situations with particular cold adjusted microorganisms in mixed cultures should enhance the biodegradation of hard-headed chemicals. Because of the high reactant effectiveness of their enzymes and their one of a kind specificity at low and direct temperatures, cold adopted microorganisms ought to be perfect for bioremediation purposes (Margesin and Schinner, 1999; Charles et al., 2000).
The treatment of waste waters debased because of human exercises would most likely be the least demanding approach to begin considering the potential utilizations of cold adopted microorganisms in bringing down the measure of lethal mixes, for instance, 42 nitrates, hydrocarbons, sweet-smelling mixes, substantial metals and biopolymers, for example, cellulase, chitin, lignin, proteins and triacylglycerols; these endeavors have as of now started (Margesin and Schinner, 1997; Margesin and Schinner, 1998; Margesin and Schinner, 1999).
The conceivable utilizations of cold active enzymes in the food industry are various. For instance, in the milk sector, β-galactosidase is utilized at low temperature to decrease the measure of lactose in charge of serious initiated bigotries in roughly 66 % of the total populace (Charles et al., 2000).
Cold active enzymes offer financial advantages through vitality reserve funds, they nullify the necessity for costly warming strides, work in frosty situations and amid the winter season, give expanded response yields, suit an abnormal state of stereospecificity, limit undesirable synthetic responses that can happen at higher temperatures and display heat lability for quickly and effectively inactivating the enzyme when required (Smal et al., 2000; Gerday et al., 2000; Ricardo et al., 2002).
Current utilizations of cold active enzymes created by psychrophilic microorganisms are financially essential in various modern applications (Cavicchioli et al., 2002; Cristóbal et al., 2011a; Hector et al., 2015), including the production of cheese, animal sustain, cowhide, indigo and material, vinegar, lager, wine and natural product juice, chemicals, and biopolymers (Egorova and Antranikian 2005; Hector et al., 2015).
Singh (2010) evaluated an incentive for enzymes in the worldwide market is about US$2.3 billion/year, an incentive in which food enzymes constitute the significant piece of the overall industry. On these bases of utilizations, proteins are disseminated to food (45 %), cleanser (34 %), agribusiness and bolsters (16 %), materials (11 %), calfskin (3 %), and mash and paper (1.2 %) ventures (Demain and Dana 2007; Singh, 2010). Really, numerous industrial microbial enzymes assume vital parts in current biotechnology, enhancing or notwithstanding supplanting already existing procedures (Cristóbal et al., 2009, 2011b; Hector et al., 2015).
Agribusiness is a standout amongst the most vital areas of the Pakistani Economy, contributing 21 % to Pakistan's GDP and utilizing 45 % of its work constrain. Besides, the absolute most essential subsector of farming is animals; including bovines, wild oxen and goats which give milk, meat, covers up and other crude 43 materials for the nearby market. The animals part alone contributes 11% of the nation's GDP, with an expected 42 billion liters of milk created per annum (Jassar Farms, 2009).
According to the Economic Survey of Pakistan 2009, Pakistan has a crowd size of around 63 million animals, which is the third biggest on the planet. Around 35 million individuals are included in dairy cultivating, inferring over 40% of their aggregate wage from domesticated animals. For these agriculturists, dairy animals give milk to household utilization and in addition small pay through the offer of milk. In provincial Pakistani culture domesticated animals is a segment of riches. It is seen as imperative social capital and offers protection to the proprietor in times of money related trouble (Jassar Farms, 2009).
Pakistan is positioned at fifth position among the top drain creating nations of the world. In Pakistan around 42.199 million tons/year drain is delivered (GOP, 2008). Wild ox and dairy animals contribute 58 % and 35 % share, separately to add up to drain generation in the nation (GOP, 2008). Different species like sheep, goat and camel contribute 5 % in the aggregate drain creation of Pakistan. Just 3-4 % of the aggregate created is utilized by the formal procedure drain channel. The rest is dispersed as crude drain.
44
MATERIALS AND METHODS
Collection of samples
Water samples, sediments and microbial mats were taken from ponds of Pyramid Trough (78°18′S, 163°15′E) surrounded by Royal Society Mountains, Mc Murdo Dry Valleys, Antarctica (Anne et al., 2012).
Culture medium
Two culture media Marine medium (DifcoTM 2216) and R2 medium prepared in lab were used for isolation and screening purposes. R2 medium composed of (KH2PO4 0.3 g, casamino acids 0.5 g, peptone 0.5 g, NaCl 10 g , soluble starch 0.3 g, sodium pyruvate 0.3 g, MgSO4.7H2O 0.05 g, yeast extract 0.5 g. All the chemicals were of analytical grade and purchased from Merck (Germany). Marine and R2 medium were autoclaved by using Hirayama (HiclaveTM HV-50) at 121˚C for 20 minutes and pH of the medium was checked by Thermo Scientific (Orion Star A111) pH meter. After autoclaving buffered to pH 10 using Na2CO3 buffer with final concentration (50 mM). Buffer was separately sterilized by using sterile BD Plastipak (20 ml) syringe and Q- Max syringe filter pore size (0.2 µm) (Mariane et al., 2006). For solid agar plates, 1.5 % (w/v) agar was added.
Isolation of bacterial strains
Antarctic samples were taken from -20 ˚C freezer and diluted in sterilized 0.9 % NaCl (w/v) solution, then vortexed by using Holm & Halby (Vortex-2 GenieTM ) to mixed it very well. 40 µl of appropriate dilution was taken with the help of Andrea (Thermo Scientific) micropipette and spread it onto the R2 agar plates and Marine agar plates at pH 7 and pH 10 under sterile conditions in Biowizard laminar flow hood. The agar plates were incubated in Sanyo incubator for one month at 10°C.
Screening of β-galactosidase producing bacterial strains
Cultures were plated on new R2 agar medium and Marine agar medium at pH 7 and pH 10 supplemented with 20 μg /mL of 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal), 1.0 mM of IPTG (Isopropyl β-D-1-thiogalactopyranoside) Sigma(16758-1G) and lactose (0.5 % w/v) as an inducer solution. After one month incubation at 10°C, plates were divided into different parts then picked the only blue colonies and streaked them onto new plates. Finally blue colonies representing β-galactosidase 45 producing bacteria were selected and again streaked to get purified single colonies for further characterization.
Preparation of Inoculum
The sterilized R2 broth and Marine broth at pH 7 and pH 10 induced with IPTG and lactose (0.5 % w/v) was inoculated with a single strain (blue colonies) from petri dishes and incubated at 10˚C on a shaker Ika Labortechnik (KS 250 Basic) with an orbital shaking velocity of 150 rmp.
DNA Extraction (PCR Amplification for 16s rDNA Analysis)
Strains were selected for the extraction of DNA by using simple DNA extraction method (Terefework et al., 2001) in which phosphate buffer (pH 7.4) was used to wash the cells twice, centrifuged it by using Sigma 1-15P (Buch & Holm) centrifuge machine at 10000 g for 5 minutes, supernatant was removed and 100 µl of Milli-Q water was added and put it in the small incubator (Thermomixer-5436) for 10 minutes at 99 ˚C. After boiling samples were placed in the ice box for 3 minutes and finally 900 µl of ice cold MQ-water was added in it and kept it in the freezer at -20 ˚C. DNA samples were used as a template for PCR reaction by using primers 16F27 (AGAGTTTGATCMTGGCTCAG) 20 kbp and 16R1492 (TACGGYTACCTTGTTACGACTT) 22 kbp with 1465 bp under reaction system (Sigma water, PCR Buffer, Dntps (2.5 mM), 16F27 (1ng/µl), 16R1492 (1ng/µl), Templates DNA (cell lysates containing genomic DNA fragments), Takara Bio Inc Taq polymerase (5U). PCR amplification for 16s rDNA was carried out in a thermal cycler (AH Diagnostics Mastercycler) with reaction profile (Initial denaturation at 95 ˚C for 5 min; 30 cycles consisting of denaturation at 94 ˚C for 30 s, primer annealing at 57 ˚C for 60 s and elongation at 72 ˚C for 90 s. The final elongation step was extended to 10 min). Run the PCR products by using 1 % agarose gel (Sigma A-4018) in the EMBI Tec (Run OneTM Electrophoresis Cell) and then patterns were visualized using a model 2000 GelDoc imager.
UP-PCR
Same DNA templates were used for UP-PCR reaction by using primers L15/AS19 5‟- GAGGGTGGCGGCTAG-3‟ (15 bp) under PCR reaction system (Sigma water, PCR buffer, Dntps (2.5 mM), L15/AS19 (1ng/µl), DNA Templates, Takara Bio Inc Taq polymerase (5U) with PCR program: Initial denaturation at 94 ˚C for 3 min; 32 cycles 46 consisting of denaturation at 94 ˚C for 60 s, primer annealing at 53 ˚C for 60 s and elongation at 72 ˚C for 60 s. The final elongation step was extended to 3 min. 1.5 % agarose gel (Sigma A-4018) was used, then visualized the patterns by using Gel Doc imager model 2000.
Purification of PCR Product
PCR product was purified by using QIAquick Gel Extraction (Qiagen) (Annexure I). Sterilized clean and sharp scalpel was used for the cutting of DNA fragments from agarose gel. Gel slice was weighed in a colorless tube then 3 volumes of Buffer QG to 1 volume of gel (100 mg 100 l) was added and incubated at 50 ˚C for 10 min in a small incubator (Thermomixer Comfort) or until the gel slice had completely dissolved. During incubation every 2-3 min, tubes were vortexed by using Holm & Halby (Vortex-2 GenieTM). When gel slice was completely dissolved, color of the mixture was turned yellow (similar to Buffer QG without dissolved agarose). One gel volume of isopropanol was added to the sample and mixed it very well then placed a QIAquick spin column in a provided 2 ml collection tube or into a vacuum manifold. DNA binding was done by appling the sample to the QIAquick column and centrifuged for 1 min in Sigma 1-15P (Buch & Holm) centrifuge machine at 10000 rpm or applied vacuum to the manifold until all samples passed through the column. Flow-through was discarded and placed the QIAquick column back into the same tube. 0.5 ml of Buffer QG was added to QIAquick column and was centrifuged for 1 min as above. Flow through was discarded again and QIAquick column was placed back into the same tube. 0.75 ml of Buffer PE was added to QIAquick column to wash it and then centrifuged for 1 minute. Now flow through was discarded and QIAquick column was put it back into the same tube. Column in a 2 ml collection tube (provided) was centrifuged for 1 min at 17,900 x g (13,000 rmp). QIAquick column was placed into a clean 1.5 ml microcentrifuge tube. Elution of DNA was done by adding 50 µl of buffer EB (10 mM Tris.Cl, pH 8.5) or water to the center of QIAquick membrane and then column was centrifuged for 1 min. Alternatively, for increased DNA concentration, 30 µl elution buffer was added to the center of the QIAquick membrane, column stand for 1 min and then centrifugation was done for 1 min in Sigma 1-15P (Buch & Holm) centrifuge machine. Purified DNA was analysed on a gel by adding 1 volumn of Loading Dye to 5 volumes of purified DNA.
47
Extraction of DNA
Genomic DNA was isolated from cells using the Gentra Puregene Yeast/Bact kit (Qiagen) (Annexure II). Liquid culture was prepared and 500 µl of the cell culture was transferred (containing approx. 0.5-1.5 x 109 cells) to a 1.5 ml micro centrifuge tubes on ice. Centrifugation was done for 5 s at 13,000-16,000 x g for cell pellets. Carefully discarded the supernatant by pipetting or pouring. 300 µl of Cell Suspension Solution was added and pipette up and down then 1.5 µl Lytic Enzyme Solution was added and mixed it by inverting 25 times and incubated for 30 min at 37 ˚C. Centrifugation was done for 1 min at 13,000-16,000 x g to form cell pellets. Then carefully discarded the supernatant with a pipette and 300 µl of Cell Lysis solution was added and pipette up and down for cell lysation. 1.5 µl RNase A Solution was added and mixed by inverting 25 times then incubated for 15-60 min at 37 ˚C. Incubation for 1 min on ice was done to quickly cool the samples. 100 µl Protein Precipitation Solution was added and vortexed vigorously for 20 s at high speed. Centrifugation for 3 min was done at 13,000-16,000 x g. 300 µl isopropanol was pipetted into a clean 1.5 ml micro centrifuge tube and the supernatant was added from the previous step by pouring carefully. Mixing was done by inverting gently 50 times. Again centrifugation was done for 1 min at 13,000-16,000 x g. Supernatant was discarded carefully and the tube was drained by inverting on a clean piece of absorbent paper, taking care that the pellet remained in the tube. 300 µl of 70 % ethanol was added and then inverted several times to wash the DNA pellet. Centrifugation for 1 min was done at 13,000-16,000 x g. Again supernatant was discarded carefully by taking care of the pellet. It was allowed to air dry for 5 min. 100 µl of DNA Hydration Solution was added and vortexed at Holm & Halby (Vortex-2 GenieTM) for 5 sec at medium speed to mix very well. Incubation was done at 65 ˚C for 1 h to dissolve the DNA. Now incubated in a small incubator (Thermomixer-5436) at room temperature (15-25 ˚C) overnight with gentle shaking. Ensured tube cap was tightly closed to avoid leakage. Samples can then be centrifuged briefly and transferred to a storage tube.
48
Phenol Chloroform Extraction Method
(Chromosomal DNA preparation)
DNA for phylogenetic analysis and full genome sequencing were extracted using a conventional phenol chloroform extraction method (Sambrook and Russell, 2001) (Annexure III). Cells were harvested from 20 ml culture by centrifugation (15 minutes, 10000 rpm, 15 oC). Supernatant was removed and washed with 5 ml TE buffer and centrifugation was done for 20 minutes at 10000 rpm at 15 oC. Supernatant was removed again (Pellet can be stored at -20 oC). Cell pellet was resuspended in 2 ml STET buffer and 375 µl of Lysozyme was added (from a stock of 10 mg/ml in TE) then incubation was done for 1 hour at 37 oC. 500 µl of SDS was added and mixed thoroughly. Now incubation was done for ½ hour at 37 oC and then ½ hour at 65 oC then 5 ml of sterile H2O was added. 2.5 ml of phenol (phenol: chloroform: isomylalcohol, 25:24:1) was added in the Alvent D208-1 (fume hood) by wearing nitrile gloves (Ansell NBR-92-600) and was mixed carefully for 5 minutes. Centrifugation was done (10 minutes, 10000 rpm, 15 oC) and upper phase (water phase) was transferred to a new falcon tube then 2.5 ml of chloroform (chloroform: isomylalcohol, 24:1) was added and mixed carefully. Centrifugation was done again (10 minutes, 10000 rpm, 15oC) and upper phase (water phase) was transferred to a new falcon tube. Chloroform extraction was repeated 3 times (transferred upper phase to new tube). 1/10 vol of NaCl (5 M) was added and mixed carefully after adding 1 volume of isopropanol. Tube was inverted carefully a couple of times and see if DNA was precipitated as white threads (tube can be stored at -20 oC over night). Centrifugation was done (10 minutes, 10000 rpm, 5 oC) and after removing the supernatant, washed with ~1 ml 70 % EtOH (and transferred to eppendorf tube) and was centrifuged again (5 minutes, 16000 rpm, 5 oC) with Sigma 1-15P (Buch & Holm) centrifuge machine. Process was repeated two times (three washes in total). Pellet was not fully dried (EtOH should fully evaporated). 30-50 µl TE buffer was added and was transferred to new eppendorf tube. Finally quality of DNA was measured. It was measured by nanodrop (Spectrophotometer ND-1000) and then finally measured the concentration of pure genomic DNA with Qubit (Invitrogen-2.0 fluorometer) for further 16s rRNA and whole genome sequence analysis. 16s rRNA sequencing was performed in a highly automated gene sequencer by using sequencing 49 primers 16F27, 16R1492 and 16S-BAC-338F (ACTCCTACGGGAGGCAG) as a middle primer at GATC Biotech (Germany).
16s rRNA gene sequence analysis
Sequencing results were downloaded from the GATC Biotech website https://www.gatc-biotech.com/en/mygatc4/my-login.html and then EzTaxon server http://www.ezbiocloud.net/eztaxon was used to BLAST and identify the closely related species to the bacterial strains. Further these sequences were compared by using RAST database http://rast.nmpdr.org/ with existing sequences available from the NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/).
Whole genome sequencing
Whole genome sequencing of the selected bacterial strains were performed at Department of Environmental Science, Aarhus University, Roskilde, Denmark. The draft genome sequences were determined by the Illumina MiSeq 2x250 PE platform to generate a paired-end library. De novo assembly was performed with SPAdes 3.5.0, resulting in 161 contigs (>200 bp). A total of 4,822 open reading frames were predicted by the RAST server and annotated using the information from GenBank and RAST (Wang et al., 2016). This whole-genome shotgun project has been deposited at GenBank/EMBL/DDBJ under the accession no. LSRP00000000 (Annexure IV).
Phylogenetic Analysis of Sequences
Sequences were extracted in Genbank Database (BLAST) to analyze the bacterial class and its phylogeny and then were compared with other available sequencing. Further sequences were trimmed, aligned and phylogenetic trees were constructed using the alignment tool in the CLC Main Workbench 7.6.3 (Qiagen Aarhus A/S) and the phylogenetic tree function in the Mega 7 program (http://www.megasoftware. net/). In each case bootstrap values were calculated based on 1000 replications. Genome relatedness was investigated by comparing the whole genome sequences of selected strains with sequences of the closest relatives using average nucleotide identity (ANI) and the Genome-Genome Distance Calculator (GGDC) (Puławska et al., 2016).
50
Phenotypic Characterization
Reference strains were purchased from BCCM/LMG (Begian Co-ordinated Collections of Microorganisms, Belgium-Europe), HAMBI (The Culture Collection, Finland) and NBRC (NITE Biological Resource Center, Japan) (Annexure V, VI and VII respectively). Yeast Mannitol Agar (YMA) broth consisted of yeast extract 1.0 g/l, D-Mannitol (Sigma-Aldrich, 63560-250G-F) 10 g/l, K2HPO4 0.5 g/l, MgSO4.7
H2O, NaCl 0.10 g/l, CaCO3 1.0 g/l was used to retrieve the strains from culture collections. Media was sterilized in HIRAYAMA (HICLAVETM HV-50) autoclave at 121 ˚C for 15 minutes. 15 g/l of agar was added to solidify the media. Selected strains with references strains were incubated at 28 ˚C for 3 days, subcultured under the same conditions and preserved at -80 ˚C in Yeast Mannitol broth with 20 % (v/v) glycerol.
β-galactosidase Fermentation
Growth characteristics were investigated by using YMA and YMA cheese whey based medium. Cheese whey was obtained from Institute of Food Science, IFV, University of Copenhagen, Denmark. Milkoscan (MilkoScanTM FT2) was used to get the whey profile for different parameters such as lactose concentration, total fat, protein and solid content. Then it was deproteinized and adjusted to pH 7.0 after filtration with (Whatman No.1) filter paper.
Effect of Temperature
YMA broth was used to determine the temperature range and incubated at 4 ˚C in the refrigerator, 10 ˚C in SANYO incubator, 15 ˚C, 20 ˚C, 28 ˚C and 37 ˚C in the Termaks incubators respectively. Then measured OD600 of YMA broth cultures by using Holm & Halby (UV-Mini 1240) spectrophotometer.
Effect of pH
The pH was adjusted by using appropriate buffers, 0.1M citric acid / 0.2M Na2HPO4 buffer, pH 4.0-7.0; 0.2M tris / 0.2M HCl buffer, pH 8.0-9.0; 0.05M NaHCO3 / 0.1M NaOH buffer, pH 10.0-11.0. Molar solutions were prepared first then buffers were sterilized by using sterile BD Plastipak syringe and Q-Max syringe filter pore size (0.2 µm) under sterile conditions in the Biowizard laminar flow hood. pH was checked by Thermo Scientific (Orion Star A111) pH meter and incubated at 28 ˚C (Termaks Incubator) with the shaking velocity of 150 rmp by using Ika Labortechnik (KS 250 Basic) shaker (Dan et al., 2014). 51
Effect of NaCl
Effect of NaCl was investigated by preparing YMA (Yeast Mannitol Agar) broth without NaCl and then its concentration was adjusted to 0.0-5.0 % (w/v, at intervals of 1.0 %). 10 % NaCl stock solution was prepared first by using sterile filter and then used it to adjust the NaCl range from 0 to 5 % (final concentration).
Biochemical characterization
Biochemical tests were performed by using API 20 E kits (bio Me´rieux) (Annexure XIII). These tests included enzyme activities i.e. β-galactosidase, arginine dihydrolase (ADH), lysine decarboxylase (LDC), ornithine decarboxylase (ODC) then citrate utilization (CIT), urease (URE), acetoin production (VP), gelatinase (GEL), H2S production, indole production (IND) and fermantation of glucose, mannitol, inositol, sorbitol, rhamnose, saccharose, melibiose, amygdalin, arabinose. Cells were harvested from bacterial cultures in Sigma 1-15P (Buch & Holm) centrifuge machine then washed it two times with sterile 0.9 % NaCl solution and different dilutions were prepared for further use. An incubation box (tray and lid) was prepared and about 5 ml of distilled water was distributed into the honey combed wells of the tray to create a humid atmosphere. Strain reference was recorded on elongated flap of the tray, strip was removed from its packaging and then placed it in the incubation box. Distribution of bacterial suspension was done with the help of pipette into the tubes of the strip. For the CIT, VP and GEL tests, both tube and cupule were filled and for other tests only tubes were filled not cupules. For the tests ADH, LDC, ODC, H2S and URE created anaerobiosis by overlaping with mineral oil then the incubation box was closed and incubated at 28 ˚C for 18-24 hours. Strip was read after incubation period by referring to the reading table (Annexure VIII).
Effect of Nitrogen Sources
Base medium i.e. FeSO4.7H2O 0.0005 g, NaH2PO4 2.13 g, K2HPO4 1.36 g,
MgSO4.7H2O 0.2 g, CaCl2.2H2O 0.005 g and glucose 10 g was supplemented with different nitrogen sources (0.1 % m/v) including (NH4)2 SO4, KNO3, L-glutamic acid and L-valine. Media was sterilized and its pH was adjusted to 7 by using previously sterilized buffer (citric acid / Na2HPO4).
52
Antibiotic Sensitivity
Antibiotic sensitivity was checked by using disc diffusion method. YMA plates were used to check the susceptibility of antibiotics. Discs impregnated with antibiotics were used containing ampicillin (1000 mg/ml), kanamycin (25 mg/ml), streptomycin (25 mg/ml), tetracycline (10 mg/ml) and penicillin (1mg/ml). 2 µl of antibiotic was used on each disc and incubated at 20 ˚C then zone of growth inhibition was measured after 2-3 days.
Electron microscopy
Cell morphology was observed by phase-contrast microscopy (Leica DMLS). For electron microscopy samples were prepared on liquid media, sample cells were washed with PBS buffer and centrifugation was done for 5 min at 10000 g in the Sigma 1-15P (Buch & Holm) centrifuge machine then supernatant was discarded carefully. Different dilutions for samples were prepared i.e. 5 x 10-1, 5 x 10-2, 5 x 10-3 and 5 x 10-4. 1 ml of 2.5 % gluteraldehyde was added to each sample and put it at 4 ˚C for 4.5 hours. After 4.5 hours, 500 µl of 50 % ethanol was added to each sample then allowed to dry for 5 min and centrifuged for 2 min at 10000 g. Supernatant was discarded carefully and the same process was repeated with 70 %, 80 %, 90 % and 96 % ethanol respectively. 10 µl suspension for each sample was dropped on the slide (Annexure IX).
Gram Staining
The Gram Stain Set S kit (BD Difco) and the Ryu non staining KOH method (Powers, 1995) were used for testing the Gram reaction (Annexure X and XI) respectively.
Motility Test
Motility was tested by using the YMA medium supplemented with 0.5 % agar.
Catalase Test
Catalase activity was assessed by the production of bubbles in 3 % (v/v) (H2O2).
Oxidase Test
Oxidase test was performed by using the Fluka (40560 Oxidase strips) (Annexure XII).
53
Anaerobic Growth Conditions
Oxoid Anaero Gen system was used to check the growth of strains under anaerobic conditions.
Chemotaxonomic Analysis
For chemotaxonomic analysis cells were harvested by freeze-drying method. Stock culture was used on YMA plates and single colonies were transferred to 50 ml of YMA broth. 5.0 ml of liquid culture was used in 500 ml of broth in 1.5 liter biometric conical flasks with orbital shaking velocity of 150 rmp at 28 ˚C. Blue cap falcon tubes were weighed before centrifugation then liquid culture was added and centrifugation was done by using Sigma (3-8K) centrifuge machine at 4700 rmp at 4 ˚C for 10 min with rotor 11180. Supernatant was removed and centrifuged it again, after final centrifugation cells were washed with 0.9 % NaCl solution and put it at -80 ˚C (Thermo Scientific) freezer over night. Samples were placed in freeze-dryer (Hetosicc) with Edwards (High Vacuum Pump) and finally stored at -20 ˚C for further use. For fatty acids, polar lipids and respiratory quinones, approximately 300 mg freeze dried cells were shipped to identification services of Leibniz-Institut DSMZ- Deutsche Sammlung von Mikroorganismen und Zellkulturen (Germany) (Annexure XIII). Strains were deposited in DSMZ and LMG (Annexure XIV and XV).
Effect of Incubation period, Temperature, and pH on enzyme production
The effect of temperature on the production of β-galactosidase was investigated by incubating the organisms at different temperatures (5 ˚C, 20 ˚C, 28 ˚C and 37 ˚C). Effect of pH was also determined by using different pH values (6, 7 and 8) and effect of incubation time was checked by cultivating the strains at various incubation time (24, 48, 72 and 96) hours. After incubation the activity of β-galactosidase was determined in the supernatant.
Enzyme Extraction
Cells for β-galactosidase activity assays were grown in YMA broth at pH 7 for 3 days at 20 °C on a rotary shaker-Ika Labortechnik (KS 250 Basic) (rpm=150). Cells were harvested by centrifugation for 10 min at 5 ˚C by using Sigma (3-8K) centrifuge machine at 4700 rmp with rotor 11180. Supernatent was kept for further use. Cells were resuspended in 300-400 µl of 0.1 M phosphate buffer (NaH2PO4 / Na2HPO4, pH 7.4). Cells were transferred into Fast Prep Tubes and then lysed by bead beating (glass 54 beads of different diameter) in a Fast Prep shaker (Bio101/Savant Instruments, Holbrook, NY) for 3 times at speed of 5.5 for 25 sec. Samples were immediately cooled on ice for 2-3 min in-between the beating cycles. Lysates were centrifuged at 10,000 g for atleast 10 minutes at 5 °C. β-galactosidase containing supernatants were carefully removed and transferred to new clean eppendorfs. These enzyme extracts were kept at 4 ˚C for further analysis.
Enzyme Unit
One unit of β-galactosidase activity (U) is defined as the amount of enzyme that liberates 1 μmole ONP per minute under assay conditions. Results will be represented as mean of at least three experiments.
Effect of Temperature on Enzyme Activity
Reaction mixture consisted of 10 µl o-nitrophenly β-D-D-galactoprynoside ONPG (1 mM), 15 µl of 0.1 M phosphate buffer (pH 7.4), 50 µl of enzyme extract and 25 µl of autoclaved Milli-Q water was incubated at 5 ˚C, 20 ˚C, 28 ˚C and 37 ˚C for different time intervals. Reaction was stopped by adding 50 µl of 1M Na2CO3 then read the absorbance of o-nitrophenol (ONP) released from ONPG with Holm & Halby (EL- 808 Ultra Microplate Reader) at 415 nm using standard calibration curve.
Effect of pH on Enzyme Activity
Reaction mixture containing 15 µl of ONPG (20 mM), 10 µl of different buffers at pH
6, pH 7 and pH 8 adjusted the pH by using different concentration of NaH2PO4 /
Na2HPO4 and 75 l of enzyme extract was incubated at 28 ˚C for different time periods. Reaction was ended by the addition of 50 µl of 1M Na2CO3 and determined the enzyme activity by using Holm & Halby (EL-808 Ultra Microplate Reader) at 415 nm. All experiments were carried out in triplicates and results were mentioned in the mean values of all observations.
55
RESULTS
ISOLATION OF BACTERIAL STRAINS
Bacterial strains isolated from Antarctic samples (Mc Murdo, USA) and from Ikka columns, Greenland were screened for β-galactosidase activity on R2 medium at pH 10 with X-gal and incubated at 10 ˚C for twenty eight days (Annexure Table 1). There was no growth was observed for twenty four strains from Antarctic samples. Only five strains turned blue, two from Ikka columns (A & B) and three from Antarctic samples (1, 9 and 16) (Fig. 1). Ten strains showed different colors on X-gal plates from Antarctic samples. One turned green, five turned white, two turned light yellow and dark yellow respectively. Strain A showed one large and mostly small blue colonies, strain B and 1 showed medium size colonies, strain 9 showed small colonies and strain 16 had medium size colonies. Only blue colonies were transferred to liquid medium (R2 broth, pH 10) with lactose. Fig. 2 showed the growth rate of different bacterial strains on R2 medium at pH 10.
More Antarctic samples (1A, 2A, 3A, 4A and 5A) were used for the isolation of β- galactosidase producing bacterial strains from Antarctica on R2 Agar at pH 7 and incubated at 10 ˚C for thirty five days (Annexure Table 2). Five samples from Antarctica (mixture of water, sediments and microbial mats) and ten samples from Ikka columns (1K, 2K, 3K, 4K, 5K, 6K, 7K, 8K, 9K and 10K) were grown on Marine agar at pH 10 and incubated for one week. Antarctic samples showed some growth after one week (still need more incubation time) but for Ikka samples, absolutely no growth after a long incubation time (Annexure Table 3). Fig. 3. and Fig. 4. showed the growth of Antarctic and Ikka samples respectively.
Plates A1, A2, A3, A4 and A5 were divided with scale and bacterial colonies were marked with numbers. Seventy colonies were picked from plate A1, forty colonies from plate 2A, seventy five colonies from plate 3A, five colonies from plate 4A and forty five colonies from plate 5A respectively (Annexure Table 4). Total two hundred and thirty five colonies were selected from five plates. Fig. 5. showed the division of plates on marine agar at pH 10.
56
Figure 1: β-galactosidase producing bacterial strains from Antarctica on R2 agar medium with X-gal.
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 28 days.
57
25
20
15
10
5 No. of of No. Colonies (cfu) 0 No Blue Growth Green White Light Dark Yellow Yellow Color of Colonies
Figure 2: Number of β-galactosidase producing bacterial colonies (turned blue on X-gal) from Antarctica (Mc Murdo, USA) and Ikka columns (Greenland).
Environmental Conditions: R2 agar: pH 10, Temperature: 10 ˚C, Incubation Time: 28 Days.
58
Figure 3: Isolation of β-galactosidase producing bacterial strains from Antarctica on Marine agar without X-gal from Antarctic samples (1A, 2A, 3A, 4A and 5A).
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 35 days.
59
Figure 4: Isolation of β-galactosidase producing bacterial strains on Marine agar without X-gal from Ikka column samples (1K, 2K, 3K, 4K, 5K, 6K, 7K, 8K, 9K and 10K).
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 12 weeks.
60
Figure 5: Bacterial strains on Marine agar medium without X-gal supplemented with cheese whey.
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 08 days.
61
SCREENING OF β-GALACTOSIDASE PRODUCING BACTERIAL STRAINS ON X-GAL
Marine agar plates were prepared again at pH 10 with X-gal to check the β- galactosidase activity. Before inoculation, plates were divided into squares and marked with numbers then inoculated the strains with the same numbers onto new plates (Fig. 6. Fig. 7. and Fig. 8). Plate 1A had forty two blue colonies, one brown, two dark yellow, two light yellow, three pink and twenty white on X-gal respectively (Fig. 9). Plate 2A had seventeen blue, two dark yellow, four light yellow and seventeen white colonies on X-gal (Fig. 10). 3A had six blue, forty one dark yellow, three light yellow, one orange and twenty four white colonies on X-gal (Fig. 11). Plate 4A had only two light yellow and three white colonies (Fig. 12) and finally 5A had twenty eight dark yellow, sixteen light yellow and one white colony on X-gal (Fig. 13). Two hundred and thirty five strains were inoculated again on new Marine agar plates with X-gal. Only sixty five strains turned blue on all plates, it showed that they could produce β-galactosidase (Annexure Table 5).
Blue colonies were streaked again on marine agar plates at pH 10 for 27 days at 10 ˚C. Each plate was divided in 8 parts (Fig. 14, Annexure Table 6). Strains were categorized on the basis of their color with X-gal, light blue, medium blue and dark blue respectively. Plate 1A (i) had five light blue colonies, one white and two medium blue colonies, plate 1A (ii) had seven dark blue and one white colony, plate 1A (iii) had seven light blue and one pink colony, plate 1A (iv) had six light blue and two white colonies, plate 1A (v) had one medium blue, two white and six light blue colonies, plate 1A (vi) had one white, one dark blue and three medium blue colonies respectively. Plate 2A (i) showed two medium blue and one dark blue colony, plate 2A (ii) had one medium blue and seven light blue colonies, plate 2A (iii) had six light blue and two medium blue colonies respectively. Finally plate 3A (i) showed five dark blue colonies (Annexure Table 6). Total seventy colonies were streaked onto new plates, in which thirty seven turned light blue, eleven turned medium blue, fourteen turned dark blue, seven turned white and one in a pink color (Fig. 15, Annexure Table 6).
62
Figure 6: Bacterial strains on Marine agar medium supplemented with cheese whey and X-gal (Plate 1A).
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 08 days.
63
Figure 7: Bacterial strains on Marine agar medium supplemented with cheese whey and X-gal (Plate 2A and 3A).
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 08 days.
64
Figure 8: Bacterial strains on Marine agar medium supplemented with cheese whey and X-gal (Plate 4A and 5A).
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 08 days.
65
45
40
35 30 25 20 15
10 N0. of of N0. Colonies (cfu) 5 0 blue brown dark light pink white yellow yellow Color of Colonies
Figure 9: Number of β-galactosidase producing bacterial colonies from Antarctica (Mc Murdo, USA) (Plate 1A).
Environmental Conditions: Marine agar: pH 10, Temperature: 10 ˚C, Incubation Time: 08 Days.
66
18
16
14 12 10 8 6
No.of Colonies (cfu) 4 2 0 blue dark yellow light yellow white Color of Colonies
Figure 10: Number of β-galactosidase producing bacterial colonies from Antarctica (Mc Murdo, USA) (Plate 2A).
Environmental Conditions: Marine agar: pH 10, Temperature: 10 ˚C, Incubation Time: 08 Days.
67
45 40 35 30 25 20 15
No. of of No. Colonies (cfu) 10 5 0 blue dark yellow light yellow orange white Color of Colonies
Figure 11: Number of β-galactosidase producing bacterial colonies from Antarctica (Mc Murdo, USA) (Plate 3A).
Environmental Conditions: Marine agar: pH 10, Temperature: 10 ˚C, Incubation Time: 08 Days.
68
3
2.5
2
1.5
1
No. of of No. Colonies (cfu) 0.5
0 light yellow white Color of Colonies
Figure 12: Number of β-galactosidase producing bacterial colonies from Antarctica (Mc Murdo, USA) (Plate 4A).
Environmental Conditions: Marine agar: pH 10, Temperature: 10 ˚C, Incubation Time: 08 Days.
69
30
25
20
15
10
No. of of No. Colonies (cfu) 5
0 dark yellow light yellow white Color of Colonies
Figure 13: Number of β-galactosidase producing bacterial colonies from Antarctica (Mc Murdo, USA) (Plate 5A).
Environmental Conditions: Marine agar: pH 10, Temperature: 10 ˚C, Incubation Time: 08 Days.
70
Figure 14: β-galactosidase producing bacterial strains on Marine agar medium supplemented with X-gal.
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 27 days.
71
40
35 30 25 20 15
10 No. of of No. Colonies (cfu) 5 0 Light blue Medium Dark blue White Pink blue Color of Colonies
Figure 15: β-galactosidase producing bacterial strains from Antarctica were categorized on the basis of their colour with X-gal on Marine agar medium.
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 27 days.
72
SCREENING OF β-GALACTOSIDASE PRODUCING STRAINS ON DIFFERENT MEDIA AND DIFFERENT pH
R2 Agar (pH 10)
The data of Fig. 16 showed that bacterial colonies of Antarctic sample A2 with X-gal at pH 10. Colonies were categorized on the basis of their sizes. Ten small, eight medium and two large blue colonies were selected from R2 agar at pH 10 with X-gal. From plate 2A, fifteen small, three medium and two large blue colonies were selected. Finally from plate 3A, fifteen small, one medium and four large blue colonies were selected and streaked onto new R2 medium plates (60 plates) with X-gal at pH 10 (Annexure Table 7). Plates 4A and 5A showed no blue colonies. Most of the plates had small size of colonies. Fig. 17 represented the difference of colonies from plates 1A, 2A and 3A respectively.
R2 Agar (pH 7)
The data of Fig. (18) shows the growth of bacterial colonies from sample A3 on R2 agar, pH 7 with X-gal. Colonies were divided according to their size. Samples 1A, 2A, 4A and 5A showed no bacterial growth at pH 7 on R2 medium. From plate 3A, six small, one medium and five large blue colonies were selected and streaked them on new R2 Agar plates (12 plates) at pH 7 with X-gal and incubated at 10 ˚C again (Annexure Table 8).
Marine Agar (pH 7)
β-galactosidase producing bacterial strains were isolated from Antarctic samples, 1A, 2A, 3A, 4A and 5A by using Marine agar at pH 7 with X-gal. Growth of bacterial colonies for sample 2A on Marine agar at pH 7 with X-gal is shown in Fig. 19. Colonies were divided according to their size (Annexure 9). From plate 1A, eighteen small and two medium colonies were selected, from plate 2A, eighteen small and two medium, from plate 3A, five small and five large colonies and from plate 4A, twelve small and three large colonies were picked respectively and streaked on new Marine agar plates (65 plates) at pH 7 with X-gal. Again incubation was done at 10 ˚C. Fig. 20 showed the graphical representation of different sizes of colonies from different samples.
73
Figure 16: Isolation of β-galactosidase producing bacterial strains from Antarctic samples A1, A2, A3, A4 and A5 on R2 agar plates supplemented with cheese whey and X-gal.
Environmental conditions: Temperature: 10 ˚C, pH: 10, Time: 7 days.
74
16
14 12 10 8 6 4
No. of of No. Colonies (cfu) 2
0
large large
small small
Large
Small
medium medium Medium 1A 2A 3A Size of Colonies
Figure 17: β-galactosidase producing bacterial strains from Antarctica were categorized on the basis of their size with X-gal on R2 agar medium.
Environmental conditions: Temperature: 10 ˚C, pH: 10, Time: 7 days.
75
Figure 18: Isolation of β-galactosidase producing bacterial strains from Antarctic samples A1, A2, A3, A4 and A5 on R2 agar plates supplemented with cheese whey and X-gal.
Environmental conditions: Temperature: 10 ˚C, pH: 7, Incubation Time: 5 days.
76
Figure 19: Isolation of β-galactosidase producing bacterial strains from Antarctic samples A1, A2, A3, A4 and A5 on Marine agar plates supplemented with cheese whey and X-gal.
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 5 days.
77
20
18
16 14 12 10 8 6
4 No. of of No. Colonies(cfu) 2
0
Large Large Large Large
Small Small Small Small
Medium Medium Medium Medium 1A 2A 3A 4A Size of Colonies
Figure 20: β-galactosidase producing bacterial strains from Antarctica were categorized on the basis of their size with X-gal on Marine agar medium.
Environmental conditions: Temperature: 10 ˚C, pH: 7, Incubation Time: 5 days.
78
SCREENING OF BACTERIAL STRAINS WITH β-GALACTOSIDASE ACTIVITY FROM IKKA COLUMN SAMPLES
R2 Agar (pH 10)
Isolation of β-galactosidase producing bacterial strains on R2 medium with X-gal from Ikka samples I(1), I(2), I(3), I(4), I(5), I(6), I(7), I(8), I(9) and I(10) and incubated at 10 ˚C for one week (Fig. 21). Absolutely there was no growth for the other samples I(1), I(2), I(3), I(4), I(5), I(6), I(8), I(9) and I(10) (Annexure Table 10).
R2 Agar (pH 7)
Ten plates were prepared with R2 Agar medium at pH 7 with X-gal for the isolation of β-galactosidase producing bacterial strains from Ikka samples I(1), I(2), I(3), I(4), I(5), I(6), I(7), I(8), I(9) and I(10) and incubated at 10 ˚C. All the plates showed absolutely no growth.
Marine Agar (pH 7)
I(1), I(2), I(3), I(4), I(5), I(6), I(7), I(8), I(9) and I(10) (Ikka samples) were isolated on Marine agar plates at pH 7 with X-gal. Plate I(3) showed only one single blue colony and other plates had no growth at all on Marine agar at pH 7 from Ikka samples (Annexure Table 10).
COMPARISON OF GROWTH RATE OF BACTERIA AT DIFFERENT pH AND DIFFERENT MEDIA WITH X-GAL
Very good growth of sample 1A is observed on R2 agar at pH 10, Marine agar (pH 10) and Marine agar (pH 7), there is no growth on R2 agar medium at pH 7. Sample 2A was well grown on R2 agar (pH 10) and Marine agar (pH 7), good growth was observed on Marine agar (pH 10) and no growth was observed on R2 agar at (pH 7) respectively. Plate 3A had a good growth on all media with varied pH values (R2 agar (pH 10), R2 agar (pH 7), Marine agar (pH 10) and Marine agar (pH 7). 4A plate shows good growth on Marine agar at (pH 7) and no growth on R2 agar (pH 10), R2 agar (pH 7) and Marine agar at (pH 10) respectively. No growth was observed for sample 5A on R2 agar (pH 7), Marine agar (pH 10) and Marine agar (pH 7) and has some growth on R2 agar at pH 10 but with no blue colony.
Ikka column sample I(3) shows only one blue colony on Marine agar at pH 7 and no growth for rest of the media: R2 agar (pH 10), R2 agar (pH 7) and Marine agar (pH 79
10). Sample I(7) had only single colony on R2 agar (pH 10) and no growth on R2 agar (pH 7), Marine agar (pH 10) and Marine agar (pH 7) respectively (Annexure Table 11).
Marine Agar (pH 10)
Thirty six plates were prepared again with Marine agar medium at pH 10 with X-gal and incubated at 10 ˚C for 44 days. Strains 3, 5, 12 and 13 from plate 1A (i) were selected. Similarly, strains 16, 17, 20, 23 and 25 were selected from plate 1A (ii), strains 26, 27, 29 and 39 from plate 1A (iii), strains 41, 47 and 48 from plate 1A (iv), strains 52, 54, 55 and 57 from 1A (v), strains 61, 68 and 69 from 1A (iv), strains 1 and 7 from plate 2A (i), 11, 17, 22 and 26 from plate 2A (ii), strains 27, 30, 32 and 37 from 2A (iii) and finally strains 6, 32 and 75 from plate 3A were streaked onto new plates with X-gal on Marine agar at pH 10 (Annexure Table 12).
PURIFICATION OF β-GALACTOSIDASE PRODUCING STRAINS WITH DIFFERENT MEDIA AND X-GAL
R2 Agar (pH 10)
Isolated colonies were streaked again on new plates with X-gal and incubated at 10 ˚C for 40 days. Twenty colonies were selected from plate 1A: 1(1A), 2(1A), 3(1A), 4(1A), 5(1A), 6(1A), 7(1A), 8(1A), 9(1A), 10(1A), 11(1A), 12(1A), 13(1A), 14(1A), 15(1A), 16(1A), 17(1A), 18(1A), 19(1A), 20(1A). Twenty colonies were selected from plate 2A: 1(2A), 2(2A), 3(2A), 4(2A), 5(2A), 6(2A), 7(2A), 8(2A), 9(2A), 10(2A), 11(2A), 12(2A), 13(2A), 14(2A), 15(2A), 16(2A), 17(2A), 18(2A), 19(2A), 20(2A). Twenty colonies were selected from plate 3A: 1(3A), 2(3A), 3(3A), 4(3A), 5(3A), 6(3A), 7(3A), 8(3A), 9(3A), 10(3A), 11(3A), 12(3A), 13(3A), 14(3A), 15(3A), 16(3A), 17(3A), 18(3A), 19(3A), 20(3A) and one colony from sample I(7) were streaked on sixty one new plates with R2 medium at pH 10 with X-gal and incubated at 10 ˚C (Fig. 22). After incubation one strain showed no growth, thirteen showed blue color, four turned dark blue, one (green and blue), two (greenish yellow and blue), three light blue, one (light yellow and blue), one (orangish yellow and blue), one very light blue, two (very light yellow and blue), ten strains showed very small blue colonies, seven (blue and white), two (white, blue and dark blue), two yellow, eight (yellow and blue), one (yellow, blue and orange), one (yellow, green and blue) and one (yellow and light blue) (Fig. 23). Blue colonies were selected again and 80
Figure 21: Isolation of β-galactosidase producing bacterial strains on R2 agar medium with X-gal from Ikka samples I(1), I(2), I(3), I(4), I(5), I(6), I(7), I(8), I(9) and I(10).
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 7 days.
81
Figure 22: Isolated colonies were purified again on new R2 agar medium plates with X-gal.
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 43 days.
82
14 12 10 8 6 4 2 No. of of No. Colonies (cfu) 0
Color of Colonies
Figure 23: β-galactosidase producing bacterial strains from Antarctica were categorized on the basis of their color on R2 agar medium with X-gal.
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 43 days.
83 streaked on new plates to get the purified blue strains (Fig. 24). Growth rate was different for different strains, thirty four strains showed slow growth rate, twenty three showed medium, three showed good growth and there was no growth for one strain respectively. Size of colonies was also different for different strains, thirty one had small size, twenty five had medium size, four had small size and no growth for one strain (Annexure Table 13).
R2 Agar (pH 7)
Antarctic sample 3A only showed the growth on R2 agar at pH 7 with X-gal. Thirteen bacterial colonies were selected from 3A: 1(3A), 2(3A), 3(3A), 4(3A), 5(3A), 6(3A), 7(3A), 8(3A), 9(3A), 10(3A), 11(3A), 12(3A), 13(3A) and streaked on new thirteen plates (R2 medium, pH 7 with X-gal) and incubated at 10 ˚C for 43 days (Fig. 25). Strains showed different results after incubation, four strains had no growth, six strains showed very light blue color of colonies, one showed yellow, one showed (yellow and blue) and one with (yellow, blue and green) color (Fig. 26). Blue colonies were selected from the plate with (yellow, blue and green) color and streaked on the new plate to get only blue strain (Fig. 27). Growth rate was very slow on R2 agar at pH 7, no growth was observed for four strains and slow growth was observed for nine other strains. Colonies had different sizes for different strains, nine strains showed small size of colonies and four showed no growth respectively (Annexure Table 13).
Marine Agar, pH 10
Blue colonies were streaked again for repurification to get the only blue strains on new Marine agar plates at pH 10 with X-gal and incubated at 10 ˚C for 44 days. Colonies from plate 1A (i) – 3 (1A) i, 5 (1A) i, 12 (1A) i, 13 (1A) i, colonies from plate 1A (ii) – 16 (1A) ii, 17 (1A) ii, 20 (1A) ii, 23 (1A) ii, 25 (1A) ii, colonies from plate 1A (iii) – 26 (1A) iii, 27 (1A) iii, 29 (1A) iii, 39 (1A) iii, colonies from plate 1A (iv) – 41 (1A) iv, 47 (1A) iv, 48 (1A) iv, 52 (1A) iv, 54 (1A) iv, 55 (1A) iv, 57 (1A) iv and colonies from plate 1A (v) – 61 (1A) v, 68 (1A) v, 69 (1A) were selected and streaked on new plates. Colonies from plate 2A (i) – 1 (2A) i, 7 (2A) i, from plate 2A (ii) – 11 (2A) ii, 17 (2A) ii, 22 (2A) ii, 26 (2A) ii, from plate 2A (iii) – 27 (2A) iii, 30 (2A) iii, 32 (2A) iii, 37 (2A) iii and colonies from plate 3 A– 6 (3A) i, 32 (3A) i, 75 (3A) i were streaked on new plates (36 plates) with marine agar at pH 10 with X-gal and incubated at 10 ˚C again to get the purified blue colonies (Fig. 28). After 84
Figure 24: Isolated colonies were purified again on new R2 agar medium plates with X-gal.
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 43 days.
85
Figure 25: β-galactosidase producing bacterial strains from Antarctica were purified again on new R2 agar medium plates with X-gal.
Environmental conditions: Temperature: 10 ˚C, pH: 7, Incubation Time: 43 days.
86
7
6
5
4
3
2 No. of of No. Colonies (cfu) 1
0 No growth V. light blue Yellow yellow + blue yellow + blue + green Color of Colonies
Figure 26: β-galactosidase producing bacterial strains from Antarctica were categorized on the basis of their color on R2 agar medium with X-gal.
Environmental conditions: Temperature: 10 ˚C, pH: 7, Incubation Time: 43 days.
87
Figure 27: Isolated colonies were purified again on new R2 agar medium plates with X-gal.
Environmental conditions: Temperature: 10 ˚C, pH: 7, Incubation Time: 43 days.
88
Figure 28: Isolated colonies were purified again on new Marine agar plates with X-gal.
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 44 days.
89 incubation, strains showed different sizes and shades of blue colonies on X-gal. Four strains showed blue color, fifteen showed (blue and white), one turned (blue, white and yellow), one (blue and yellow), twelve dark blue, one (dark blue and white), one (very light blue and white) and one plate had only white colonies respectively (Fig. 29). Blue colonies were streaked again on new plates from blue and white strain collection (Fig. 30). On the basis of growth rate, twenty strains showed slow growth rate, seven showed medium and nine strains had a good growth on marine agar at pH 10. Colony size was also different for different plates, fifteen plates had small colonies, nine with medium colonies and twelve plates exhibited large colony size (Annexure Table 13).
Marine Agar ( pH 7)
Sixty six new plates were prepared with marine agar at pH 7 with X-gal. Twenty colonies were picked from plate 1 A–1 (1A), 2 (1A), 3 (1A), 4 (1A), 5 (1A), 6 (1A), 7 (1A), 8 (1A), 9 (1A), 10 (1A), 11 (1A), 12 (1A), 13 (1A), 14 (1A), 15 (1A), 16 (1A), 17 (1A), 18 (1A), 19 (1A), 20 (1A). Twenty colonies were selected from plate 2 A–1 (2A), 2 (2A), 3 (2A), 4 (2A), 5 (2A), 6 (2A), 7 (2A), 8 (2A), 9 (2A), 10 (2A), 11 (2A), 12 (2A), 13 (2A), 14 (2A), 15 (2A), 16 (2A), 17 (2A), 18 (2A), 19 (2A), 20 (2A). Ten colonies from plate 3 A–1 (3A), 2 (3A), 3 (3A), 4 (3A), 5 (3A), 6 (3A), 7 (3A), 8 (3A), 9 (3A), 10 (3A) and fifteen colonies from plate 4 A–1 (4A), 2 (4A), 3 (4A), 4 (4A), 5 (4A), 6 (4A), 7 (4A), 8 (4A), 9 (4A), 10 (4A), 11 (4A), 12 (4A), 13 (4A), 14 (4A), 15 (4A) with one colony from Ikka sample I (3) were selected and streaked on new plates with Marine agar at pH 7 with X-gal and incubated at 10 ˚C for 38 days (Fig. 31). Plates showed different combination of bacterial strains, five with blue color, one with (blue and white), three with dark blue, two with green, two with (light blue and yellow), two with orange, eight with (orange and blue), one with (orange, blue and white), one with (orange and light blue), three with white, fourteen with (blue and white), twelve with (white and light blue), eight with (yellow and blue), three with (yellow, blue and orange) and one with no growth (Fig. 32). Blue colonies were transferred again on new plates to get purified blue strains (Fig. 33). Out of sixty six strains, sixty two showed slow growth, three had medium growth and there was no growth for one strain. Variation in size was also found among sixty six strains. Fifty one found in small size, twelve in medium size, two in large size and no growth for one strain was observed (Annexure Table 13). 90
16
14
12 10 8 6
4 No. of of No. Colonies (cfu) 2 0 Blue Blue+ blue + Blue+ Dark dark blue v. light white white white + yellow blue + white blue + yellow white Color of Colonies
Figure 29: β-galactosidase producing bacterial strains from Antarctica were categorized on the basis of their color on Marine agar medium with X- gal.
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 44 days.
91
Figure 30: Isolated colonies were purified again on new Marine agar plates with X-gal.
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 44 days.
92
Figure 31: Isolated colonies were purified on new Marine agar plates with X-gal. Environmental conditions: Temperature: 10 ˚C, pH: 7, Incubation Time: 38 days.
93
16 14 12 10 8 6 4 2 No. of of No. Colonies (cfu) 0
Color of Colonies
Figure 32: β-galactosidase producing bacterial strains were categorized on the basis of their color on Marine agar medium with X-gal.
Environmental conditions: Temperature: 10 ˚C, pH: 7, Incubation Time: 38 days.
94
Figure 33: Isolated colonies were screened again on new Marine agar plates with X-gal.
Environmental conditions: Temperature: 10 ˚C, pH: 7, Incubation Time: 38 days.
95
COLLECTION OF BEST BLUE STRAINS ON DIFFERENT MEDIA AND DIFFERENT pH
R2 Agar (pH 10)
R2 agar medium was prepared at pH 10 with X-gal and plates were prepared to inoculate the samples (1-19) – 18 (1A), 19 (1A), 20 (1A), 1 (3A), 10 (3A), 7 (3A), 6 (3A), 1 (2A), 2 (2A), 4 (2A), 7 (2A), 8 (2A), 9 (2A), 10 (2A), 13 (2A), 15 (2A), 16 (2A), 18 (2A) and 20 (2A) by streaking method and incubated at 10 ˚C for 42 days. After incubation pure blue colonies were obtained (Fig. 34). Strains showed different shades of blue color on X-gal with variation in the size of colonies. Five strains were in light blue color, seven in dark blue and seven were in very dark blue color. Five plates showed small size of colonies, eleven showed medium and three showed large size of colonies respectively (Annexure Table 14).
Marine Agar (pH 7)
Ninteen plates were prepared again with Marine agar at pH 7 with X-gal. Strains (20- 38) – 4 (1A), 8 (1A), 9 (1A), 12 (1A), 13 (1A), 14 (1A), 15 (1A), 16 (1A), 17 (1A), 18 (1A), 19 (1A), 20 (1A), 10 (2A), 11 (2A), 12 (2A), 15 (2A), 18 (2A), 19 (2A) and 2 (3A) were selected, streaked on new plates with marine agar pH 7 and incubated for 42 days at 10 ˚C. Nine strains represented the light blue color of colonies, nine with dark blue color and one with very dark blue color. Difference in sizes was also observed. Fifteen plates showed small colonies, one medium and three plates had large colonies respectively (Annexure Table 14).
Marine Agar (pH 10)
Strains (38-57) – 5 (1A) i, 16 (1A) ii, 17 (1A) ii, 20 (1A) ii, 68 (1A) ii, 1 (2A) i, 11 (2A) ii, 17 (2A) ii, 22 (2A) ii, 26 (2A) ii, 37 (2A) ii, 26 (1A) iii, 27 (1A) iii, 29 (1A) iii, 54 (1A) iv, 48 (1A) v, 75 (3A) i, 32 (3A) i and 16 (3A) were inoculated (streaking) again on Marine agar at pH 10 with X-gal and incubated at 10 ˚C for 42 days. After incubation, results were excellent, all 19 strains showed very dark blue colonies and all in a large size (Annexure Table 14).
96
R2 Agar (pH 7)
Only two plates were prepared with R2 agar at pH 7 with X-gal and inoculated with two samples (58, 59) – 4 (3A) and 7 (3A) then incubated at 10 ˚C for 42 days. With both strains found very dark blue and large colonies (Annexure Table 14).
Ikka Samples
Two Ikka samples (60 and 61) – I (7) and I (3) were streaked on new plates with R2 agar, pH 10 and Marine agar, pH 7 with X-gal respectively. Incubated at 10 ˚C for more than one month (42 days). Light blue and small colonies were observed after incubation for both strains (Annexure Table 14).
DNA AMPLIFICATION FOR UP-PCR
DNA extraction was done for the selected 61 strains. DNA templates were used in diluted (10 times) and undiluted form. Strains 1, 2 and 3 showed bands with undiluted DNA templates and no bands with ten times diluted templates. Strains 4 and 5 had no bands, both in diluted and undiluted form. Strains 6, 7, 8, 9 and 10 had bands with undiluted templates and no bands with diluted templates. Strains 11 and 12 showed weak bands with undiluted templates and no bands with diluted templates (Fig. 35). Strains 13 and 14 also showed no bands with both diluted and undiluted form. Strain 15 had bands with undiluted template and no band with diluted template, strain 16 showed weak bands with undiluted template and no band with diluted template. Strains 17, 18 and 19 showed bands with undiluted templates and no bands with diluted ones. Strain 20 showed bands with undiluted template but also had weak bands with diluted template as well. Strain 21 showed bands with undiluted template and no band with diluted DNA template. Strain 22 showed bands with both templates (undiluted and diluted). Strains 23 and 24 showed weak bands with undiluted form of DNA template and no bands with diluted templates (Fig. 36). Strain 25 showed weak bands with undiluted template and no bands with diluted template. Strains 26, 27, 28 and 29 showed no bands with both undiluted and diluted templates respectively. Strain 30 showed no band with undiluted template and had weak bands with diluted template. No bands for strains 31, 32, 33 and 34 for both undiluted and diluted DNA templates. Strain 35 showed weak bands for undiluted template and no bands for diluted template. Strain 36 had no bands for both diluted and undiluted DNA templates (Fig. 37). Strain 37 had also no bands with both diluted and undiluted 97
Figure 34: β-galactosidase producing bacterial strains (purified colonies) were obtained on R2 agar medium with X-gal.
Environmental conditions: Temperature: 10 ˚C, pH: 10, Incubation Time: 42 days.
98
Figure 35: Band patterns of diluted (10 times) and undiluted DNA templates used for the amplification of PCR reaction. Strains 1-12 were used as undiluted (1st column) and diluted (2nd column) form in the PCR reaction
99
Figure 36: Diluted (10 times) and undiluted DNA templates were used for the amplification of PCR reaction. Strains 12-24 were used as undiluted (1st column) and diluted (2nd column) form in the PCR reaction, band patterns were shown in the figure.
100
Figure 37: Diluted (10 times) and undiluted DNA templates were used for the amplification of PCR reaction. Strains 24-36 were used as undiluted (1st column) and diluted (2nd column) form in the PCR reaction, band patterns were shown in the figure.
101 templates. Strain 38 showed strong bands with undiluted template and weak bands with diluted one and strain 39 had weak bands with undiluted template and very strong band with diluted DNA template. Strains 40, 41 and 42 showed very weak bands with undiluted templates and no bands with diluted templates. Strain 43 had no bands with undiluted template and showed bands with diluted template. Strain 44 showed weak bands with undiluted template and comparatively strong bands with diluted template. Strain 45 had weak bands with undiluted DNA template and no bands with diluted template. Strain 46 showed no bands with undiluted template and weak bands with diluted template. Strain 47 had no bands at all for both diluted and undiluted DNA templates. Strain 48 showed no bands for undiluted DNA template and weak bands for diluted template (Fig. 38). No bands were observed for strains 49, 50, 51 and 52 respectively with both undiluted and diluted DNA templates. Strains 53 and 54 showed weak bands with undiluted templates and no bands with diluted DNA templates. Strains 55 and 56 showed bands for both undiluted and diluted templates and strain 57 showed no bands with both templates (diluted and undiluted). Strains 58 and 59 had bands for both diluted and undiluted templates and strain 60 showed bands for undiluted template and no bands for diluted template (Fig. 39). Finally strain 61 had no bands for both (diluted and undiluted) DNA templates (Fig. 40).
UP-PCR
Twenty strains (1, 8, 10, 15, 18, 19, 20, 21, 22, 30, 38, 39, 43, 53, 55, 56, 58, 59, 60 and 61) were selected and DNA templates were used for further analysis from UP- PCR. Strains 1, 8, 10, 15, 18 and 19 showed different band patterns while strains 20 and 21 showed almost the same band patterns. Strain 22 also showed different band pattern. There was no band for strain 30. Strain 38 also showed different band pattern (Fig. 41). No bands for strains 39 and 43 respectively. Different band patterns were observed for strains 53, 55 and 56 respectively. Strains 58 and 59 showed almost the same band patterns while strain 60 had different band pattern and no bands for strain 61 (Fig. 42).
Amplification of DNA templates for 16s Analysis
14 strains (1, 8, 9, 16, 19, 20, 21, 22, 38, 53, 55, 56, 58 and 59) were selected and used their DNA as a template for the amplification of 16s analysis. Strains 8 and 19 102 showed weak bands while strains 1, 9, 16, 20, 21, 22, 38, 53, 55, 56, 58 and 59 had strong bands. There was no band for strain 55 (Fig. 43).
16s Sequencing
Finally 14 strains from Antarctic and Ikka samples (A, B, 1, 9, 16, 8, 20, 21, 22, 38, 53, 56, 58 and 59) were used for 16s sequencing. Five samples (9, 8, 38, 53 and 56) did not work and nine samples (A, B, 1, 16, 20, 21, 22, 58 and 59) were successfully amplified. Results were presented in Table 1. Origin of sample A and B was Ikka column and growth occurred at 10˚C on R2 agar at pH 10. Closest relative of strain A, was Pseudomonas pelagia CL- AP6(T) with the sequence similarity of 97.15 %. Strain B showed the highest sequence similarity with Pseudomonas pelagia CL- AP6(T) with 73.32 % identity. Strains 1 and 16 belonged to Mc Murdo, USA and growth occurred at 10˚C on R2 agar, pH 10. Strain 1 showed the highest sequence similarity with Pseudomonas pelagia CL- AP6(T) as well but with 98.50 % identity. Strain 16 had the highest similarity with Marinilactibacilus piezotolerans LT20(T) with 94.89 % identity.
Strains 20, 21and 22 were originated from Canary pond and isolated on Marine agar at pH 7 at 10 ˚C. Closest sequence similarity for strain 20 was found with Alkalibacterium subtropicum O24-2(T) with 94.66 % identity. Strain 21 showed the closest relative, Alkalibacterium putridalgicola T129-2-1(T) with 92.00 % identity. Closest relative for strain 22 was Marinobacter psychrophilus 20041(T) with 96.11 % identity. Origion of strains 58 and 59 was Kingfisher pond and growth occurred at pH 7 on R2 agar at 10 ˚C. Both strains were similar according to 16s sequencing and found highest similarity with Rhizobium herbae CCBAU83011(T) with 95.22 % and 96.66 % identity respectively.
Whole Genome Sequencing
Five strains (B, 20, 22, 58 and 59) were used for whole genome sequencing. Results from the whole genome sequencing were tabulated in Table 2. DNA concentration (ng/µl) used for whole genome sequencing were 30, 30, 83.8, 33, 48.9 (ng/µl) for strains B, 20, 22, 58 and 59 respectively. Strain B showed the highest whole genome sequences similarity with Pseudomonas pelagia CL-AP6(T) with 99.66 % identity. Strain 20 showed 97.83 % identity with Alkalibacterium subtropicum O24-2(T). Closest relative for strain 22 was Marinobacter psychrophilus 20041(T) with 99.32 % 103
Figure 38: Diluted (10 times) and undiluted DNA templates were used for the amplification of PCR reaction. Strains 37-48 were used as undiluted (1st column) and diluted (2nd column) form in the PCR reaction, band patterns were shown in the figure.
104
Figure 39: Diluted (10 times) and undiluted DNA templates were used for the amplification of PCR reaction. Strains 49-60 were used as undiluted (1st column) and diluted (2nd column) form in the PCR reaction, band patterns were shown in the figure.
105
Figure 40: Diluted (10 times) and undiluted DNA templates were used for the amplification of PCR reaction. Strains 1-12 were used as undiluted (1st column) and diluted (2nd column) form in the PCR reaction, band patterns were shown in the figure.
106
Figure 41: β-galactosidase producing strains were selected for UP-PCR reaction. Band patterns were used to differentiate between bacterial strains as shown in the figure.
107
Figure 42: β-galactosidase producing strains were selected for UP-PCR reaction. Band patterns were used to differentiate between bacterial strains as shown in the figure.
108
Figure 43: Band patterns for different strains PCR reaction (amplification of 16s rDNA) of 14 strains (1, 8, 9, 16, 19, 20, 21, 22, 38, 53, 55, 56, 58 and 59) were shown in the figure.
109
Table 1. Showing the highest sequence similarity with other bacterial strains.
Bacterial Origion Media pH Temp 16s rDNA Sequencing Similarity isolates ◦C (Closest Match) (%) A Ikka R2 10 10 Pseudomonas pelagia CL-AP6(T) 97.15 column Agar B Ikka R2 10 10 Pseudomonas pelagia CL-AP6(T) 73.32 column Agar 1 Mc R2 10 10 Pseudomonas pelagia CL-AP6(T) 98.50 Murdo Agar 16 Mc R2 10 10 Marinilactibacilus piezotolerans 94.89 Murdo Agar LT20(T) 20 Canary Marine 07 10 Alkalibacterium subtropicum 94.66 pond Agar O24-2(T) 21 Canary Marine 07 10 Alkalibacterium putridalgicola 92.00 pond Agar T129-2-1(T) 22 Canary Marine 07 10 Marinobacter psychrophilus 96.11 pond Agar 20041(T) 58 Kingfisher R2 07 10 Rhizobium herbae 95.22 pond Agar CCBAU83011(T) 59 Kingfisher R2 07 10 Rhizobium herbae 96.66 pond Agar CCBAU83011(T)
110 identity. Closest match for strains 58 and 59 was Rhizobium giardinii H152(T) with 98.2 % identity (Table 2). From the whole genome sequencing two strains of Pararhizobium sp. (NAQVI-58 and NAQVI-59) were selected for further analysis. Both showed the highest sequence similarity with Rhizobium giardinii H152(T) (98.2 %) identity. Growth occured at 10 ˚C on R2 agar, pH 7 from Kingfisher pond, Antarctica (Table 3).
GENOMIC ANALYSIS
Genomic distribution was done on the basis of their function for strain NAQVI-58 (5,309,041 bp) with contigs 162 and NAQVI-59 (5,457,546 bp) with contigs 161 were represented in (Fig. 44). Three hundred and two genes were involved in the functioning of cofactors, vitamins, prosthetic groups and pigments. One hundred and twenty eight genes were involved in cell wall and capsule function. Eighty three were in virulence, disease and defence, seventeen in potassium metabolism, five in photosynthesis, forty one in miscellaneous, thirty one in (phages, prophages, transposable elements, plasmids), one hundred and eighty five in membrane transport, fifty nine in iron acquisition and metabolism, one hundred and forty three in RNA metabolism, one hundred and fifteen in nucleosides and nucleotides, two hundred and sixty one in protein metabolism, twenty four in cell division and cell cycle, one hundred and twenty two in motility and chemotaxis, one hundred and two in regulation and cell signaling, seven in secondary metabolism, ninty eight in DNA metabolism, one hundred and forty eight in (fatty acids, lipids and Isoprenoids), forty eight in nitrogen metabolism, one in dormancy and sporulation, one hundred and forty four for respiration, one hundred and eighty five for stress response, forty three for metabolism of aromatic compounds, four hundred and seventy one for amino acids and derivatives, thirty nine for sulfur metabolism, sixty five for phosphorus metabolism and five hundred and forty six for carbohydrates respectively.
Comparison of genomic data between two organisms
Rhizobium sp. 58 (6666666.166359) (NAQVI-58) was used as a reference organism to compare with Rhizobium sp. 59 (6666666.166360) (NAQVI-59). It showed the percent protein sequence identity with bidirectional best hit and unidirectional best hit. Table showed the number of contigs, genes and length of reference organism
111
Table 2. Whole genome sequencing results showing the highest sequence similarity with other bacterial strains. Sample Media pH Temp Gram DNA Whole Genome Sequencing Similarity No. ◦C Staining Conc. (Closest Match) (%) (ng/µ) B R2 10 10 -ve 30 Pseudomonas pelagia CL- 99.66 Agar AP6(T) 20 Marine 07 10 +ve 30 Alkalibacterium 97.83 Agar subtropicum O24-2(T) 22 Marine 07 10 -ve 83.8 Marinobacter psychrophilus 99.32 Agar 20041(T) 58 R2 07 10 -ve 33 Rhizobium giardinii H152 (T) 98.2 Agar 59 R2 07 10 -ve 48.9 Rhizobium giardinii H152 (T) 98.2 Agar
Table 3. Description of new Pararhizobium sp.
Sample Species name Whole Similarity Media Temp pH Origion No. genome (%) ˚C sequencing NAQVI- Pararhizobium Rhizobium 98.2 R2 10 7 Kingfisher 58 sp. (i) giardinii Agar pond H152 (T) NAQVI- Pararhizobium Rhizobium 98.2 R2 10 7 Kingfisher 59 sp. (ii) giardinii Agar pond H152 (T)
112
NAQVI-(58) 5,309,041 bp , 162 Contigs
NAQVI-(59) 5,457,546 bp , 161 Contigs
Figure 44: Distribution of genes on the basis of their function for strain NAQVI- 58 (5,309,041 bp) with contigs 162 and NAQVI-59 (5,457,546 bp) with contigs 161 were shown in the figure.
113
(NAQVI-58) with hits of No. of contigs and genes of test organism (NAQVI-59) respectively (Fig. 45).
Gene cluster containing β-galactosidase
The gene cluster for strain NAQVI-58 contained (ABC transporter sugar binding protein, Glycerol-3-phosphate ABC transporter permease protein and β-galactosidase producing gene) with Ribose ABC transporter, oxidoreductase and β-galactosidase (Fig. 46).
PHYLOGENETIC ANALYSIS
16s rRNA Gene Sequence Analysis
16s rRNA Sequences were extracted; trimmed, aligned and phylogenetic trees were constructed. The 16S rRNA gene sequence analysis indicated that strain NAQVI-58 and NAQVI-59 belonged to the family Rhizobiaceae of the order Rhizobiales, class Alphaproteobacteria. Strain NAQVI-58 and NAQVI-59 formed a distinct subclade within the genus Pararhizobium in the phylogenetical tree (Fig. 47). Sequence similarity calculations (over 1400 bp) showed that strains NAQVI-58 and NAQVI-59 were closely related to Pararhizobium giardinii H152T (98.1 %), Pararhizobium herbae CCBAU 83011T (98.1 %) and P. polonicum F5.1T CCNWGS 0238T (98.1 %) (Annexure Table 15. Fig. 47).
Phylogenetic Analysis of house keeping genes
(atpD Gene Sequence Analysis)
Maximum likelihood tree showed the phylogenetic relationship between atpD sequences of strains NAQVI 58 and NAQVI 59 and related species. The tree is based on sequences of atpD shown in Annexure Table 16. Bootstrap values expressed as percentages of 1000 replications were shown next to the nodes. Bar indicates estimated substitutions per site. Tree showed the phylogenetic relationship of strains (NAQVI 58 and NAQVI 59) with other Rhizobium species. Sequence similarity of atpD house keeping gene showed that strains NAQVI-58 and NAQVI-59 were closely related to Pararhizobium capsulatum DSM 1112T, P. polonicum F5.1T, and Pararhizobium herbae CCBAU 83011T respectively (Annexure Table 16. Fig. 48).
114
(recA Gene Sequence Analysis)
Maximum likelihood tree was constructed by using recA gene sequences of NAQVI- 58, NAQVI-59 and related Rhizobium species. The tree was based on sequences of recA shown in Annexure Table 16. Phylogenetic relationship of recA (house keeping gene) sequences of NAQVI-58 and NAQVI-59 with related Rhizobium species showed that strains were closely related to Pararhizobium capsulatum DSM 1112T, Pararhizobium herbae CCBAU 83011T and then Pararhizobium polonicum F5.1T (Annexure Table 16. Fig. 49).
(rpoB Gene Sequence Analysis )
Sequences of rpoB were extracted and tree was constructed by using Maximum likelihood method. Sequences of rpoB were given in Annexure Table 16. Phylogenetic tree based on rpoB genes showed the closest relatives of strains NAQVI-58 and NAQVI-59 were Pararhizobium capsulatum DSM 1112T, Pararhizobium helanshanense CCNWQTX14T and Pararhizobium giardinii H152T (Annexure Table 16. Fig. 50).
Phylogenetic Analysis of Three House Keeping Genes
(atpD, recA and rpoB)
The phylogenetical relationship was confirmed by analyses of the three housekeeping genes atpD, recA and rpoB from strains NAQVI-58 and NAQVI-59 and related species within the Rhizobiaceae (Annexure Table 16). Phylogenetical analysis of concatenated atpD, recA, and rpoB sequences confirmed that strains NAQVI-58 and NAQVI-59 formed a separate clade in the combined phylogenetic tree (Fig. 51). Maximum likelihood tree showing the phylogenetic relationship between strains NAQVI-58, NAQVI-59 and related Rhizobium species. The tree was based on concatenated sequences of atpD, recA, and rpoB (Annexure Table 16). Combined tree showed that NAQVI-58 and NAQVI-59 were closely related to Pararhizobium giardinii H152T, Pararhizobium herbae CCBAU 83011T and P. polonicum F5.1T respectively (Annexure Table 16, Fig. 51).
115
Figure 45: Figure showed the comparison of data between two organisms (number of contigs, genes and length of reference organism (NAQVI- 58) were compared with number of contigs and genes of test organism (NAQVI-59) respectively).
116
β-Galactosidase
ABC transporter sugar binding protein Glycerol-3-phosphate ABC transporter,permease protein
Ribose ABC Oxidoreductase transporter β-Galactosidase
Figure 46: Gene cluster for strain NAQVI-58 and NAQVI-59 was the same and contained (ABC transporter sugar binding protein, Glycerol-3- phosphate ABC transporter permease protein and β-galactosidase producing gene) with Ribose ABC transporter, oxidoreductase and β- galactosidase as shown in the figure.
117
Figure 47. Maximum likelihood tree showing the phylogenetic relationship between strains NAQVI 58 and NAQVI 59T and related species based on 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 500 replications) are given at the nodes. GenBank accession numbers are given in parantheses. Bar shows estimated nucleotide substitutions per site.
118
Figure 48. Maximum likelihood tree showing the phylogenetic relationship between atpD sequences of strains NAQVI 58 and NAQVI 59 and related species. The tree is based on sequences of atpD shown in Annexure Table 16. Bootstrap values expressed as percentages of 1000 replications are shown next to the nodes. Bar indicates estimated substitutions per site.
119
Figure 49. Maximum likelihood tree showing the phylogenetic relationship between recA sequences of strains NAQVI 58 and NAQVI 59 and related species. The tree is based on sequences of recA shown in Annexure Table 16. Bootstrap values expressed as percentages of 1000 replications are shown next to the nodes. Bar indicates estimated substitutions per site.
120
Figure 50. Maximum likelihood tree showing the phylogenetic relationship between rpoB sequences of strains NAQVI 58 and NAQVI 59T and related species. The tree is based on sequences of rpoB shown in Annexure Table 16. Bootstrap values expressed as percentages of 1000 replications are shown next to the nodes. Bar indicates estimated substitutions per site.
121
Figure 51. Maximum likelihood tree showing the phylogenetic relationship between strains NAQVI 58 and NAQVI 59 and related species. The tree is based on concatenated sequences of atpD, recA, and rpoB. Bootstrap values expressed as percentages of 1000 replications are shown next to the nodes. Bar indicates estimated substitutions per site.
122
ANI (Average Nucleotide Identity)
The ANI values for strains NAQVI-58 and NAQVI-59 when compared to the closely related species, P. giardinii H152T and P. polonicum F5.1T were 80.5 % and 81.1 %, respectively.
GGDC Analysis
GGDC (Genome to Genome Distance Calculator) showed that the strains NAQVI-58 and NAQVI-59 were 22.1 % and 22.6 % identical to P. giardinii H152T and P. polonicum F5.1T, respectively.
PHENOTYPIC CHARACTERIZATION
Growth on Yeast Mannitol Agar
Strains NAQVI-58 and NAQVI-59 and three reference strains (Pararhizobium giardinii H152T, Parahizobium herbae CCBAU 83011T and P. sphaerophysae CCNWGS 0238T) were used for further characterization. YMA (Yeast Mannitol Agar) was used to retrieve the strains with pH 7 at 28 ˚C for 2-3 days.
Morphology of bacterial strains
Morphology of strains NAQVI-58 and NAQVI-59 was observed after 3 days of incubation at 28 ˚C (pH 7). Colonies were creamy white, circular, smooth and half transparent with diameter 3 mm for NAQVI-58 and 2 mm for NAQVI-59 respectively (Table 4, Fig. 52 and Fig. 53).
Effect of Temperature on the growth of bacterial strains
Strains NAQVI-58 and NAQVI-59 were inoculated in YMA broth and incubated at different temperatures (4 ˚C, 10 ˚C, 15 ˚C, 20 ˚C, 28 ˚C and 37 ˚C) then measured its
OD600 (Optimal Density). Duplicate set of each strain was prepared for each temperature. Strains NAQVI-58 and NAQVI-59 showed the maximum values of
OD600 at 20 ˚C and showed no growth at 37 ˚C (Annexure Table 17).
Effect of pH on the growth of bacterial strains pH range of the broth was adjusted from 4-11 (4, 5, 6, 7, 8, 9, 10, 11) for strains NAQVI-58 and NAQVI-59 and incubated at 28 ˚C. For each sample duplicate set was prepared and then measured its OD600. Maximum value was observed at pH (9) and pH (7) for strains NAQVI-58 and NAQVI-59 respectively (Annexure Table 18). 123
Table 4. Morphological characteristics of selected bacterial isolates (NAQVI-58 and NAQVI-59).
Cell Morphology NAQVI-58 NAQVI-59 Configuration Circular Circular Margin Entire Entire Elevation Convex Convex Surface Smooth Smooth Pigment Creamy white Creamy white Opacity Translucent Translucent Cell shape Rods Rods Arrangement Occurred singly/paired Occurred singly/paired
124
Figure 52: Growth of bacterial strain NAQVI-58 on YMA at pH 7 and incubated at 28 ˚C for 3 days. Colonies were creamy white, circular, smooth and half transparent with diameter of 3 mm for NAQVI-58 as shown in the figure.
125
Figure 53: Growth of bacterial strain NAQVI-59 on YMA at pH 7 and incubated at 28 ˚C for 3 days. Colonies were creamy white, circular, smooth and half transparent with diameter of 2 mm for NAQVI-59 as shown in the figure.
126
Effect of NaCl on the growth of bacterial strains
YMA broth was prepared without NaCl then adjusted its range from 0-5 % (0, 1, 2, 3, 4 and 5) (w/v) for strains (NAQVI-58, NAQVI-59) and incubated at 28 ˚C. Duplicate set was prepared for each sample. Maximum value of OD600 was found at 0 % for strain NAQVI-58 and 1 % for strain NAQVI-59 respectively (Annexure Table 19).
Gram-Staining of bacterial strains
Gram-Staining showed negative results for both strains (NAQVI-58 and NAQVI-59) then performed the KOH test for both strains to cross check the Gram-staining results. KOH test also showed that they are Gram-negative (Table 5).
Motility Test of bacterial strains
Semi-solid agar (Yeast Mannitol Agar) was used to perform the motility test. Test showed positive results after 3 days incubation at 20 ˚C (Table 5, Fig. 54).
Catalase Test of bacterial strains
Catalase test was performed for both strains NAQVI-58 and NAQVI-59. Presence of bubbles showed the positive results (Table 5, Fig. 55).
Aerobic Test of bacterial strains
Absolutely no growth was observed for strains NAQVI-58 and NAQVI-59 in an anaerobic jar. Results showed that they are strictly aerobic (Table 5, Fig. 56).
Spore Formation for bacterial strains
No spore forming genes and no spores were found for strains (NAQVI-58 and NAQVI-59) (Table 5).
Resistance to Antibiotics
Five antibiotics, Kanamycin 25 mg/ml (MQ- H2O), Penicillin 1 mg/ml, Tetracyklin 10 mg/ml (70 % Et OH), Ampicilin 1000 mg/ml (70 % Et OH) and Streptomycin 25 mg/ml (MQ- H2O) were tested for strains NAQVI-58 and NAQVI-59. Results showed that both strains were resistant to Penicillin, Ampicilin and Streptomycin and sensitive to Kanamycin and Tetracyklin (Table 5, Fig. 57, 58, 59 and 60).
127
Table 5. Distinctive features of strains NAQVI-58 and NAQVI-59 are represented in the table.
Reactions were scored as: +, positive; -, negative. All data were obtained in this study. (1) Strain NAQVI-58, (2) Strain NAQVI-59. NAQVI-58 NAQVI-59 Characteristic 1 2 Growth Temp optimum, °C 20 20 Temp range, °C 4-37 4-37 pH optimum 9 7 pH range 4-11 4-11 NaCl optimum, % 0 1 NaCl, range % 0-5 0-5 Resistance to antibiotics
Kanamycin 25mg/ml (MQ- H2O) - - Penicillin 1mg/ml + + Tetracyklin 10 mg/ml (70% Et OH) - - Ampicilin 1000 mg/ml (70% Et OH) + +
Streptomycin 25mg/ml (MQ- H2O) + + Gram-Staining - - Motility + + Catalase test + + Aerobic + + Spore formation - - DNA G+C mol% 60.63 60.67
128
Figure 54: Strains NAQVI-58 and NAQVI-59 were inoculated in the sterile semi solid agar (YMA) at pH 7 and incubated at 20 ˚C for 3 days. Growth were spread across the semi solid medium (0.5 % agar) and showed the motility of the strains.
129
Figure 55: Catalase activity for the strains NAQVI-58 and NAQVI-59 was determined by the production of bubbles in 3 % (v/v) (H2O2) as shown in the figure.
130
Figure 56: Strains NAQVI-58 and NAQVI-59 were inoculated on YMA (Yeast Mannitol Agar) at pH 7 and incubated at 20 ˚ for 3 days. Absolutely no growth was observed for both strains in an anaerobic jar as shown in the figure.
131
Figure 57: YMA (Yeast Mannitol Agar) plates at pH 7 were used to check the susceptibility of antibiotics. Discs impregnated with 2 µl of antibiotic, Kanamycin 25 mg/ml (MQ- H2O) were used and zone of growth inhibition was measured after incubating at 20 ˚C for 3 days. Both strains (NAQVI-58 and NAQVI-59) were sensitive to antibiotic Kanamycin (Km) as shown in the figure.
132
Figure 58: YMA (Yeast Mannitol Agar) plates at pH 7 were used to check the susceptibility of antibiotics. Discs impregnated with 2 µl of antibiotic, Penicillin 1 mg/ml were used and zone of growth inhibition was measured after incubating at 20 ˚C for 3 days. Both strains (NAQVI-58 and NAQVI-59) were resistant to antibiotic Penicillin (Pen) as shown in the figure.
133
Figure 59: YMA ((Yeast Mannitol Agar) plates at pH 7 were used to check the susceptibility of antibiotics. Discs impregnated with 2 µl of antibiotic, Tetracyklin 10 mg/ml (70 % Et OH) were used and zone of growth inhibition was measured after incubating at 20 ˚C for 3 days. Both strains (NAQVI-58 and NAQVI-59) were sensitive to antibiotic Tetracyklin (Tet) as shown in the figure.
134
Figure 60: YMA (Yeast Mannitol Agar) plates at pH 7 were used to check the susceptibility of antibiotics. Discs impregnated with 2 µl of antibiotic, Ampicilin 1000 mg/ml (70 % Et OH) were used and zone of growth inhibition was measured after incubating at 20 ˚C for 3 days. Both strains (NAQVI-58 and NAQVI-59) were resistant to antibiotic Ampicilin (Amp) as shown in the figure.
135
BIOCHEMICAL CHARACTERIZATION
Utilization of Enzymes
Biochemical tests were performed using API 20E kits for strains NAQVI-58, NAQVI-59 and three reference strains (Pararhizobium giardinii H152T, Parahizobium herbae CCBAU 83011T and P. sphaerophysae CCNWGS 0238T). Strains (NAQVI-58, NAQVI-59) showed positive results for β-galactosidase (ONPG) whereas Pararhizobium giardinii H152T, Parahizobium herbae CCBAU 83011T and P. sphaerophysae CCNWGS0238T showed negative results for ONPG (β- galactosidase). Arginine dihydrolase (ADH), Lysine decarboxylase (LDC), Ornithine decarboxylase (ODC), Citrate utilization (CIT) were positive for NAQVI-58, NAQVI-59, Pararhizobium giardinii H152T and Parahizobium herbae CCBAU T T 83011 while negative for P. sphaerophysae CCNWGS 0238 . H2S production (H2S) was negative for NAQVI-58, NAQVI-59, Pararhizobium giardinii H152T, and P. sphaerophysae CCNWGS 0238T and positive for Parahizobium herbae CCBAU 83011T. Urease (URE) test was negative for P. sphaerophysae CCNWGS 0238T and positive for NAQVI-58, NAQVI-59, Pararhizobium giardinii H152T and Parahizobium herbae CCBAU 83011T. Tryptophane deaminase (TDA) was positive for (NAQVI-58, NAQVI-59) and negative for the other reference strains (Pararhizobium giardinii H152T, Parahizobium herbae CCBAU 83011T and P. sphaerophysae CCNWGS 0238T). Indole production (IND) was negative for all the test strains. Acetoin production (VP) was positive for NAQVI-58, NAQVI-59, Pararhizobium giardinii H152T and Parahizobium herbae CCBAU 83011T and only negative for P. sphaerophysae CCNWGS 0238T. Gelatinase (GEL) test was positive for all the test strains (NAQVI-58, NAQVI-59, Pararhizobium giardinii H152T, Parahizobium herbae CCBAU 83011T and P. sphaerophysae CCNWGS 0238T) (Table 6, Fig. 61).
Fermentation of Carbon Sources
Glucose (GLU), Mannitol (MAN), Inositol (INO), Sorbitol (SOR), Rhamnose (RHA) and Melibiose (MEL) tests were negative for all strains, NAQVI-58, NAQVI-59, Pararhizobium giardinii H152T, Parahizobium herbae CCBAU 83011T and P. sphaerophysae CCNWGS 0238T. Saccharose (SAC) was negative for NAQVI-58, NAQVI-59 and P. sphaerophysae CCNWGS 0238T whereas positive for 136
Table 6. Biochemical characteristics of strains NAQVI-58, NAQVI-59 and three reference strains.
(1) Strain NAQVI-58, ( 2) Strain NAQVI-59, (3) P. giardinii H152T, (4) P. herbae CCBAU 83011 T , (5) P. sphaerophysae CCNWGS 0238 T. Reactions were scored as: +, positive; -, negative. All data were obtained in this study. NAQVI- NAQVI- P. P. P. herbae 58 59T giardinii sphaerophysae Characteristic 1 2 3 4 5 Utilization of β- galactosidase - (ONPG) + + - - Arginine dihydrolase - (ADH) + + + + Lysine decarboxylase - (LDC) + + + + Ornithine - decarboxylase (ODC) + + + + Citrate utilization - (CIT) + + + + H2S production (H2S) - - - + - Urease (URE) + + + + - Tryptophane - deaminase (TDA) + + - - Indole production - (IND) - - - - Acetoin production - (VP) + + + + Gelatinase (GEL) + + + + + Fermentation of Glucose (GLU) - - - - - Mannitol (MAN) - - - - - Inositol (INO) - - - - - Sorbitol (SOR) - - - - - Rhamnose (RHA) - - - - - Saccharose (SAC) - - + + - Melibiose (MEL) - - - - - Amygdalin (AMY) - - + - - Arabinose (ARA) - - + - - Oxidase (OX) + + - - + Utilization of Nitrogen Sources (NH4)2 SO4 + + + + + KNO3 + + + + + L- Glutamic Acid + + + + + L- Valine - - + + +
137
Figure 61: Biochemical tests were performed using API 20E kits for strains NAQVI-58, NAQVI-59 and three reference strains (Pararhizobium giardinii H152T, Parahizobium herbae CCBAU 83011T and P. sphaerophysae CCNWGS 0238T).
138
Pararhizobium giardinii H152T and Parahizobium herbae CCBAU 83011T. Amygdalin (AMY) and Arabinose (ARA) were negative for NAQVI-58, NAQVI-59, Parahizobium herbae CCBAU 83011T and P. sphaerophysae CCNWGS 0238T and only positive for Pararhizobium giardinii H152T (Table 6, Fig. 60). Oxidase test showed positive results for NAQVI-58, NAQVI-59 and P. sphaerophysae CCNWGS 0238T while negative for Pararhizobium giardinii H152T and Parahizobium herbae CCBAU 83011T (Table 6, Fig. 62).
Fermentation of Nitrogen Sources
(NH4)2 SO4, KNO3 and L- Glutamic Acid showed positive results for all the test strains. L- Valine was negative for strains (NAQVI-58, NAQVI-59) and positive for Pararhizobium giardinii H152T, Parahizobium herbae CCBAU 83011T and P. sphaerophysae CCNWGS0238T (Table 6).
CHEMOTAXONOMIC ANALYSIS
Fatty Acids
Cellular fatty acid composition of strains NAQVI-58 and NAQVI-59 was shown in Table 7. Data were percentages of total fatty acids. The major fatty acid was determined to be C18:1 ω7c. Values were found 60.5 and 42.9 for strain NAQVI-58 and NAQVI-59 respectively (Table 7).
Polar Lipids
The polar lipid profile for both strains consisted of phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmethanolamine and in addition to three unknown lipids, one unknown glycolipid, and five unidentified phospholipids (Fig. 63).
Respiratory Quinones
Ubiquinone (Q-10) was 100% for both strains.
ELECTRON MICROSCOPY
Transmission electron microscopy for strains NAQVI-58 and NAQVI-59 showed the size, shape and arrangement of cells. It showed 1 µm long rod shaped cells, mostly occurred in pairs (Fig. 64).
139
Table 7. Cellular fatty acid (%) composition of strains NAQVI-58 and NAQVI-59.
Fatty acid NAQVI-58 (%) NAQVI-59 (%)
C15:1ω8c - 0.8
C16:0 8.4 10.4
C16:0 3-OH - 0.9
C18:0 5.8 8.9
C18:0 3-OH 1.1 1.9
C18:1 ω7c 60.5 42.9
C18:1 ω7c 11-methyl 6.2 13.6
C19:0 10-methyl 2.8 4.2 Summed feature 2* 6.0 9.0 Summed feature 3* 9.2 7.2
Summed features are groups of two or three fatty acids that cannot be separated by GLC using the MIDI system. * Summed feature 2 = C14:0 3-OH/C16:1 iso I * Summed feature 3 = C16:1 ω7c/C16:1 ω6c
140
Figure 62: Strains NAQVI-58, NAQVI-59 and three reference strains (Pararhizobium giardinii H152T, Parahizobium herbae CCBAU 83011T and P. sphaerophysae CCNWGS 0238T) were grown on YMA at pH 7 and incubated at 28 ˚C for 3 days. Oxidase test was performed by using strips, change in color showed that strains were positive for Oxidase test.
141
NAQVI 58 NAQVI 59
Figure 63: Two dimensional thin layer chromatography (TLC) of polar lipids of strains NAQVI-58 and NAQVI-59. L, lipid; GL, glycolipid; AL, aminolipid; PL, phospholipid; PG, phosphatidylglycerol; PE, phosphatidylethanolamine and PME, phosphatidylmethanolamine.
142
Figure 64: Transmission electron microscopy for strains NAQVI-58 and NAQVI- 59 showed the size, shape and arrangement of cells. It showed 1 µm long rod shaped cells, mostly occurred in pairs as shown in the figure.
143
ENZYME PRODUCTION
Effect of Temperature on the production of β-galactosidase
Production of β-galactosidase from NAQVI-58 was 0.1 U/ml and from NAQVI-59 was 0.16 U/ml was determined at 5 ˚C respectively. At 20 ˚C enzyme activity was measured as 0.15 U/ml from NAQVI-58 and 0.2 U/ml from NAQVI-59 respectively. The highest activity of enzyme was measured at 28 ˚C for both strains (NAQVI-58 and NAQVI-59 with values measured as 0.21 U/ml for NAQVI-58 and 0.33 U/ml for NAQVI-59 respectively. Values for strains 58 and 59 were 0.15 U/ml and 0.23 U/ml accordingly (Fig. 65).
Effect of pH on the production of β-galactosidase
The highest enzyme activity was found at pH 7 with values 0.19 U/ml for strain NAQVI-58 and 0.29 U/ml for strain NAQVI-59 respectivley. At pH 6, Enzyme activity for strain NAQVI-58 was 0.16 U/ml and for strain NAQVI-59 was 0.25 U/ml. The decrease in activity was found at pH 8 with values for strain NAQVI-58 (0.09 U/ml) and NAQVI-59 (0.1 U/ml) (Fig. 66).
Effect of Incubation Time on the production of β-galactosidase
The maximum activity was measured after 48 hrs of incubation with values 0.21 U/ml for starin NAQVI-58 and 0.34 U/ml for strain NAQVI-59. After 24 hours of incubation enzyme activity for NAQVI-58 was 0.15 U/ml and for NAQVI-59 was 0.2 U/ml. After 72 hours of incubation activity was measured as 0.11 and 0.15 for NAQVI-58 and NAQVI-59 respectively. Enzyme production decreased after 96 hours of incubation with 0.09 U/ml for NAQVI-58 and 0.11 U/ml for NAQVI-59 accordingly (Fig. 67).
ENZYME ASSAY
Effect of temperature on enzyme activity
Three strains were selected for enzyme assay (NAQVI-58, NAQVI-59 and NAQVI- 22). Assays were run in triplicates and incubated at different temperatures (5 ˚C, 20 ˚C, 28 ˚C and 37 ˚C) with different time intervals. NAQVI-58 and NAQVI-59 were incubated at 5 ˚C for 60 minutes and NAQVI-22 was incubated at 5 ˚C for 15 min. Strains (NAQVI-58 and NAQVI-59) incubated at 20 ˚C for 45 min and incubation for strain NAQVI-22 was done at 20 ˚C for 15 minutes. At 28 ˚C strains (NAQVI-58, 144
NAQVI-59) were incubated for 40 min and NAQVI-22 was incubated for 15 minutes respectively. Strains NAQVI-58 and NAQVI-59 were incubated at 37 ˚C for 40 min and NAQVI-22 was incubated at the same temperature for 20 minutes. Graph showed the maximum enzyme activity for strains NAQVI-58 and NAQVI-59 at 37 ˚C while maximum enzyme activity for strain NAQVI-22 was observed at 28 ˚C (Annexure Table 20, Fig. 68).
Effect of pH on enzyme activity
Enzyme Assay was run at different pH values (6, 7, 8) and incubated at 28 ˚C. Strains NAQVI-58 and NAQVI-59 were incubated at 28 ˚C for 50 minutes and NAQVI-22 was incubated for 40 min at the same temperature. Triplicate assays were performed for each pH value. Maximum enzyme activity was observed at pH 7 for NAQVI-58 and NAQVI-59 but maximum enzyme activity for strain NAQVI-22 was shown at pH 8 (Annexure Table 21, Fig. 69).
145
0.25 0.35
0.3 0.2 0.25
0.15 0.2
0.15 59 0.1 58 0.1
Enzyme Enzyme Activity(U/ml) 0.05 0.05
0 0 5 20 28 37 Temperature (ºC)
Figure 65: Effect of temperature on the production of β-galactosidase from strains NAQVI-58 and NAQVI-59 respectively.
Environmental Conditions: Temperature: 5 ˚C, 20 ˚C, 28 ˚C, 37 ˚C, pH: 7.
146
0.2 0.35 0.18 0.3
0.16 0.14 0.25 0.12 0.2 0.1 0.15 59 0.08 58 0.06 0.1
Enzyme Enzyme Activity(U/ml) 0.04 0.05 0.02 0 0 6 7 8 pH
Figure 66: Effect of pH on the production of β-galactosidase from strains NAQVI-58 and NAQVI-59 respectively.
Environmental Conditions: Temperature: 28 ˚C, pH: 6, 7, 8.
147
0.25 0.4
0.35
0.2 0.3
0.15 0.25 0.2 59 0.1 0.15 58 0.1 Enzyme Activity (U/ml) 0.05 0.05
0 0 24 48 72 96 Incubation Time (Hrs)
Figure 67: Effect of Incubation time on the production of β-galactosidase from strains NAQVI-58 and NAQVI-59 respectively.
Environmental Conditions: Temperature: 28 ˚C, pH: 7, Incubation Time: 24 hrs, 48 hrs, 72 hrs, 96 hrs.
148
120
100
80
60 58 59
40 22
RelativeActivity (%)
20
0 0 10 20 30 40 Temperature (˚C)
Figure 68: Graph showing the maximum enzyme activity for strains NAQVI-58 and NAQVI-59 at 37 ˚C while maximum enzyme activity for strain NAQVI-22 was observed at 28 ˚C as shown in the figure.
149
120
100
80
60 22 59
40 58 Relative ActivityRelative (%)
20
0 0 1 2 3 4 5 6 7 8 9 pH
Figure 69: Graph showing the maximum enzyme activity for strains NAQVI-58 and NAQVI-59 at pH 7 while maximum enzyme activity for strain NAQVI-22 was observed at pH 8 as shown in the figure.
150
DISCUSSION
The hydrolysis of lactose by enzyme β-galactosidase into galactose and glucose assume an essential part in biotechnology, pharmaceutical and nourishment preparing ventures. The aim of present study was centered around the isolation of noval bacterial strains and preparation of low cost medium which is based on whey for maximum β-galactosidase production. β-galactosidase activity of isolated bacterial strains were done by ONPG assay. The bacterial strain (Pararhizobium sp. nov.) demonstrated optimum enzyme activity when incubated with whey permeate (pH 7.0), temperature 20 ˚C after incubating 48 hours. The utilization of dairy industrial waste (cheese whey) was observed to be financially suitable for β-galactosidase production by utilizing separated bacterial cells. This enzyme is likewise known for its transgalactosylation reaction and orchestrated lactose based subordinates including lactulose and galactooligosaccarides, which can be additionally utilized as a part of probiotic sustenances. These outcomes from this study are as per the consequences of (Kumari et al., 2011).
A total of 235 bacterial isolates were obtained from water, ice and microbial mats of Antarctica. Antarctic samples were chosen in this study due to extreme environment of the sampling site. All bacterial isolates were screened for the capability of β- galactosidase production by inoculating on plates with X-gal at 10 °C for one month. Favier et al., revealed a strategy to recognize bacteria with β-galactosidase activity by X-gal (Favier et al., 1996). Our outcomes identified by various biochemical techniques (ONPG, X-gal) were similar to previous work (Favier et al., 1996; Elmira et al., 2010).
As a result, 61 isolates out of 235 bacterial isolates were able to produce β- galactosidase at different culture media (R2 agar and Marine agar medium). Out of sixty one, twenty blue strains were selected for UP-PCR reaction. It was observed that 14 isolates were well amplified in PCR reaction then nine strains worked very well for 16s rRNA amplification.
Strain A, B and 1 showed highest similarity with Pseudomonas pelagia CL-AP6(T) 97.15 %, 73.32 % and 98.50 % respectively. The results of (Chung et al., 2009) are in accordance with our hypothesis because these species were also isolated from Antarctic green alga Pyramimonas gelidicola from Antarctica. 151
Chung et al. (2009) described maximum growth for Pseudomonas pelagia at 25 ˚C (4-33˚C) with pH range from 7.5-8.1. Our strains were also from extreme environments (Arctic and Antarctic samples) so from 16s rRNA sequence analysis there was a possibility that strains A, B and 1 might be a noval Pseudomonas sp.
Strain 16 showed the highest similarity with Marinilactibacilus piezotolerans LT 20(T) (94.89 %). Marinilactibacilus piezotolerans LT 20(T) was a mesophilic, marine lactic acid bacterium, isolated from a deep sub-seafloor sediment core and maximum growth was observed at 37-50 ˚C with pH range from 7.0-8.0. Studies of (Laurent et al., 2005) showed the different results for temperature range as expected because the present study was aimed for the strains which can grow at low temperatures. Strain 20 had the highest sequence similarity with Alkalibacterium subtropicum O24-2(T) with 94.66 % identity. Alkalibacterium subtropicum O24-2(T) were marine lactic acid bacteria, isolated from decaying marine algae collected from a subtropical area of Japan. (Morio et al., 2011) described optimum growth of strains at pH 8.0-8.5.
The temperature range for growth was 15-40 ˚C, with the optimum at 20-30 ˚C. Results from (Morio et al., 2011) are interesting because strains had a wide range of growth for temperature and pH. Strain 21 had the closest relative Alkalibacterium putridalgicola T129-2-1(T) with the percentage identity 92.00 %. The optimum pH for Alkalibacterium putridalgicola T129-2-1(T) is 8.0-9.0, with a range of 6.5–10.0. Growth occurs between 40-45 ˚C, with an optimum temperature of 37–40 ˚C.
Results from (Morio et al., 2009) showed high pH range which might be of our interest but temperature range was also quite high as compared to present studies. Maximum percentage identity of strain 22 was with Marinobacter psychrophilus 20041(T) 96.11%. Marinobacter psychrophilus 20041(T) might be interesting because it was isolated from the extreme environment (sea-ice of the Canadian Basin). Temperature range for growth was tested for Marinobacter psychrophilus 20041(T) from 0-22 ˚C with maximum growth occurred at 16-18 ˚C and pH range tested from 5.0-10.0 with approximately pH 6.0-9.0.
Results from (Zhang et al.,2008) was in accordance with the results of present study because the growth of strains was observed at low temperature and at high pH. Strains 58 and 59 showed the 100 % 16s rRNA sequence similarity with each other and closest match with Rhizobium herbae CCBAU83011(T) (Ren et al., 2011). 152
Strain 58 showed 95.22 % and strain 59 showed 99.66 % sequence similarity with Rhizobium herbae CCBAU83011(T). Growth occurred between 10-40 ˚C with optimum growth at 28 ˚C and pH range was 7-9 with maximum growth at pH 7.0. In the present study these two strains might be highly interesting because they can also grow at low temperatures with high pH values and were the same according to16s rRNA analysis.
Results from whole genome sequencing of DNA showed different percentage identity with the closest relatives as compared to 16s rRNA sequence analysis. Strain B showed 99.66 % similarity with Pseudomonas pelagia CL-AP6(T) (Chung et al., 2009). More than 99 % identity had a very high probability that strain might be the same Pseudomonas sp. as described by (Chung et al., 2009). Strain 20 showed 97.83 % identity with Alkalibacterium subtropicum O24-2(T). This strain might be significant for further characterization because results from (Morio et al., 2011) described optimum growth of strains at higher pH values (8.0-8.5).
The optimum temperature range for growth was also from 20-30 ˚C. Highest whole genome sequence similarity for strain 22 was 99.32 % with Marinobacter psychrophilus 20041(T). Sequence similarity more than 99 % had the low probability of being noval species. Results from (Zhang et al., 2008) indicated that optimum temperature for growth was 37–40 ˚C so this strain might be of less interest. Strains 58 and 59 were at first priority because both had the 100 % similarity with each other and showed the 98.2 % similarity with Rhizobium giardinii H152 (T) (Amarger et al., 1997).
Now finally two strains (58 and 59) were selected for further analysis and changed their names as NAQVI-58 and NAQVI-59 respectively. Therefore it is thought that the two bacterial isolates could be newly isolated β-galactosidase producing bacteria from Antarctica and optimization should be conducted in future to improve the β- galactosidase production of the bacterial isolates. The 16S rRNA gene sequence analysis indicated that strain NAQVI-59 and NAQVI-58 belonged to the family Rhizobiaceae of the order Rhizobiales, class Alphaproteobacteria. Strain NAQVI-58 and NAQVI-59 formed a distinct subclade within the genus Pararhizobium (Mousavi et al., 2015; Oren and Garrity, 2016). 153
Sequence similarity calculations (extracted from whole genome data) showed that strains NAQVI-58 and NAQVI-59 were closely related to Pararhizobium giardinii H152T (98.1 %) (Amarger et al., 1997), P. herbae CCBAU 83011T (98.1 %) (Ren et al., 2011) and “P. polonicum” F5.1T (98.1%) (Puławska et al., 2016). This phylogenetical relationship was confirmed by analyses of the three house-keeping genes atpD, rpoB and recA from strains NAQVI 58 and NAQVI 59 and related species within the Rhizobiaceae. Phylogenetical analysis of concatenated atpD, rpoB, and recA sequences confirmed that strains NAQVI 58 and NAQVI 59 formed a separate clade in the combined phylogenetic tree, an observation that was further confirmed by phylogenetical trees of the individual genes.
Genome relatedness was investigated by comparing the genome sequences of strains NAQVI 58 and NAQVI 59 with sequences of the closely related Pararhizobium species using average nucleotide identity (ANI) using the Kostas Lab platform and the Genome-Genome Distance Calculator (GGDC) program (Meier et al., 2013). These novel methods for comparing whole genome sequences in pair wise relatedness analyses have been shown to be as good as or better than the traditional DNA-DNA hybridization method because they are reproducible, easier to perform, and compatible with genome sequences (Figueras et al., 2014; Goris et al., 2007; Stropko et al., 2014; Mc Ginnis et al., 2015; Jung et al., 2015).
The ANI values for strains NAQVI 58 and NAQVI 59 when compared to the closest related species, P. giardinii H152T and “P. polonicum” F5.1T (accession no. LGLV01000000) were 80.5 % and 81.1 % (accession no. ARBG00000000), respectively. These values are below the 95-96 % threshold that is accepted for species delineation (Goris et al., 2007; Richter and Rosselló, 2009). Similarly, GGDC analyses (Auch et al., 2010; Meier et al., 2013) with Blast for strains NAQVI 58 and NAQVI 59 compared to P. giardinii H152T and “P. polonicum” F5.1T using equation two in the GGDC program, gave DNA-DNA homology values of 22.1 % and 22.6 %. The 70 % DNA-DNA hybridization value has been used as a gold standard for bacterial species delineation (Wayne et al., 1987). A BLAST search for nodC, nifH and other genes involved in nodule formation and nitrogen formation remained unsuccessful. A similar analysis of the genome sequence of P. giardinii H152T by (Laguerra et al., 2001) showed the presence of nodC but not of nifH. 154
Enzyme production by NAQVI-58 and NAQVI 59 was studied under different environmental conditions i.e. temperature, pH and incubation time. The temperature was one of most imperative element which impacts the activity of metabolic enzyme. The elevated enzyme activity was seen at the temperature of 28 °C and found as ideal temperature for the production of β-galactosidase. Similarly, the temperature of 28-30 ˚C has likewise been watched for β-galactosidase generation by (Ku and Hang, 1992) and (Artolozaga et al., 1998). Matheus and Rivas (2003) have revealed 30 ˚C as an optimum temperature for β-galactosidase production by K. lactis.
The change in enzyme activity as a component of temperature demonstrates that, there is an increase in β-galactosidase activity with an elevation in temperature up to 30 ˚C. However diminish in enzyme activity was seen with an expansion in temperature which may be because of the halfway inactivation of enzyme or cell lysis at high temperature. The highest enzyme activity was seen at the temperature of 28-30 ˚C (Ku and Hang, 1992; Artolozaga et al., 1998).
The hydrogen ion concentration of condition has the greatest impact of the microbial development and enzyme generation. The impact of various starting pH in the β- galactosidase generation was investigated. β-galactosidase generation increment focus upto a range of 7.0 and diminishing in enzyme production in regard to increment in pH was observed. pH 5.5 has been seen as ideal for the production of β-galactosidase by Rajoka et al., 2003 (Hin et al., 1986; Rojoka et al., 2003).
As per the outcomes taken at consistent interims, the most optimum enzyme activity was obtained at 48th hour of incubating. Past this, the enzyme productivity was stayed constant and no further elevation underway in yield was observed. It may be because of abatement in the supplement accessibility in the medium, or catalobic constraint of enzyme (Gupta and Nair, 2010).
β-galactosidase activity of cells expanded with the expansion in incubating time period upto 28 hour which may be because of increment in biomass, after that there is diminishing of β-galactosidase production because of further hindrance of cells development after the stationary stage has come to. The maximum yield of yeast extricates focus and incubation time for highest β-galactosidase activity was seen at 26-28 hours, individually. 155
Rajoka et al. (2003) considered the β-galactosidase production K. marxianus revealed that the optimum production of β-galactosidase reached after 30-40 hours and the production of enzyme was growth related.
Incubation time is administered by attributes of the way of life and furthermore in light of growth rate and production of enzyme. Results demonstrated that the ideal incubation time period for production of β-galactosidase was found to 72 hours. Contrasting the results with the literature there is a wide incubation time detailed for Bacillus strains (Chen et al., 2003; Clerck and Vos, 2004; Konsoula and Liakopoulou, 2007). In view of these information our strains fit in this interim. A long incubation time more than this period did not eleviate the enzyme yield. The explanation behind this may have been because of the denaturation of enzyme caused by the connection with different parts in the medium and presumably because of exhaustion of supplements accessible to microorganism (Ramesh and Lonsane, 1987; Akcan et al., 2011).
The generation of extracellular β-galactosidase by Bacillus sp. was enhanced in a submerged fermentation. The impact of incubation time, temperature and pH of the medium were improved. The enzyme production was observed to be optimum at the 48th hour of inoculation at 30°C and pH 7 (Mukesh et al., 2012). Our results were in accordance with results observed in previous studies of (Mukesh et al., 2012).
The nature and quantity of carbon source in culture media is essential for the development and production of extracellular β-galactosidase in microbes (bacteria). Carbon source directs biosynthesis of β-galactosidase in different microorganisms (Nagy et al., 2001; Akolkar et al., 2005; Hsu et al., 2005; Konsoula and Liakopoulou, 2007; Alazzeh et al., 2009). All showed that the part of carbon source in the biosynthesis of β-galactosidase may fluctuate and rely upon the microorganisms tried.
Kim and Rajagopal (2000) portrayed that galactose was the best carbon hotspot for the biosynthesis of β-galactosidase by L. crispatus, while addition of glucose or lactose to the growth medium curbed the production of β-galactosidase. In the present investigation, strains NAQVI-58 and NAQVI 59 additionally demonstrated the negative outcomes for glucose however demonstrated the elevated production of β- galactosidase with the option of lactose in the fermentation medium. In this way, our outcomes affirmed the Vasiljevic and Jelen outcomes and were similar to the results 156 that they have reported about increased production of β-galactosidase with the addition of lactose in the medium.
However, (Hsu et al., 2005) found that the last practical populace of B. longum CCRC 15708 was increased in cultures containing either glucose or lactose as the sole carbon source with the most astounding β-galactosidase activity recognized with lactose taken after by galactose and the least activity with glucose as the carbon source. We watched these results recommend that β-galactosidase productionm from B. licheniformis ATCC 12759 is initiated by some promptly metabolisable sugars. Results from (Hsu et al., 2005) were the same as found in the present investigation.
Less expensive wellsprings of both carbon and nitrogen sources are the key fascination for commercialization of the enzyme production and subsequently, capacity of the microbial agent to develop and deliver enzymes utilizing these sources has been seemingly a state of intrigue (Patel et al., 2005).
Natural and inorganic nitrogen supplements were analyzed to decide their impact on the production of enzyme. The increase enzyme production has been seen with the addition of nitrogen in the culture medium, which might be because of the vital part of nitrogen (main component of protein and nucleic acids) in development of microorganisms (Mukhtar et al., 2010).
In many microorganisms, both inorganic and natural types of nitrogen are used to deliver amino acids, nucleic acids, proteins, and cell wall componenets. Nitrogen sources may influence microbial biosynthesis of β-galactosidase (Rao and Dutta, 1977; Shaikh et al., 1997; Hsu et al., 2005). All inorganic and organic nitrogen sources brought about a lessening in β-galactosidase generation.
These outcomes demonstrate that the expansion of natural and inorganic nitrogen sources in the medium was no adequate to fortify the β-galactosidase production from B. licheniformis ATCC 12759. In the present study addition of L-Valine to the medium indicated negative results and stifled the growth of test living beings (Pararhizobium sp.) as depicted for B. licheniformis ATCC 12759. While addition of
(NH4)2 SO4, KNO3 and L-Glutamic Acid improved the growth of NAQVI-58 and NAQVI-59. Notwithstanding, different works detailed that better β-galactosidase production within the sight of nitrogen sources (Konsoula and Liakopoulou, 2007; Nizamuddin et al., 2008). 157
Hsu et al. (2005) announced that yeast extract vital for β-galactosidase production, while casein, peptone and beef extricate curbed β-galactosidase development. Yeast extricate is the principle element of the R2 medium which is utilized as a part of the present study so it demonstrated an indistinguishable outcome from exhibited by (Hsu et al., 2005) where as peptone demonstrated the positive results in our study as compared to (Hsu et al., 2005). Supplementation of the L-tryptophane in fermentation medium was improved β-galactosidase production. It is as per the consequences of present examination. Konsoula and Liakopoulou (2007) announced that glycine was found to upgrade the enzyme production from Bacillus subtilis. Be that as it may, the part of amino compounds was thought to be neither as nitrogen nor as a carbon source, however as stimulators of enzyme production and discharge (Gupta et al., 2003). The enzyme synthesis by a few microorganisms has been connected to the nearness or nonattendance of various nitrogen sources and different amino acids in the development medium. The distinctions in nourishing prerequisites of different enzyme delivering creatures or microbial strains could be ascribed to the distinction in their genetics (Rasooli et al., 2008).
The β-galactosidase production increased when production medium was supplemented with MgSO4. The growth medium utilized as a part of the present examination was likewise supplemented with MgSO4 and demonstrated an indistinguishable outcomes as described by Rao and Dutta (1977). This demonstrated that Mg2+ was fundamental for enzyme stabilization. Constructive outcomes of metal salts including Mg2+ and Mn2+ on β-galactosidase production have likewise been exhibited by (Rao and Dutta, 1977).
β-galactosidase was an intracellular enzyme segregated from Streptococcus thermophilus developed in whey. β-galactosidase which is obtained in our study was also intracellular and isolated from Pararhizobium sp. nov. grown on medium which was supplemented with cheese whey. So results from our investigation were the same as portrayed by (Princely et al., 2013). Because of the intracellular location of the enzyme, the distinctive cell interruption methods have been contemplated for the extraction. Distinctive strategies for cell interruption have additionally been researched by (Bansal et al., 2008) for the isolation of β-galactosidase from Kluyveromyces marxianus MTCC 1388. 158
One of the significant impediments to the whey use is lactose content, which causes crystallization at low temperatures, low sweetness, and poor absorbability when utilized as sustenance (Princely et al., 2013). These issues can be understood if whey lactose is hydrolyzed to galactose and glucose. Already, β-galactosidase biosynthesis by microorganisms, yeasts, and moulds has been accounted for by different agents (Itoh et al., 1982; Nagy et al., 2001).
The ideal pH for the hydrolysis of ONPG by the investigated enzyme in the present study was 7.0. Comparative outcomes have been accounted for a few β-galactosidases from yeast and microbes which have ideal pH in the range 6.5-7.5 (Pisani et al., 1990; Khare et al., 1988). The maintenance of around 70% of maximal activity at pH values of 5.0 and 7.4 shows that the enzyme is reasonable for hydrolysis of lactose in milk and sweet whey. The ideal temperature was observed to be 40°C (Princely et al., 2013).
The enzyme production by microorganism is extraordinary accomplishment in the field of fermentation technology. It is apparent that enzyme production emphatically dependant on the selection of isolates (Swati et al., 2012).
The physicochemical examination of whey demonstrated that it contained high measure of lactose (4.89 ± 0.11 %, w/v), trailed by protein (0.48 ± 0.33 %, w/v), fat (0.18 ± 0.08 %, w/v) and minerals tarces. The β-galactosidase production was conveyed by utilizing novel yeast isolate Kluyveromyces marxianus WIG2 using whey as a substrate under the optimal conditions (Rupinder et al., 2015).
Examination of whey which is utilized as a part of the present study demonstrated the estimations of lactose as 5.49 %, fat 0.056 %, protein 0.95 % and traces of minerals as 7.01 %. Rupinder et al. (2015) did the same work with the noval yeast strain as it was presented in our study with noval Pararhizobium sp. by using dairy industrial waste (cheese whey) as a substrate under optimum conditions.
Phenotypic characteristics of strains NAQVI-58 and NAQVI-59 and four reference strains. P. giardinii H152T (Amarger et al., 1997; Ren et al., 2011); P. herbae CCBAU 83011T (Ren et al., 2011); P. capsulatum DSM 1112 (Hirsch and Müller, 1985); “P. sphaerophysae” CCNWGS 0238 (Xu et al., 2011) were studied. It was found that in contrast to other species of Pararhizobium, strains NAQVI-58 and NAQVI-59 display β-galactosidase and tryptophan deaminase activity and are unable 159 to use L-valine as nitrogen source. Other phenotypic characteristics are positive for catalase, oxidase, arginine dihydrolase, lysine carboxylase, ornithine decarboxylase, citrate utilization, urease, acetoin production, gelatinase, negative on H2S production and indole production (Amarger et al., 1997; Hirsch and Müller, 1985; Ren et al., 2011; Xu et al., 2011).
The isolate NAQVI-59 was found negative for fermentation of glucose, mannitol, inositol, sorbitol, rhamnose, saccharose, melibiose, amygdalin, arabinose. Utilization of (NH4)2 SO4, KNO3, L- glutamic acid was positive for all strains. The phenotypical, phylogenetical and genome analyses presented here support our hypothesis that strain NAQVI-59 represents a novel species of the genus Pararhizobium, for which the name Pararhizobium antarcticum is proposed. The type strain is NAQVI 59T (DSMZ 103442 = LMG 29675).
CONCLUSION
A noval bacterial strain isolated from Antarctica (Pararhizobium sp.) is discovered by using dairy industrial waste cheese whey as a substrate which is further used for the production of β-galactosidase. On the basis of phenotypic, phylogenetic, genomic and chemotaxonomic data, the strains represent a novel species of the genus Pararhizobium, for which the name Pararhizobium antarcticum sp. nov. is proposed. In this research work an industrially important enzyme was produced by using an environmental pollutant as a raw material which reduce the cost of production and environmental load on the ecosystem.
160
REFERENCES
Adekambi, T. and Drancourt, M. 2004. Dissection of phylogenetic relationships among 19 rapidly growing Mycobacterium species by 16S rRNA, hsp65, sodA, recA and rpoB gene sequencing. International Journal of Systematic and Evolutionary Microbiology. 54: 2095 - 2105.
Aislabie, J. M., Jordan, S. and Barker, G. M. 2008. Relation between soil classification and bacterial diversity in soils of the Ross Sea region, Antarctica. Geoderma, 144: 9 - 20.
Akbache, A., Lamiot, É., Moroni, O., Turgeon, S., Gauthier, S. F., Pouliot, Y. 2009. Use of membrane processing to concentrate TGF-β2 and IGF-I from bovine milk and whey. Journal of Membrane Science, 326: 435 - 440.
Akcan, N., Uyar, F., Guven, A. 2011. Alpha-amylase production by Bacillus subtilis RSKK 96 in submerged cultivation. af as niversitesi eteriner a ltesi ergisi, 17: 17 - 22.
Akolkar, S. K., Sajgure, A., Lele, S. S. 2005. Lactase production from Lactobacillus acidophilus. World Journal of Microbiology and Biotechnology, 21: 1119 - 1122.
Alm, L., Lactose intolerance. 2003. In: Roginski, H., Fuquay, J. W., Fox, P. F., ed. Encyclopedia of Dairy Sciences. Academic Press, London. pp. 1533 - 1539.
Alvarenga, A. E., Cristóbal, H. A. and Abate, C. M. 2011. Cold-active enzymes: potential use in biotechnology. In: Berhardt, L. V. ed. Advances in medicine and biology, vol. 45. Nova, Commack, NY.
Amarger, N., Macheret, V., Laguerre, G. 1997. Rhizobium gallicum sp. nov. and Rhizobium giardinii sp. nov., from Phaseolus vulgaris nodules. International Journal of Systematic and Evolutionary Microbiology. 47: 996 - 1006.
Ana, R. P., Fátima, C. and Javier, R. 2012. Cheese whey management: A review. Journal of Environmental Management, 110: 48 - 68. 161
Anna, L., Alexander, W. and Svein, V. 2013. Metagenomics of microbial life in extreme temperature environments. Current Opinion in Biotechnology, 24: 516 - 525.
Anne, D. J., Susanna, A. W., Ian, H., Jenny, W. B. and Colin, H. 2012. The Pyramid Trough Wetland: environmental and biological diversity in a newly created Antarctic protected area. FEMS Microbiol Ecology, 82: 356 - 366.
Archana, P., Yiqiong, J., Beth, M., Michael, C. and David, C. B. 2015. Incorporation of whey permeate, a dairy effluent, in ethanol fermentation to provide a zero waste solution for the dairy industry. Journal of Dairy Science, 99: 1859 - 1867.
Arijit, N., Ranjana, C., Chiranjib, B. and Madhumita, M. 2016. Production of β-galactosidase in a Batch Bioreactor Using Whey through Microbial Route - Characterization of Isolate and Reactor Model. Periodica Polytechnica Chemical Engineering. 60 (4): 298 - 312.
Ariosvana, F. L., Kenia, F. C., Maria, F. M. F., Tigressa, H. S. R., Maria, V. P. R., Luciana, R. B. G. 2013. Comparative biochemical characterization of soluble and chitosan immobilized β-galactosidase from Kluyveromyces lactis NRRL Y1564. Process Biochemistry, 48: 443 - 452.
Artolozaga, M. J., Jones, R., Schneider, A. L., Furlan, S. A. and Carvallo, J. M. P. 1998. One step partial purification of and β-D-galactosidase from Kluyveromyces marxianus CDB002 using streamline-deae. Bioseparation, 7: 137 - 143.
Artolozaga, M. J., Jones, R., Schneider, A. L., Furlan, S. A., Carvallo, J. M. F. 1998. Bioseparation, 7: 137 - 143.
Asraf, S. S., Gunasekaran, P. 2010. Current trends of β-galactosidase research and application. In: Mendez, V. A. ed. Current research, technology and education topics in applied microbiology and microbial biotechnology, 2nd ed. Formatex Research Center, Spain. pp. 880 - 889.
Ayesha, K., Zainab, B., Afsheen, A., Shah, A. U. Qader. 2016. Lactose hydrolysis approach: Isolation and production of β-galactosidase from newly 162
isolated Bacillus strain B-2. Biocatalysis and Agricultural Biotechnology, 5: 99 - 103.
Banaszewska, A., Cruijssen, F., Claassen G. D. H., Van der Vorst., J. G. A. J. 2014. Effect and key factors of byproducts valorization: the case of dairy industry, Journal of Dairy Science, 97: 1893 - 1908.
Bansal, S., Oberoi, H. S., Dhillon, G. S. and Patil, R. T. 2008. Production of β- galactosidase by Kluyveromyces marxianus MTCC 1388 using whey and effect of four different methods of enzyme extraction on β-galactosidase activity, Indian Journal of Microbiology, 48: 337 - 341.
Barbara, G. H. M. N., Stefanie, W. H. A. N., Cindy, L., Roman, K., Geir, M., Vincent, G.H. E., Dietmar, H., Thu, H. N. 2016. From by-product to valuable components: Efficient enzymatic conversion of lactose in whey using β- galactosidase from Streptococcus thermophilus. Biochemical Engineering Journal, 116: 45 - 53.
Bentley, S. D., Parkhill, J. 2004. Comparative genomic structure of prokaryotes. Annual Reviwes of Genetics, 38: 771 - 792.
Berger, F., Normand, P. and Potier, P. 1997. capA, a cspA-like gene that encodes a cold acclimation protein in the psychrotrophic bacterium Arthrobacter globiformis SI55. Journal of Bacteriology, 179: 5670 - 5676.
Berruga, M.I., Jaspe, A., SanJose, C. Selection of yeast strains for lactose hydrolysis in dairy effluents. 1997. International Biodeterioration and Biodegradation, 40 (2 - 4): 119 -123.
Biddle, J.F., Fitz, G. S., Schuster, S. C., Brenchley, J. E., House, C. H. 2008. Metagenomic signatures of the Peru Margin sub seafloor biosphere show a genetically distinct environment. Proceedings of the National Academy of Sciences of the United States of America, 105: 10583 - 10588.
Binnewies, T. T., Motro, Y., Hallin, P. F., Lund, O., Dunn, D., La, T., Hampson, D. J., Bellgard, M., Wassenaar, T. M., Ussery, D. W. 2006. Ten years of bacterial genome sequencing: comparative-genomics-based discoveries. Functional and Integrative Genomics, 6: 165 - 185. 163
Bohlin, J., Snipen, L., Hardy, S. P., Kristoffersen, A. B., Lagesen, K., Donsvik, T., Skjerve, E., Ussery, D. W. 2010. Analysis of intra-genomic GC content homogeneity within prokaryotes. BMC Genomics, 11.
Boon, M. A., Janssen, A. E. M., Van, R. K. 2000. Effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides. Enzyme and Microbial Technology, 26: 271 - 281.
Brayam, L. B. P., Harádia, C. M. S., Maikon, K., Giannini, P. A., Ana, P. T. P., Andréa, L. S. S. 2013. Production of β-Galactosidase from Cheese Whey Using Kluyveromyces marxianus CBS 6556. Chemical Engineering Transactions. 32: 991 - 996.
Bull, A. T. and Stach, J. E. 2007. Marine actinobacteria: new opportunities for natural product search and discovery. Trends in Biotechnology, 15: 491 - 499.
Bury, D., Jelen, P. 2000. Lactose hydrolysis using a disrupted dairy culture: Evaluation of technical and economical feasibility. Canadian Agricultural Engineering, 42: 75 - 80.
Bury, D., Jelen, P., Kalab, M. 2001. Disruption of Lactobacillus delbrueckii ssp. bulgaricus 11842 cells for lactose hydrolysis in dairy products: a comparison of sonication, high-pressure homogenization and bead milling. Innovative Food Science and Technology, 2: 23 - 29.
Carla, O., Pedro, M. R. G., Lucília, D. 2011. Recombinant microbial systems for improved β-galactosidase production and biotechnological applications. Biotechnology Advances, 29: 600 - 609.
Cary, S. C., Mc Donald, I. R., Barrett, J. E. and Cowan, D. A. 2010. On the rocks: the microbiology of Antarctic Dry Valley soils. Nature Reviews in Microbiology, 8: 129 - 138.
Cavicchioli, R., Siddiqui, K. S., Andrews, D., Sowers, K. R. 2002. Low temperature extremophiles and their applications. Current Opinion in Biotechnology, 13: 253 - 261.
Charles, G., Mohamed, A., Mostafa, B., Jean, P. C., Paule, C., Tony, C., Salvino, D. A., Joëlle, D., Geneviève, G., Daphné, G., Anne, H., Thierry, L., 164
Marie, A. M. and Georges, F. 2000. Cold-adapted enzymes: from fundamentals to biotechnology. Trends in Biotechnology, 18: 103 - 107.
Chen, C. W., Yang, C. C., Yeh, C. W. 2003. Synthesis of galacto-oligo saccharides and transgalactosylation modeling in reverse micelles. Enzyme and Microbial Technology, 33: 497 - 507.
Cheng, C. C., Yu, M. C., Cheng, T. C., Sheu, D. C., Duan, K. J. and Tai, W. L. 2006. Production of high-content galacto-oligosaccharide by enzyme catalysis and fermentation with Kluyveromyces marxianus. Biotechnology Letters, 28: 793 -797.
Chistoserdova, L. 2001. Recent progress and new challenges in metagenomics for biotechnology. Biotechnology Letters, 32: 1351 - 1359.
Cho, J. C. and Tiedje, J. M. 2001. Bacterial species determination from DNA- DNA hybridization by using genome fragments and DNA microarrays. Appl Environ Microbiol, 67: 3677- 3682.
Christensen, H., Kuhnert, P., Olsen, J. E. and Bisgaard, M. 2004. Comparative phylogenies of the housekeeping genes atpD, infB and rpoB and the 16S rRNA gene within the Pasteurellaceae. International Journal of Systematic and Evolutionary Microbiology. 54: 1601 - 1609.
Clerck, E. D. and Vos, P. D. 2004. Genotypic diversity among Bacillus licheniformis strains from various sources. FEMS Microbiology Letters, 231: 91 - 98.
Coenye, T., Gevers, D., Van de Peer, Y., Vandamme, P. and Swings, J. 2005. Towards a prokaryotic genomic taxonomy. FEMS Microbiology Reviews, 29: 147 - 167.
Comeau, Y., Lamarre, D., Roberge, F., Terrier, M., Desjardins, G. and Hadet, C. 1996. Biological nutrient removal from a phosphorus-rich pre-fermented industrial wastewater. Water Science and Technology, 34 (1 - 2): 169 - 77.
Cook, H., Ussery, D. W. 2013. Sigma factors in a thousand E. coli genomes. Environ Microbiol, 15: 3121 - 3129. 165
Corral, M. A., Montori, V. M., Somers, V. K., Korinek, J., Thomas, R. J., Allison, T. G., Mookadam, F. and Lopez, J. F. 2006. Association of bodyweight with total mortality and with cardiovascular events in coronary artery disease: a systematic review of cohort studies. Lancet, 368 (9536): 666 - 678.
Cortes, G., Trujillo, R. M. A., Ramirez, O. T. and Galindo, E. 2005. Production of β-galactosidase by Kluyveromyces marxianus under oscillating dissolved oxygen tension. Process Biochemistry. 40: 773 - 778.
Cristóbal, H. A., Alvarenga, A. E. and Abate, C. M. 2011a. Isolation and molecular characterization of marine bacteria isolated from the Beagle Channel, Argentina. The marine environment: ecology, management and conservation. Nova, Commack, NY. pp. 87 - 118.
Cristóbal, H. A., López, A.M., Erica, K. and Abate, C. M. 2011b. Diversity of protease-producing marine bacteria from sub-Antarctic environments. Journal of Basic Microbiology. 51: 1 - 11.
Cristóbal, H. A., Schmidt, A., Kote, E., Breccia, J. and Abate, C. M. 2009. Characterization of inducible cold-active β-glucosidases from the psychrotolerant bacterium Shewanella sp. G5 isolated from a Sub Antarctic Ecosystem. Enzyme and Microbial Technology, 45: 498 - 506.
Čurda, L., Rudolfová, J., Tovarová, I., Brmčevová, D. 2001. Vliv reakčních podmínek na prubeh enzýmové hydrolýzy laktosy. Mliekarenstvo, 32: 28.
Dakhmouche, S. D., Aoulmi, Z. G., Meraihi, Z. and Bennamoun, L. 2006. Application of a statistical design to the optimization of culture medium for α- amylase production by Aspergillus niger ATCC 16404 grown on orange waste powder. Journal of Food Engineering, 73: 190 - 197.
D'Amico, S., Claverie, P., Collins, T., Georlette, D., Gratia, E., Hoyoux, A., Meuwis, M. A., Feller, G. and Gerday, C. 2002. Molecular basis of cold adaptation. Philosophical Transactions of the Royal Society of London Series B-Biological Sciences, 357: 917 - 924.
De Lajudie, P., Laurent, F. E., Willems, A., Torck, U., Coopman, R., Collins, M. D., Kersters, K., Dreyfus, B. L. and Gillis, M. 1998. Allorhizobium 166
undicola gen.nov., sp. nov., nitrogen-fixing bacteria that efficiently nodulate Neptunia natansin Senegal. International Journal of Systematic Bacteriology, 48: 1277 - 1290.
De Lajudie, P., Willems, A., Pot, B., Dewettinck, D., Maestrojuan, G., Neyra, M., Collins, M. D., Dreyfus, B. L., Kersters, K. and Gillis, M. 1994. Polyphasic taxonomy of Rhizobia: emendation of the genus Sinorhizobium and description of Sinorhizobium meliloti comb. nov., Sinorhizobium saheli sp. nov., and Sinorhizobium teranga sp. nov. International Journal of Systematic Bacteriology, 44: 715 - 733.
De Long, E. F. and Pace, N. R. 2001. Environmental diversity of bacteria and archaea. International Journal of Systematic Bacteriology, 50: 470 - 478.
De Vrese, M., Stegelmann, A., Richter, B., Fenselau, S., Laue, C. and Schrezenmeir, J. 2001. Probiotics compensation for lactase insufficiency. American Journal of Clinical Nutrition, 73: 421 - 429.
De Wit, J. N. 2001. Lecturer’s Handboo on Whey and Whey Products, 1st ed. European Whey Products Association, Brussels, Belgium.
Degbagli, S. and Y. Goksungur. 2008. Optimization of β-D-galactosidase production using Kluyveromyces lactis NRRL Y-8279 by response surface methodology. Electronic Journal of Biotechnology, 11: 1 - 12.
Demain, A. L. and Dana, C. A. 2007. The business of biotechnology. General Pub incorporation, 3(3): 269 - 283.
Demirel, B., Yenigun, O. and Onay, T. T. 2005. Anaerobic treatment of dairy waste waters: a review. Process Biochemistry, 40 (8): 2583 - 2595.
Domingues, L., Guimarães, P. M. and Oliveira, C. 2010. Metabolic engineering of Saccharomyces cerevisiae for lactose/whey fermentation. Bioengineered Bugs, 1: 164 - 171.
Domingues, L., Lima, N. and Teixeira, J. A. 1999. Novas Metodologias para a Fermentação Alcoólica do Soro de Queijo. In: Actas da 6.a Conferência Nacional sobre a Qualidade do Ambiente, Universidade Nova de Lisboa, Lisboa, Portugal. 3: 271 - 280. 167
Domingues, L., Lima, N., Teixeira, J. A. 2005. Aspergillus niger β- galactosidase production by yeast in a continuous high density reactor. Process Biochemistry, 40: 1151 - 1154.
Egorova, K. and Antranikian, G. 2005. Industrial relevance of thermophilic Archaea. Current Opinion in Microbiology, 8: 649 - 655.
Eliwa, E. S. and El-Hofi, M. 2010. β-galactosidase (β-Gal) from the yeast Rhodotorulaingeniosa and its utilization in ice milk production. Electronic Journal of Polish Agricultural Universities, 13 (1): 2 - 10.
Elmira, G., Fariba, H., Bahareh, K. S., Jamileh, N. and Farahnaz, M. 2010. Study on β-galactosidase enzyme produced by isolated lactobacilli from milk and cheese. African Journal of Microbiology Research. 4 (6): 454 - 458.
Ergüder, T. H., Tezel, U., Güven, E. and Demirer, G. N. 2001. Anaerobic biotransformation and methane generation potential of cheese whey in batch and UASB reactors. Waste Management, 21 (7): 643 - 650.
Farizoglu, B., Keskinler, B., Yildiz, E. and Nuhoglu, A. 2007. Simultaneous removal of C, N, P from cheese whey by jet loop membrane bioreactor (JLMBR). Journal of Hazardous Materials, 146 (1 - 2): 399 - 407.
Fátima, C., Ana, R. P. and Javier, R. 2013. Cheese whey wastewater: Characterization and treatment. Science of the Total Environment, 385 - 396.
Feller, G. and Gerday, C. 1997. Psychrophilic enzymes: molecular basis of cold adaptation. Cellular and Molecular Life Sciences, 53: 830 - 841.
Feller, G. and Gerday, C. 2003. Psychrophilic enzymes: Hot topics in cold adaptation. Nature Reviews Microbiology, 1: 200 - 208.
Fernandes, S., Geueke, B., Delgado, O., Coleman, J. and Hatti, K. R. 2004. β- galactosidase from a cold-adapted bacterium: purification, characterization and application for lactose hydrolysis. Applied Microbiology and Biotechnology, 58: 313 - 321.
Fernandez, M., Margolles, A., Suarez, J. E. and Mayo, B. 1999. Duplication of the β-galactosidase gene in some Lactobacillus plantarum strains. International Journal of Food Microbiology, 48: 113 - 123. 168
Ferrer, M., Golyshina, O., Beloqui, A., Golyshin, P. N. 2007. Mining enzymes from extreme environments. Current Opinion in Biotechnology, 10: 207- 214.
Fleischmann, R. D., Adams, M. D., White, O., Clayton, R. A., Kirkness, E. F., Kerlavage, A. R., Bult, C. J., Tomb, J. F., Dougherty, B. A. and Merrick, J. M. 1995. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 269: 496 - 512.
Francis, F., Sabu, A., Nampoothiri, K. M., Ramachandran, S., Ghosh, S., Szakacs, G. and Pandey, A. 2003. Use of response surface methodology for optimizing process parameters for the production of α-amylase by Aspergillus oryzae. Biochemical Engineering Journal, 15: 107 - 115.
Fraser, C. M., Gocayne, J. D., White, O., Adams, M. D., Clayton, R. A., Fleischmann, R. D., Bult, C. J., Kerlavage, A. R., Sutton, G. and Kelley, J. M. 1995. The minimal gene complement of Mycoplasma genitalium. Science, 270: 397 - 404.
Fred, C. M. 2002. What are bacterial species? Annual Review of Microbiology, 56: 457 -487.
Furlan, S. A., Schneider, A. L. S., Merkle, R., Carvalho, J. M. F. and Jonas, R. 2002. Formulation of lactose-free, low cost culture medium for production of β-galactosidase by Kluyveromyces marxianus. Biotechnology Letters, 22: 589 - 593.
Gangadharan, D., Sivaramakrishnan, S., Nampoothiri, K. M., Sukumaran, R. K. and Pandey, A. 2008. Response surface methodology for the optimization of alpha amylase production by Bacillus amyloliquefaciens. Bioresource Technology, 99: 4597- 4602.
Gannoun, H., Khelifi, E., Bouallagui, H., Touhami, Y. and Hamdi, M. 2008. Ecological clarification of cheese whey prior to anaerobic digestion in up flow anaerobic filter. Bioresource Technology. 99 (14): 6105 - 6111.
Ganzle, M. G., Haase, G. and Jelen, P. 2008. Lactose: crystallization, hydrolysis and value-added derivatives. International Dairy Journal, 18: 685 - 694. 169
Garman, J., Coolbear, T. and Smart, J. 1996. The effect of cations on the hydrolysis of lactose and the transferase reactions catalysed by β-galactosidase from six strains of lactic acid bacteria. Applied Microbiology and Biotechnology, 46: 22 - 37.
Garrity, G. M., Winters, M., Kuo, A. W. and Searles, D. 2002. Taxonomic outline of the prokaryotes. Bergey’s manual of systematic bacteriology, 2nd ed. Springer, New York. pp. 49 - 66.
Gekas, V. and Lopez, L. M. 1985. Hydrolysis of lactose. Process Biochemistry, 2: 2 - 12.
Gerday, C., Aittaleb, M., Bentahir, M., Chessa, J. P., Claverie, P., Collins, T., D‟Amico, S., Dumont, J., Garsoux, G. and Georlette, D. 2000. Cold-adapted enzymes: from fundamentals to biotechnology. Trends in Biotechnology, 18: 103 - 107.
Ghaly, A. E. and Kamal, M. A. 2004. Submerged yeast fermentation of acid cheese whey for protein production and pollution potential reduction. Water Research, 38 (3): 631 - 644.
Ghaly, A. E. and Singh, R. K. 1989. Pollution potential reduction of cheese whey through yeast fermentation. Applied Biochemistry Biotechnology, 22 (2): 181 - 203.
Ghosh, M., Pulicherla, K. K., Rekha, V. P., Raja, P. K. and Sambasiva, R. K. R. 2012. Cold active β-galactosidase from Thalassospira sp. 3SC-21 to use in milk lactose hydrolysis: a novel source from deep waters of Bay-of-Bengal. World Journal of Microbiology and Biotechnology, 28 (9): 2859 - 2869.
GOLD (2014) Genomes On Line Database. https://gold.jgi-psf.org/.
Gonzales-Siso, M. I. 1996. The biotechnological utilization of cheese whey. A review. Bioresearch Technology, 57: 1 - 11.
González, M. I., Álvarez, S., Riera, F. and Álvarez, R. 2007. Economic evaluation of an integrated process for lactic acid production from ultra filtered whey. Food and engineering, 80 (2): 553 - 561. 170
Gopal, G., Raol, B. V., Raol, V. S., Prajapati., Nirav, H. and Bhavsar. 2015. Utilization of agro-industrial waste for β-galactosidase production under solid state fermentation using halotolerant Aspergillus tubingensis GR1 isolate. Biotechnology, 5: 411 - 421.
Gosling, A., Stevens, G. W., Barber, A. R., Kentish, S. E. and Gras, S. L. 2010. Food Chemistry, 121 (2): 307 - 318.
Grazina, J., Daiva, Z., Elena, B. and Dovile, K. 2016. Application of acid tolerant Pedioccocus strains for increasing the sustainability of lactic acid production from cheese whey. LWT - Food Science and Technology, 72: 399 - 406.
Greenberg, N. A. and Mahoney, R. R. 1982. Production and characterization of β-galactosidase from Streptococcus thermophilus. Journal of Food Science, 47: 1824 - 1828.
Gueimonde, M., Corzo, N., Vinderola, G., Reinheimer, J. and De Los, R. G. 2002. Journal of Dairy Research, 69: 125-137.
Guimarães, P. M. R., Teixeira, J. A. and Domingues, L. 2010. Research review paper: fermentation of lactose to bio-ethanol by yeasts as part of integrated solutions for the valorisation of cheese whey. Biotechnology Advances, 28 (3): 375-384.
Gupta, A. M. and Nair, J. S. 2010. β-Galactosidase production and ethanol fermentation from whey using Kluyveromyces marxianus NCIM 3551. Journal of scientific and industrial Research, 69: 85 - 859.
Gupta, R., Beg, Q. K. and Lorenz, P. 2002. Bacterial alkaline proteases: molecular approaches and industrial applications. Applied Microbiology and Biotechnology, 59: 15 - 32.
Gupta, R., Gigras, P., Mohapatra, H., Goswami, V. K. and Chauhan, B. 2003. Microbial α-amylases: a biotechnological perspective. Process Biochemistry, 38: 1599 - 1616.
Hagström, A., Pommier, T., Rohwer, F., Simu, K., Stolte, W., Svensson, D. and Zweifel, U. L. 2002. Use of 16S ribosomal DNA for delineation of marine 171
bacterioplankton species. Applied and Environmental Microbiology, 68: 3628 - 3633.
Haider, T. and Husain, Q. 2007. Calcium alginate entrapped preparation of Aspergillus oryzae β-galactosidase: its stability and application in the hydrolysis of lactose. Journal of Biological Macromolecules, 41: 72 - 80.
Handelsman, J. 2004. Metagenomics: application of genomics to uncultured microorganisms. Microbiology and Molecular Biology Reviews, 68: 669 - 685.
Harju, M. 1987. Lactose hydrolysis. Bulletin of International Dairy Federation, 212: 50 - 54.
Harju, M., Kallioinen, H. and Tossavainen, O. 2012. Lactose hydrolysis and other conversions in dairy products: Technological aspects. International Dairy Journal, 22: 104 - 109.
He, T., Priebe, M. G., Zhong, Y., Huang, C., Harmsen, H. J., Raangs, G. C., Antoine, J. M., Welling, G. W. and Vonk, R. J. 2008. Effects of yogurt and bifidobacteria supplementation on the colonic microbiota in lactose intolerant subjects. Journal of Applied Microbiology, 104 (2): 595 - 604.
Héctor, A., Cristóbal., Juliana, B., Gustavo, A., Lovrich., Carlos, M. and Abate. 2015. Phylogenentic and enzymatic characterization of psychrophilic and psychrotolerant marine bacteria belong to γ-Proteobacteria group isolated from the sub-Antarctic Beagle Channel, Argentina. Folia Microbiologica, 60: 183 - 198.
Heyman, M. B. 2006. The Committee on Nutrition. Lactose intolerance in infants, children, and adolescents. Journal of Pediatrics, 118: 1297 - 1286.
Hildebrandt, P., Wanarska, M. and Kur, J. 2009. A new cold-adapted β-D- galactosidase from the Antarctic Arthrobacter sp. 32c–gene cloning, overexpression, purification and properties. BMC Microbiology, 9 (151): 1 - 11.
Hin, C., Chien, A., His, L., Yeh, W., Hsueh, C. and Chin, F. 1986. Applied and Environmental Microbiology, 52 (5): 1147 - 1152. 172
Hirsch, P. and Müller, M. 1985. Blastobacter aggregatus sp. nov., Blastobacter cap-sulatus sp. nov. and Blastobacter denitrificans sp. nov., new budding bacteria from fresh water habitats. Systematic and Applied Microbiology, 6: 281 - 286.
Holmes, D. E., Nevin, K. P. and Lovley, D. R. 2004. Comparison of 16S rRNA, nifD, recA, gyrB, rpoB and fusA genes within the family Geobacteraceae fam. nov. International Journal of Systematic and evolutionary Microbiology. 54: 1591 - 1599.
Holzapfel, W.H. and Schillinger, U. 2002. Introduction to pre- and probiotics. Food Research International, 35: 109 - 116.
Hoyoux, I., Jennes, P., Dubois, S., Genicot, F., Dubail, J. M., François, E., Baise, G., Feller. and Gerday, C. 2001. Cold-Adapted β-Galactosidase from the Antarctic Psychrophile Pseudoalteromonas haloplanktis. Applied and Environmental Microbiology, 67 (4): 1529 - 1535.
Hsu, C. A., Yu, R. C. and Chou, C. C. 2005. Production of β-galactosidase by bifidobacteria as influenced by various culture conditions. International Journal of Food Microbiology, 104: 197 - 206.
Hugenholtz, P., Goebel, B. M. and Pace, N. R. 1998. Impact of culture- independent studies on the emerging phylogenetic view of bacterial diversity. Journal of Bacteriology, 180: 4765 - 4774.
Husain, Q. 2010. Beta galactosidases and their potential applications: a review. Critical Reviews in Biotechnology, 30: 41 - 62.
Iqbal, S., Nguyen, T. H., Nguyen, T. T., Maischberger, T. and Haltrich, D. 2010. β-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides. Carbohydrate Research, 345 (10): 1408 - 1416.
Itoh, T., Suzuki, M. and Adachi, S. 1982. Production and characterization of β-Galactosidase from lactose fermenting Yeasts. Agricultural and biological Chemistry, 46: 899 - 904. 173
Jacobson, R. H., Zhang, X. J., Du Bose, R. F. and Matthews, B. W. 1994. Three dimensional structure of β-galactosidase from E. coli. Nature, 369: 761 - 766.
Jayashree, N. C., Christobell, D. J., Mukesh, K. M. D., Balakumaran, M., Ravi, K. and Kalaichelvan P. T. 2012. Isolation and Characterization of β- galactosidase Producing Bacillus sp. from Dairy Effluent. World Applied Sciences Journal, 17 (11): 1466 - 1474.
Jelen, P. 2003. Whey processing. In Roginski, H., Fuquay, J. W. and Fox, F. P. eds. Encyclopedia of dairy sciences. Academic Press, London. pp. 2739 - 2751.
Jelen, P. 2009. Dried whey, whey proteins, lactose and lactose derivative products in Dairy Powders and Concentrated Products. Tamime, A. Y. ed. Wiley-Blackwell, Oxford, U. K. pp. 255 - 267.
Jokar, A. and Karbassi, A. 2009. Determination of proper conditions for the production of crude β-galactosidase using Lactobacillus delbrueckii sp bulgaricus. Journal of Agricultural Science and Technology, 11: 301 - 308.
Jones, P. G., Mitta, M., Kim, Y., Jiang, W. N. and Inouye, M. 1996. Cold shock induces a major ribosomal-associated protein that unwinds double- stranded RNA in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 93: 76 - 80.
Jordan, D. C. 1984. Family III Rhizobiaceae. In Bergey’s Manual of Systematic Bacteriology, Edited by Krieg, N. R. and Holt, J. G. Baltimore: William & Wilkins. vol. 1, pp. 234 - 244.
Joshi, Y., Senatore, B. and Poletto, M. 2011. Kluyveromyces marxianus biofilm in cheese whey fermentation for bioethanol production, Chemical Engineering Transactions. 24: 493 - 498.
Junge, K., Imhoff, F., Staley, T. and Deming, W. 2002. Phylogenetic diversity of numerically important arctic sea-ice bacteria cultured at sub-zero temperature. Microbial Ecology, 43: 315 - 328. 174
Jurado, E., Camacho F., Luzon G. and Vicaria J. M. 2002. A new kinetic model proposed for enzymatic hydrolysis of lactose by a β-galactosidase from Kluyveromyces fragilis. Enzyme and Microbial Technology, 31: 300 - 309.
Karasova, P., Spiwok, V., Mala, S., Kralova, B. and Russell, N. J. 2002. Beta- galactosidase activity in psychrophic microorganisms and their potential use in food industry. Czech Journal of Food Sciences, 20: 43 - 47.
Karpinets, T. V., Park, B. H. and Uberbacher, E. C. 2012. Analyzing large biological datasets with association networks. Nucleic Acids Research, 40.
Kennedy, J., Marchesi, R. and Dobson, D. 2008. Marine metagenomics: strategies for the discovery of novel enzymes with biotechnological applications from marine environments. Microbial Cell Factories, 7: 27.
Kim, J. W. and Rajagopal, S. N. 2000. Isolation and characterization of β- galactosidase from Lactobacillus crispatus. Folia Microbiologica, 45: 29 - 34.
Kisaalita, W. S., Lo, K. V. and Pinder, K. L. 1990. Influence of whey protein on continuous acidogenic degradation of lactose. Biotechnology and Bioengineering, 36 (6): 642 - 646.
Konsoula, Z. and Liakopoulou, M. 2007. Co-production of α-amylase and β- galactosidase by Bacillus subtilis in complex organic substrates. Bioresource Technology, 98: 150 - 157.
Konstantinidis, K. T. and Tiedje, J. M. 2005. Genomic insights that advance the species definition for prokaryotes. Proceedings of the National Academy of Sciences of the United States of America, 102: 2567 - 2572.
Konstantinidis, K. T., Ramette, A. and Tiedje, J. M. 2006. Toward a more robust assessment of intraspecies diversity, using fewer genetic markers. Applied and Environmental Microbiology, 72: 7286 - 7293.
Kreft, M. E. and Jelen P. 2000. Stability and activity of β-galactosidase in sonicated cultures of Lactobacillus delbrueckii ssp. bulgaricus 11842 affected by temperature and ionic environments. Journal of Food Science, 65: 1364 - 1368. 175
Ku, M. A. and Hang, Y. D. 1992. Production of yeast lactase from sauerkraut brine. Biotechnology letters, 14: 925 - 928.
Kumar, D. J. M., Sudha, M., Devika, S., Balakumaran, M. D., Kumar M. R. and Kalaichelvan, P. T. 2012. Production and Optimization of β-galactosidase by Bacillus Sp. MPTK 121, Isolated from Dairy Plant Soil. Annal Biological Research, 3 (4): 1712 - 1718.
Kumari, S., Panesar, S. P. and Panesar, R. 2011. Production of β- Galactosidase using novel yeast isolate from whey. International journal of Dairy Science, 6 (2): 150 - 157.
Kushwaha, J. P., Srivastava, V. C. and Mall, I. D. 2010. Treatment of dairy waste water by inorganic coagulants: parametric and disposal studies. Water Resources, 44 (20): 5867 - 5874.
Kyrpides, N. C., Hugenholtz, P., Eisen, J. A., Woyke, T., Göker, M., Parker, C. T., Amann, R., Beck, B. J., Chain, P. S. G. and Chun, J. 2014. Genomic encyclopedia of bacteria and archaea: sequencing a myriad of type strains. PLOS Biology, 12: 920 - 1001.
Ladero, M., Perez, M. T. and Garcia, O. F. 2003. Hydrolysis of lactose by free and immobilized β-galactosidase from Thermus sp. strain T2. Biotechnology and Bioengineering, 81: 241 - 252.
Lagesen, K., Ussery, D. W. and Wassenaar, T. M. 2010. Genome update: the 1000th genome - a cautionary tale. Microbiology, 156: 603 - 608.
Lee, H. S., Kwon, K. K., Kang, S. G., Cha, S. S., Kim, S. J. and Lee, J. H. 2010. Approaches for novel enzymes discovery form marine environments. Current Opinion in Biotechnology, 21: 353 - 357.
Lee, H., Song, M., Yu, Y. and Hwang, S. 2003. Production of Ganoderma lucidum mycelium using cheese whey as an alternative substrate: response surface analysis and biokinetics. Biochemical Engineering Journal, 15 (2): 93 - 99.
Lee, L. H., Cheah, Y. K., Nurul, S. A. M., Shiran, M. S., Tang, Y. L., Lin, H. P. and Hong, K. 2012. Analysis of Antarctic proteobacteria by PCR 176
fingerprinting and screening for antimicrobial secondary metabolites. Genetics and Molecular Research, 11 (2): 1627 - 1641.
Linko, S., Enwald, S., Zhu, Y. H. and Mayna, M. A. 1998. Production of β- galactosidase by Streptococcus salivarus subsp. thermophilus 11F. Journal of Industrial Microbiology and Biotechnology, 20: 215 - 219.
Litchfield, C. D. 2011. Potential for industrial products from the halophilic Archaea. Journal of Industrial Microbiology and Biotechnology, 38: 1635 - 1647.
Macfarlane, G. T., Steed, H. and Macfarlane, S. 2008. Bacterial metabolism and health-related effects of galacto-oligosaccharides and other prebiotics. Journal of Applied Microbiology, 104: 305 - 344.
Mahoney, R. R. 1985. Modification of lactose and lactose-containing dairy products with β-galactosidase. Developments in Dairy Chemistry, Fox, P. F. (ed). Elsevier Applied Science Publishers, New York. Vol. 3, pp. 69 - 110.
Mahoney, R. R. 1998. Galactosyl-oligasaccharide formation during lactose hydrolysis: a review. Food Chemistry, 63 (2): 147 - 154.
Maity, M., Sanyal, S., Bhowal, J. and Bhattacharyya, D. K. 2013. Studies on Isolation and Characterization of Lactase Produced from Soil Bacteria. Research Journal of Recent Sciences. 2 (8): 92 - 94.
Malaspina, F., Cellamare, C. M., Stante, L. and Tilche, A. 1996. Anaerobic treatment of cheese whey with a down flow-up flow hybrid reactor. Bioresource Technology, 55 (2): 131 - 139.
Maldonado, J., Gil, A., Narbona, E. and Molina, J. A. 1998. Special formulas in infant nutrition: a review. Early Human Development, 58: 23 - 32.
Manera, A. P., Ores, J. C., Ribeiro, V. A., Rodrigues, M. I., Kalil, S. J. and Filho, F. M. 2011. Use of agro industrial residues in biotechnological process by beta-galactosidase production from Kluyveromyces marxianus CCT 7082, Acta Scientiarium Technology, 33: 155 - 161.
Manera, A. P., Ores, J. D. C., Ribeiro, V. A., Burkert, C. A. V and Kalil, S. J. 2008. Optimization of the culture medium for the productionof β-galactosidase 177
from Kluyveromyces marxianus CCT 7082. Food Technology and Biotechnology, 46: 66 - 72.
Margesin, R. and Schinner, F. 1997. Efficiency of indigenous and inoculated cold-adapted soil microorganisms for biodegradation of diesel oil in alpine soils. Applied and Environmental Microbiology, 63: 2660 - 2664.
Margesin, R. and Schinner, F. 1998. Low-temperature bioremediation of a waste water contaminated with anionic surfactant and fuel oil. Applied Microbiology and Biotechnology, 49: 482 - 486.
Margesin, R. and Schinner, F. 1999. Biodegradation of organic pollutants at low temperatures. In Biotechnological Applications of Cold adapted Organisms. Margesin, R. and Schinner, F. eds. Springer. pp. 271 - 289.
Maria, D. F., Somerlate, B., Daison, O. S., Adao, J. R. P., Walter,V. G. and Arnaldo, C. B. 1985. Production of β-D-galactosidase from Kluyveromyces fragilis grown in cheese whey. Dairy Science, 68 (7): 1618.
Mariane, S. and Peter, S. 2010. Identification, cloning and expression of a cold-active β-galactosidase from a novel Arctic bacterium, Alkalilactibacillus ikkense. Environmental Technology, 31 (10): 1107 - 1114.
Mariane, S., Anders, P. and Peter, S. 2006. Bacterial diversity in permanently cold and alkaline ikaite columns from Greenland. Extremophiles, 10: 551 - 562.
Marinelli, F., Brunati, M., Sponga, F. and Ciciliato, I. 2004. Biotechnological Exploitation of Heterotrophic Bacteria and Filamentous Fungi Isolated from Benthic Mats of Antarctic Lakes. In: Microbial Genetic Resources and Biodiscovery. Kurtböke, I. and Swings, J. eds. Queensland Complete Printing Services, Queensland. pp. 163 - 184.
Marrakchi, M., Dzyadevych, S. V., Lagarde, F., Martelet, C. and Jaffrezic, R. N. 2008. Conductometric biosensor based on glucose oxidase and beta- galactosidase for specific lactose determination in milk. Material Science and Engineering C, 28: 872 - 875. 178
Marteau, P. R., de Vrese, M., Cellier, C. J. and Schrezenmeir, J. 2001. Protection from gastrointestinal diseases with the use of probiotics. 73: 430S - 436S.
Martinez, R. C., Fullana, N., Musto, H. and Castro, S. S. 2012. Antarctic DNA moving forward: genomic plasticity and biotechnological potential. FEMS Microbiology Letters, 331: 1 - 9.
Marwaha, S. S. and Kennedy, J. F. 1988. Whey pollution problem and potential utilization. Journal of Food Science and Technology, 23: 323 - 336.
Matheus, A. O. R. and Rivas, N. 2003. Production and partial characterization of β-D-galactosidase from Kluyveromyces marxianus grown in deproteinized whey. Archivos latinoamericanos de nutrición, 53: 194 - 201.
Mawson, A. J. 1994. Bioconversions for whey utilization and waste abatement. Bioresource Technology, 47 (3): 195 - 203.
Mende, D. R., Waller, A. S., Sunagawa, S., Jarvelin, A. I., Chan, M. M., Arumugam, M., Raes, J. and Bork, P. 2012. Assessment of metagenomic assembly using simulated next generation sequencing data. PLOS ONE, 7.
Miet, M., Peter, D., Renata, C., Monique, G., Paul, D. V. and Anne, W. 2008. Advantages of multilocus sequence analysis for taxonomic studies: a case study using 10 housekeeping genes in the genus Ensifer (including former Sinorhizobium). International Journal of Systematic and Evolutionary Microbiology, 58: 200 - 214.
Miller, G., Jarvis, J. and Mc Bean, L. D. 1995. Lactose Intolerance. Handbook of Dairy Foods and Nutrition. CRC Press, Boca Raton. FL. pp. 187 - 220.
Minhalma, M., Magueijo, V., Queiroz, D. P. and Pinho, M. N. 2007. Optimization of „„Serpa‟‟ cheese whey nano filtration for effluent minimization and by-products recovery. Journal of Environmental Management, 82 (2): 200 - 206.
Miriam, L., Loren, H., Se-Ran, Jun., Intawat, N., Michael, R., Leuze, T. H. A., Tatiana, K., Ole, L., Guruprased, K., Trudy, W., Suresh, P., David, W. U. 2015. Insights from 20 years of bacterial genome sequencing. Functional and Integrative Genomics, 15: 141 - 161. 179
Mizrahi, M. O., Davenport, E. R. and Gilad, Y. 2013. Taxonomic classification of bacterial 16S rRNA genes using short sequencing reads: evaluation of effective study designs. PLOS ONE, 8.
Moncheva, P, Tishkov, S., Dimitrova, N. and Chipeva, V. 2002. Characteristics of soil actinomycetes from Antarctica. Journal of Culture Collections, 3: 3 -14.
Montanari, G., Zambonelli, C., Grazia, L., Benevelli, M. and Chiavari, C. 2000. Release of β-galactosidase from Lactobacilli. Food Technology and Biotechnology, 38 (2): 129 - 133.
Mozumder, N. H. M. R., Akhtaruzzaman, A., Bakr, M. A. and Tuj-Zohra, F. 2012. Study on Isolation and Partial Purification of Lactase (β-Galactosidase) Enzyme from Lactobacillus Bacteria Isolated from Yogurt, Journal of Scientific Research, 4 (1): 239 - 249.
Muhammad, R. T., Aysha, S., Muhammad, I. K., Nuzhat, H. and Adeela, Y. 2013. Nutritional and therapeutic properties of whey. Annals of Food Science and Technology, 19 - 26.
Mukhtar, K., Asgher, M., Afghan, K., Hussain, K. and Zia-ul-Hussnain, S. 2010. Comparative study on two commercial strains of Saccharomyces cerevisiae for optrimm ethanol production on industrial scale. Journal of Biomedicine and Biotechnology,1 - 5.
Nagy, Z., Keresztessy, Z., Szentirmai, A. and Biro, S. 2001. Carbon source regulation of β-galactosidase biosynthesis in Penicillium chrysogenum. Journal of Basic Microbiology, 41: 351 - 362.
Nam, E. S. and Ahn, J. K. 2011. Isolation and characterization of cold adapted bacteria producing lactose hydrolyzing enzyme isolated from soils of Nome area in Alaska. International Research Journal of Microbiology, 2 (9): 348 - 355.
Naser, S. M., Thompson, F. L., Hoste, B., Gevers, D., Dawyndt, P., Vancanneyt, M. and Swings, J. 2005. Application of multilocus sequence analysis (MLSA) for rapid identification of Enterococcus species based on rpoA and pheS genes. Microbiology. 151: 2141 - 2150. 180
Natarajan, J., Christobell, C., Mukesh., Kumar, D. J., Balakumaran, M. D., Ravi, K. M., Kalaichelvan, P. T. 2012. Isolation and Characterization of β- Galactosidase Producing Bacillus sp. from Dairy Effluent. World Applied Sciences Journal. 17 (11): 1466 - 1474.
NCBI (2014) National Center for Biotechnology Information Genome Browser. http://www.ncbi.nlm.nih.gov/genome/browse/.
Nguyen, H. T., Voza, F., Ezzeddine, N. and Frasch, M. 2007. Drosophila mind bomb2 is required for maintaining muscle integrity and survival. Journal of Cell Biology, 179 (2): 219 - 227.
Nizamuddin, S., Sridevi, A. and Narasimha, G. 2008. Production of β- galactosidase by Aspergillus oryzae in solid-state fermentation. African Journal of Biotechnology, 7: 1096 - 1100.
Nor, Z. M., Tamer, M. I., Mehrvar, M., Scharer, J. M., Moo-Young, M. and Jervis, E. J. 2001. Improvement of intracellular β-galactosidase production on fed-batch culture of Kluyveromyces fragilis. Biotechnology Letters, 23: 845 - 849.
Novalin, S., Neuhaus, W. and Kulbe, K. D. 2005. A new innovative process to produce lactose reduces skim milk. Journal of Biotechnology, 119: 212 - 218.
Nurullah, K. 2011. High level production of extracellular β-galactosidase from Bacilluslicheniformis ATCC 12759 in submerged fermentation. African Journal of Microbiology Research. 5 (26): 4615 - 4621.
OECD-FAO. 2014. Organization for Economic Co-operation and Development, Food and Agriculture Organization of the United Nations. OECD-FAO Agricultural Outlook 2014–2023: Dairy. Accessed Jun. 16, 2015.
OECD-FAO. OECD-FAO Agricultural Outlook 2008–2017 Highlights. Paris: Organization for Economic Co-operation and Development - Food and Agriculture Organization of the United Nations, 2008.
Ok, S. K., Yong, J. C., Kihyun, L., Seok, H. Y., Mincheol, K., Hyunsoo, Na., Sang, C. P., Yoon, S. J., Jae-Hak, L., Hana, Y., Sungho, W. and Jongsik, C. 2012. Introducing EzTaxon-e: A prokaryotic 16S rRNA gene sequence 181
database with phylotypes that represent uncultured species. International Journal of Systematic and Evolutionary Microbiology. 62: 716 - 721.
Onishi, N., Yamashiro, A. and Yokozeki, K. 1995. Production of galactooligosaccharide from lactose by Stengmatomyces elviae CBS8119. Applied and Environmental Microbiology, 61: 4022 - 4025.
Outinen, M., Heino, A. and Uusi, R. J. 2010. Pretreatment methods of Edam cheese milk. Effect on the whey composition. LWT - Food Science and Technology, 43: 647 - 654.
Pakistan Dairy Development Company (PDDC). 2006. The White Revolution “ hoodh arya”, Retrieved May 4, 2012, from http://www.pddc.com.pk/Dairy Pakistan-Publication.
Panesar, P. S., Panesar, R., Singh, R. S., Kennedy, J. F. and Kumar, H. 2006. Microbial production, immobilization and applications of β-D-galactosidase. Journal of Chemical Technology and Biotechnology, 81: 530 - 543.
Panesar, P. S., Shweta, K. and Panesar, R. 2010. Potential application of immobilized and β-galactosidase in food processing industries. Enzyme Research, 1 - 16.
Park, A. R. and Oh, D. K. 2010b. Galacto-oligosaccharide production using microbial beta-galactosidase: current state and perspectives. Applied Microbiology and Biotechnology, 85: 1279 - 1286.
Patel, R., Dodia, M. and Singh, S. P. 2005. Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp. production and optimization. Process Biochemistry, 40: 3569 - 3575.
Pattnaik, R., Yost, R. S., Porter, G., Masunaga, T. and Attanandana, T. 2007. Improving multi-soil-layer (MSL) system remediation of dairy effluent. Ecological engineering, 32 (1): 1 - 10.
Pinheiro, R., Belo, I. and Mota, M. 2003. Growth and β-galactosidase activity in cultures of Kluyveromyces marxianus under increased air pressure. Letters in Applied Microbiology, 37: 438 - 442. 182
Pisani, F. M., Rella, R., Raia, C. A., Rozzo, C., Nucci, R. 1990. European Journal of Biochemistry, 187: 321 - 328.
Pivarnik, L. F., Senacal, A. G. and Rand, A. G. 1995. Hydrolytic and transgalactosylic activities of commercial β-galactosidase (lactase) in food processing. Advances in Food and Nutrition Research, 38: 1 - 102.
Pouliot, Y. 2008. Membrane processes in dairy technology from a simple idea to worldwide panacea. International Dairy Journal, 18: 735 - 740.
Pray, W. S. 2000. Lactose Intolerance: The Norm Among the World‟s Peoples American Journal of Pharmaceutical Education, 64: 205 - 207.
Prescott, L. M., Harley, J. P. and Klein, D. A. 1990. Microbiology. Wm. C. Brown Publishers. Duburque. IA.
Princely, S., Saleem, B. N., John, K. J. and Dhanaraju, M. D. 2003. Biochemical characterization, partial purification, and production of an intracellular beta-galactosidase from Streptococcus thermophilus grown in whey. European Journal of Experimental Biology. 3 (2): 242 - 251.
Qin, W., Deng, Z. S., Xu, L., Wang, N. N. and Wei, G. H. 2012. Rhizobium helanshanense sp. nov., a bacterium that nodulates Sphaerophysa salsula (Pall.) DC. in China. Archives of Microbiology. 194: 371 - 378.
Qing, D., Xufan, Y., Minhui, Z. and Ziwen, Y. 2014. Characterization of an extremely thermostable but cold-adaptive β-galactosidase from the hyperthermophilic archae on Pyrococcus furiosus for use as a recombinant aggregation for batch lactose degradation at high temperature. Journal of Bioscience and Bioengineering. 117 (6): 706 - 710.
Rajakal, P. and Selvi, P. K. 2006. The effect of pH, temperature and alkali metal ions on the hydrolysis of whey lactose catalysed by β-galactosidase from KLuyveromyces marxiansus. International Journal of Dairy Science, 1: 167 - 172.
Rajeshwari, K. V., Balakrishnan, M., Kansal, A., Lata, K. and Kishore, V. V. N. 2000. State of-the-art of anaerobic digestion technology for industrial wastewater treatment. Renewable Sustainable Energy Reviews, 4 (2): 135 - 156. 183
Rajoka, M. I., Khan, S. and Shahid, R. 2003. Kinetics and regulation studies of the production of β-D-galactosidase from Kluyveromyces marxianus grown on different substrates. Food Technology and Biotechnology, 41: 315 - 320.
Ram, K. D., Syed, I. A. and Lokendra, S. 2005. Characterization of β- galactosidase from Antarctic Bacillus sp. Indian Journal of Biotechnology. 4: 227 - 231.
Ramesh, M. V. and Lonsane, B. K. 1987. Solid state fermentation for production of α-amylase by Bacillus megaterium 16M. Biotechnology Letters, 9 (5): 323 - 328.
Rao, R. M. V. and Dutta, S. M. 1977. Production of β-galactosidase from Streptococcus thermophilus grown in whey. Applied and Environmental Microbiology, 34: 185- 188.
Rasooli, I., Astaneh, S. D. A., Borna, H. and Barchini, K. A. 2008. Thermostable α-amylase producing natural variant of Bacillus spp. isolated from soil in Iran. American journal of Agricultural and Biological Sciences, 3: 591 - 596.
Rastall, R. A., Gibson G. R., Gill, H. S., Guarner, F., Klaenhammer, T. R., Pot, B., Reid, G., Rowland I. R. and Sanders, M. E. 2005. Modulation of the microbial ecology of the human colon by probiotics, prebiotics and synbiotics to enhance human health: an overview of enabling science and potential applications. FEMS Microbiology Ecology, 52: 145 - 152.
Rech, R., Cassini, C. F., Secchi, A. and Ayub, M. A. Z. 1999. Utilization of protein-hydrolyzed cheese whey for production of β-galactosidase by Kluyveromyces marxianus. Journal of Industrial Microbiology & Biotechnology, 23: (2) 91 - 96.
Ren, D. W., Wang, E. T., Chen, W. F., Sui, X. H., Zhang, X. X., Liu, H. C. and Chen, W. X. 2011. Rhizobium herbae sp. nov. and Rhizobium giardinii related bacteria, minor microsymbionts of various wild legumes in China. International Journal of Systematic and Evolutionary Microbiology, 61: 1912 - 1920. 184
Reyhan, G. G., Kemal, G., Annarita, P. and Barbara, N. 2007. Purification and some properties of a β-galactosidase from the thermoacidophilic Alicyclobacillus acidocaldarius sub sp. rittmannii isolated from Antarctica. Enzyme and Microbial Technology. 40: 1570 - 1577.
Ricardo, C., Khawar, S. S., David, A. and Kevin, R. S. 2002. Low-temperature extremophiles and their applications. Current Opinion in Biotechnology. 13: 253 - 261.
Richmond, M. L., Gray, J. I. and Stine, C. M. 1981. Beta-galactosidase: Review of recent research related to technological application, nutritional concerns, and immobilization. Journal of Dairy Science, 64: 1759 - 1771.
Rivas, J., Prazeres, A. R. and Carvalho, F. 2011. Aerobic biodegradation of pre-coagulated cheese whey wastewater. Journal of Agricultural and Food Chemistry, 59 (6): 2511 - 2517.
Rivas, J., Prazeres, A. R., Carvalho, F. and Beltrán, F. 2010. Treatment of cheese whey wastewater: combined coagulation-flocculation and aerobic biodegradation. Journal of Agricultural and Food Chemistry, 58 (13): 7871 - 7877.
Rojoka, M. J., Khan, S. and Shahid, R. 2003. Food Technology and Biotechnology, 41: 315 - 320.
Rossello´, M. R. and Amann, R. 2001. The species concept for prokaryotes. FEMS Microbiology Reviews, 25: 39 - 67.
Rupinder, K., Parmjit, S., Panesar. And Ram, S. S. 2015. Utilization of Whey for the Production of β-Galactosidase Using Yeast and Fungal Culture. International Journal of Biological, Biomolecular, Agricultural, Food and Biotechnological Engineering, 9 (7): 739 - 743.
Sambrook, J. F. and Russell, D. W. 2001. 3rd ed. Cold Spring Harbor Laboratory Press, Molecular Cloning: A Laboratory Manual. Vol. 1, 2 and 3.
Santos, A., Ladero, M. and Garcia, O. F. 1998. Kinetic modeling of lactose hydrolysis by a β-galactosidase from Kluyveromyces fragilis. Enzyme and Microbial Technology, 22: 558 - 567. 185
Savaiano, D. A. and Levitt, M. D. 1987. Milk intolerance and microbe-
containing dairy foods. Journal of Dairy Science. 70 (2): 397 - 406.
Schmidt, T. M., De Long, E. F. and Pace, N. R. 1991. Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing. Journal of Bacteriology, 173: 4371 - 4378.
Scholz, M. B., Lo, C. C. and Chain, P. S. G. 2012. Next generation sequencing and bioinformatic bottlenecks: the current state of metagenomic data analysis. Current Opinion in Biotechnology, 23: 9 - 15.
Seyed, A. M., Anne, W., Xavier, N., Philippe, D. L. and Kristina, L. 2015. Revised phylogeny of Rhizobiaceae: Proposal of the delineation of Pararhizobium gen. nov., and 13 new species combinations. Systematic and Applied Microbiology, 38: 84 - 90.
Shaikh, S. A., Khire, J. M. and Khan, M. I. 1997. Production of β- galactosidase from thermophilic fungus Rhizomucor sp. Journal of Industrial Microbiology and Biotechnology, 19: 239 - 245.
Singh, B. K. 2010. Exploring microbial diversity for biotechnology: the way forward. Trends in Biotechnology, 28: 111 - 116.
Siso, M. I. G. 1996. The biotechnological utilization of cheese whey: a review. Bioresource Technology, 57 (1): 1 - 11.
Sittig, M. and Hirsch, P. 1992. Chemotaxonomic investigation of budding and orhyphal bacteria. Systematic and Appied Microbiology, 15: 209 - 222.
Smal, A. O., Leiros, H. K. S., Os, V. and Willassen, N. P. 2000. Cold-adapted enzymes. Biotechnology Annual Review, 6: 1 - 57.
Smithers, G. W. 2008. Whey and whey proteins - from „gutter-to-gold‟. International Dairy Journal, 18: 695 - 704.
Somkuti, G. A., Dominiecki, M. E. and Steýnberg, D. H. 1998. Permeabilization of Streptoccus thermophilus and Lactobacillus delbrueckii sub sp. bulgaricus with ethanol. Current Microbiology, 36: 202 - 206. 186
Somyos, O. and Phimchanok J. 2009. Isolation and characterization of β- galactosidase from the thermophile B1.2. Asian Journal of Food and Agro- Industry, 2 (04): 135 - 143.
Soon, W. K., Jin, Y. P., Jong, S. K., Jun, W. K., Yang, H. C., Chun, K. L., Matthew, A. P. and Gil, B. L. 2005. Phylogenetic analysis of the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium on the basis of 16S rRNA gene and internally transcribed spacer region sequences. International Journal of Systematic and Evolutionary Microbiology, 55: 263 - 270.
Sossna, R. 2014a. 2014. The world of whey got together: 7th International Whey Conference. International Dairy Magazine. 10: 22 - 23.
Sossna, R. 2014b. 2014. 3A Business Consulting: WHEY BOOK. International Dairy Magazine. 10: 20 - 21.
Sreekumar, G. and Soundarajan K. 2010. Isolation and characterization of probiotic Bacillus subtilis SK09 from dairy effluent. Indian Journal of Science and Technology, 3: 863 - 866.
Stackebrandt, E. and Goebel, B.M. 1994. Taxonomic Note: A Place for DNA- DNA Re-association and 16S rRNA Sequence Analysis in the Present Species Definition in Bacteriology. International journal of systematic bacteriology, 846 - 849.
Swati, S. and Sanjay, T. 2012. Biotechnological utilization of dairy waste to solve environmental problem. Journal of Environmental Research and Development. 6: 721 - 726.
Szajewska, H. and Horvath, A. 2010. Meta-analysis of the evidence for a partially hydrolyzed 100 % whey formula for the prevention of allergic diseases. Current Medical Research and Opinion, 26: 423 - 437.
Tae, H. A., Tatiana, K., Ole, L., Guruprased, K., Trudy, W., Suresh, P. and David, W. U. 2015. Insights from 20 years of bacterial genome sequencing. Functional and Integrative Genomics, 15: 141 - 161.
Taton, A., Grubisic, S., Ertz, D. and Hodgson, D. A. 2006. Polyphasic study of Antarctic cyanobacterial strains. Journal of Phycology, 42: 1257 - 1270. 187
Tejayadi, S. and Cheryan, M. 1995. Lactic acid from cheese whey permeate. Productivity and economics of a continuous membrane bioreactor. Applied Microbiology and Biotechnology, 43 (2): 242 - 248.
Terefework, Z., Kaijalainen, S. and Lindstrom, K. 2001. AFLP fingerprinting as a tool to study the genetic diversity of Rhizobium galegae isolated from Galega orientalis and Galega officinalis. Journal of Biotechnology, 91: 169 - 180.
Thigiel, A. A. and Deak, T. 1989. Selection of strains and extraction procedures for optimum production of β-D-galactosidase from Kluyveromyces strains. Zentralblatt für Mikrobiologie, 144: 465 - 471.
Thompson, F. L., Gevers, D., Thompson, C. C., Dawyndt, P., Naser, S., Hoste, B., Munn, C. B. and Swings, J. 2005. Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis. Applied and Environmental Microbiology, 71: 5107 - 5115.
Tighe, S. W., de Lajudie, P., Dipietro, K., Lindström, K., Nick, G. and Jarvis, B. D. 2000. Analysis of cellular fatty acids and phenotypic relationships of Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium species using the Sherlock Microbial Identification System. International Journal of Systematic and Evolutionary Microbiology, I50: 787 - 801.
Timmis, K. N. and Pieper, D. H. 1999. Bacteria designed for bioremediation. Trends in Biotechnology, 17: 201 - 204.
Tindall, B. J. 2004. Prokaryotic diversity in the Antarctic: the tip of the ice berg. Microbial Ecology, 47: 271 - 283.
Tindall, B. J., Rossello´, M. R., Busse, H. J., Ludwig, W. and Kampfer, P. 2010. Notes on the characterization of prokaryote strains for taxonomic purposes. International Journal of Systematic and Evolutionary Microbiology, 60: 249 - 266.
Tomoyuki, N., Ryoko, I., Masataka, U., Tatsuro, M., Katsumi, T. and Noboru, T. 2006. Cold-active acid β-galactosidase activity of isolated psychrophilic 188
basidiomycetous yeast Guehomyces pullulans. Microbiological Research, 161: 75 - 79.
Trincone, A. 2011. Marine biocatalysts: enzymatic features and applications. Marine Drugs, 9: 478 - 499.
Troelsen, J. T. 2005. Adult-type hypolactasia and regulation of lactase expression. Journal of Biochimica et Biophysica Acta, 1723: 19 - 32.
Tutino, M. L. 1999. Aspartate amino transferase from Moraxella TAC 125: an unusual psychrophilic enzyme. In Cold-adapted Organisms (Margesin, R. and Schinner, F. eds. Springer. pp. 305 - 316.
Tyson, G. W., Chapman, J., Hugenholtz, P., Allen, E. E., Ram, R. J., Richardson, P. M., Solovyev, V. V., Rubin, E. M., Rokhsar, D. S. and Banfield, J. F. 2003. Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 2004, 428: 37 - 43.
USDEC. 2003. Reference manual for US whey and lactose products. www.usdec.org.
van Berkum, P. and Fuhrmann, J. J. 2000. Evolutionary relationships among the soybean bradyrhizobia reconstructed from 16S rRNA gene and internally transcribed spacer region sequence divergence. International Journal of Systematic and Evolutionary Microbiology, 50: 2165 - 2172.
Van, D. B., Revallier, W. J. G. and Van, D. S. L. C. 1950. Preparation of lactase from Saccharomyces fragilis. Netherlands Milk and Dairy Journal, 4: 96 - 114.
Van, D. V., Goiris, K., Syryn, E., Van, D. B. C. and Aerts, G. 2014. Evaluation of the cold-active Pseudoalteromonas haloplanktis β-galactosidase
enzyme for lactose hydrolysis in whey permeate as primary step of D-tagatose production. Process Biochemistry, 49 (12): 2134 - 2140.
Vandamme, P., Pot, B., Gillis, M., De Vos, P. and Swings, J. 1996. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiological Reviews, 60: 407 - 438. 189
Vasiljevic, T. and Jelen, P. 2001. Production of β-galactosidase for lactose hydrolysis in milk and products using thermophilic lactic acid bacteria. Journal of Innovative Food Science and Emerging Technologies, 2: 75 - 85.
Vasiljevic, T. and Jelen, P. 2002. Lactose hydrolysis in milk as affected by neutralizers used for the preparation of crude β-galactosidase extracts from Lactobacillus bulgaricus 11842. Innovative Food Science and Emerging Technologies, 3: 175 - 184.
Venter, J. C., Remington, K., Heidelberg, J. F., Halpern, A. L., Rusch, D., Eisen, J. A., Wu, D., Paulsen, I., Nelson, K. E. and Nelson, W. 2003. Environmental genome shotgun sequencing of the Sargasso Sea. Science, 304: 66 - 74.
Vinderola, C. G. and Reinheimer, J. A. 2003. Lactic acid starter and probiotic bacteria, a comparative “in vitro” study of probiotic characteristics and biological barrier resistance. Journal of Food Research International, 36: 895 - 904.
Voget, C. E., Flores, M. V., Faloci, M. M. and Ertola, R. J. J. 1994. Effects of the ionic environment on the stability of Kluyveromyces lactis β-galactosidase. Lebensmittel Wissenschaft and Technologie, 27 (4): 324 - 330.
Wallenfels, K. and Malhotra, O. P. 1961. “Galactosidases.” Advances in Carbohydrate Chemistry, 16: 239 - 298.
Wang, D. Z., Xie, Z. X. and Zhang, S. F. 2014. Marine metaproteomics: current status and future directions. Journal of Proteome Research, 97: 27 - 35.
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C. and Murray, R. G. E. 1987. International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. International Journal of Systematic Bacteriology, 37: 463 - 464.
Welderufael, F. and Jauregui, P. 2010. Development of an integrated process for the production of bioactive peptides from whey by proteolytic commercial mixtures. Separation Science and technology, 45: 2226 - 2234. 190
Wertz, J. E., Goldstone, C., Gordon, D. M. and Riley, M. A. 2003. A molecular phylogeny of enteric bacteria and implications for a bacterial species concept. Journal of Evolutionary Biology, 16: 1236 - 1248.
Westermann, A., Gorski, S. and Vogel, J. 2012. Dual RNA-seq of pathogen and host. Nature Reviews Microbiology, 10: 618 - 630.
Whitman, W. B., Coleman, D. C. and Wiebe, W. J. 1998. Prokaryotes: the unseen majority. Proceedings of the National Academy of Sciences of the United States of America, 95: 6578 - 6583.
Willems, A., Coopman, R. and Gillis, M. 2001b. Comparison of sequence analysis of 16S–23S rDNA spacer regions, AFLP analysis and DNA–DNA hybridizations in Bradyrhizobium. International Journal of Systematic and Evolutionay Microbiology, 51: 623 - 632.
William, P., Hanage, C. F. and Brian G. S. 2006. Sequences, sequence clusters and bacterial species. Philosophical Transactions of the Royal Society B: Biological Sciences, 361: 1917 - 1927.
Woese, C. R. 1987. Bacterial evolution. Microbiological Reviews, 51: 221 - 271.
Woese, C. R., Kandler, O. and Wheelis, M. L. 1990. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proceedings of the National Academy of Sciences of the United States of America, 87: 4576 - 4579.
Wolosowska, S. and Synowiecki, J. 2004. Thermostable β-glucosidase with a broad substrate specifity suitable for processing of lactose-containing products. Food Chemistry, 85: 181 - 187.
Woo, P. C. Y., Lau, S. K. P., Teng, J. L. L., Tse, H. and Yuen, K. Y. 2008. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology Laboratories. Clinical Microbiology and Infection, 14: 908 - 934.
Xanthopoulos, V., Ztaliou, I., Gaier, W., Tzanetakis, N. and Litopoulou, T. E. 1999. Differentiation of Lactobacillus isolates from infant faeces by SDS- 191
PAGE and rRNA-targeted oligonucleotide probes. Journal of Applied Microbiology, 87: 743 - 749.
Xisheng, W., Guoqiang, C., Mei, S., Shuqin, Z., Jinhua, L., Yun, C., Ling, T., Youliang, S., Junling, P. and Zhenyu, Y. 2016. Millennial-scale Asian summer monsoon variations in South China since the last deglaciation. Earth and Planetary Science Letters. 451: 22 - 30.
Xu, L., Shi, J. F., Zhao, P., Chen, W. M., Qin, W., Tang, M. and Wei, G. H. 2011. Rhizobium sphaerophysae sp. nov., a novel species isolated from root nodules of Sphaerophysa salsula in China. Antonie van Leeuwenhoek Journal of Microbiology, 99: 845 - 854.
Yang, S. T. and Silva, E. M. 1995. Novel products and new technologies for use of a familiar carbohydrate, milk lactose. Journal of Dairy Science, 78: 2541- 2562.
Yergeau, E., Hogues, H., Whyte, L. G. and Greer, C. W. 2010. The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses. ISME Journal, 4: 1206 - 1214.
Zadow, J. G. 1984. Lactose - properties and uses. Journal of Dairy Science, 67: 2654 - 2679.
Zadow, J. G. 1986. Lactose hydrolysed dairy products. Food Technology in Australia, 38: 460-462, 471.
Zadow, J. G. 1994. Utilization of milk components In: Robinson, R. K. ed. Modern dairy technology, advances in milk processing, 3rd ed. Chapman and Hall London, UK.
Zhao, S. R., Fung, L. W. P., Bittner, A., Ngo, K. and Liu, X. J. 2014. Comparison of RNA-Seq and microarray in transcriptome profiling of activated T cells. PLOS ONE. 9.
Zhou, Q. Z. K. and Chen, X. D. 2001. Effects of temperature and pH on the catalytic activity of the immobilized β-galactosidase from Kluyveromyces lactis. Biochemistry and Engineering Journal, 9: 33 - 40. 192
Zuzana, M. and Michal, R. 2006. Current trends of β-galactosidase application in food technology. Journal of Food and Nutrition Research, 45: 47 - 54.
ANNEXURE TABLE 1
Strain p Temperature Colour with X- Size of Name Resource Person Origion Media H (◦C) Colour without X-gal gal Incubation Time Growth Colonies
Mediu Larg Small m e
A Mikkel- Schultz Ikka Column Greenland R2 Agar 10 10 Red Blue 26-05-15 to 22-06-15 Yes Yes
B Mikkel-Schultz Ikka Column Greenland R2 Agar 10 10 White Blue 26-05-15 to 22-06-16 Yes Yes
1 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal Blue 26-05-15 to 22-06-17 yes yes
2 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal Green 26-05-15 to 22-06-18 yes yes
3 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-19 no
4 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal white 26-05-15 to 22-06-20 yes yes
5 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal light yellow 26-05-15 to 22-06-21 yes yes
6 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-22 no
7 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-23 no
8 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-24 no
9 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal blue 26-05-15 to 22-06-25 yes yes
10 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-26 no
11 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-27 no
12 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal white 26-05-15 to 22-06-28 yes yes
13 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal dark yellow 26-05-15 to 22-06-29 yes yes
14 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal white 26-05-15 to 22-06-30 yes yes
15 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal dark yellow 26-05-15 to 22-06-31 yes yes
16 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal blue 26-05-15 to 22-06-32 yes yes
xiii
17 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-33 no
18 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-34 no
19 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-35 no yes
20 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal light yellow 26-05-15 to 22-06-36 yes
21 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-37 no
22 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-38 no
23 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-39 no yes
24 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-40 no
25 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-41 no
26 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal white 26-05-15 to 22-06-42 yes
27 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-43 no
28 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-44 no
29 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-45 no
30 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-46 no yes
31 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal white 26-05-15 to 22-06-47 yes
32 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-48 no
33 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-49 no
34 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-50 no
35 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-51 no
36 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-52 no yes
37 Morten Jepsen Antarctic Mc-Murdo R2 Agar 10 10 No plate without x-gal no 26-05-15 to 22-06-53 no
xiv
ANNEXURE TABLE 2
Sample Sample No. By Mikkel Sample No. By Kalsoom Sample Ponds date pH Glaring Naqvi
Terrestrial ponds, Miers valley/near lower Koettlitz glacier (i) Canary Pond 1/15/2010 10.02 Antarctic. 2 1A (ii) Macaw-Pond Mat Orange 1/15/2010 10.41 Antarctic. 3 2A (iii) Kingfisher Pond 1/15/2010 10.07 Antarctic. 5 3A Terrestrial ponds, near Upper Koettlitz Glacier (iv) Little Blister Pond 1/9/2010 10.08 Antarctic. 9 4A Terrestrial ponds, Pyramid Trough (v) Colin Pond Deep 1/15/2010 10.63 Antarctic. 11 5A
xv
ANNEXURE TABLE 3
Sample Name Resource Person Sample Site Media pH Incubation Time Growth 1A Mikkel Glaring Canary Pond Marine 10 08-06-15 to 15-06-15 yes 2A Mikkel Glaring Macaw Pond Marine 10 08-06-15 to 15-06-16 yes 3A Mikkel Glaring Kingfisher Pond Marine 10 08-06-15 to 15-06-17 yes 4A Mikkel Glaring Little Blister Pond Marine 10 08-06-15 to 15-06-18 yes 5A Mikkel Glaring Colin Pond Deep Marine 10 08-06-15 to 15-06-19 yes 1K Mikkel Schultz Ikka Column Marine 10 08-06-15 to onward no 2K Mikkel Schultz Ikka Column Marine 10 08-06-15 to onward no 3K Mikkel Schultz Ikka Column Marine 10 08-06-15 to onward no 4K Mikkel Schultz Ikka Column Marine 10 08-06-15 to onward no 5K Mikkel Schultz Ikka Column Marine 10 08-06-15 to onward no 6K Mikkel Schultz Ikka Column Marine 10 08-06-15 to onward no 7K Mikkel Schultz Ikka Column Marine 10 08-06-15 to onward no 8K Mikkel Schultz Ikka Column Marine 10 08-06-15 to onward no 9k Mikkel Schultz Ikka Column Marine 10 08-06-15 to onward no 10k Mikkel Schultz Ikka Column Marine 10 08-06-15 to onward no
xvi
ANNEXURE TABLE 4
Sample Name Strain No. Colour without X-gal Media pH Temperature (◦C) Incubation Time 1A 1 brown Marine 10 10 15-06-15 to 23-06-15 2 dark yellow Marine 10 10 15-06-15 to 23-06-15 3 dark yellow Marine 10 10 15-06-15 to 23-06-15 4 dark yellow Marine 10 10 15-06-15 to 23-06-15 5 light yellow Marine 10 10 15-06-15 to 23-06-15 6 light yellow Marine 10 10 15-06-15 to 23-06-15 7 light yellow Marine 10 10 15-06-15 to 23-06-15 8 light yellow Marine 10 10 15-06-15 to 23-06-15 9 light yellow Marine 10 10 15-06-15 to 23-06-15 10 light yellow Marine 10 10 15-06-15 to 23-06-15 12 light yellow Marine 10 10 15-06-15 to 23-06-15 13 light yellow Marine 10 10 15-06-15 to 23-06-15 14 light yellow Marine 10 10 15-06-15 to 23-06-15 15 light yellow Marine 10 10 15-06-15 to 23-06-15 16 light yellow Marine 10 10 15-06-15 to 23-06-15 17 light yellow Marine 10 10 15-06-15 to 23-06-15 18 light yellow Marine 10 10 15-06-15 to 23-06-15 19 orange Marine 10 10 15-06-15 to 23-06-15 20 pink Marine 10 10 15-06-15 to 23-06-15 21 pink Marine 10 10 15-06-15 to 23-06-15 22 pink Marine 10 10 15-06-15 to 23-06-15 23 pink Marine 10 10 15-06-15 to 23-06-15 24 pink Marine 10 10 15-06-15 to 23-06-15 25 pink Marine 10 10 15-06-15 to 23-06-15 26 pink Marine 10 10 15-06-15 to 23-06-15
xvii
27 pink Marine 10 10 15-06-15 to 23-06-15 28 pink Marine 10 10 15-06-15 to 23-06-15 29 pink Marine 10 10 15-06-15 to 23-06-15 30 pink Marine 10 10 15-06-15 to 23-06-15 31 pink Marine 10 10 15-06-15 to 23-06-15 32 pink Marine 10 10 15-06-15 to 23-06-15 33 pink Marine 10 10 15-06-15 to 23-06-15 34 pink Marine 10 10 15-06-15 to 23-06-15 35 pink Marine 10 10 15-06-15 to 23-06-15 36 pink Marine 10 10 15-06-15 to 23-06-15 37 white Marine 10 10 15-06-15 to 23-06-15 38 white Marine 10 10 15-06-15 to 23-06-15 39 white Marine 10 10 15-06-15 to 23-06-15 40 white Marine 10 10 15-06-15 to 23-06-15 41 white Marine 10 10 15-06-15 to 23-06-15 42 white Marine 10 10 15-06-15 to 23-06-15 43 white Marine 10 10 15-06-15 to 23-06-15 44 white Marine 10 10 15-06-15 to 23-06-15 45 white Marine 10 10 15-06-15 to 23-06-15 46 white Marine 10 10 15-06-15 to 23-06-15 47 white Marine 10 10 15-06-15 to 23-06-15 48 white Marine 10 10 15-06-15 to 23-06-15 49 white Marine 10 10 15-06-15 to 23-06-15 50 white Marine 10 10 15-06-15 to 23-06-15 51 white Marine 10 10 15-06-15 to 23-06-15 52 white Marine 10 10 15-06-15 to 23-06-15 53 white Marine 10 10 15-06-15 to 23-06-15 54 white Marine 10 10 15-06-15 to 23-06-15
xviii
55 white Marine 10 10 15-06-15 to 23-06-15 56 white Marine 10 10 15-06-15 to 23-06-15 57 white Marine 10 10 15-06-15 to 23-06-15 58 white Marine 10 10 15-06-15 to 23-06-15 59 white Marine 10 10 15-06-15 to 23-06-15 60 white Marine 10 10 15-06-15 to 23-06-15 61 white Marine 10 10 15-06-15 to 23-06-15 62 white Marine 10 10 15-06-15 to 23-06-15 63 white Marine 10 10 15-06-15 to 23-06-15 64 white Marine 10 10 15-06-15 to 23-06-15 65 white Marine 10 10 15-06-15 to 23-06-15 66 white Marine 10 10 15-06-15 to 23-06-15 67 white Marine 10 10 15-06-15 to 23-06-15 68 white Marine 10 10 15-06-15 to 23-06-15 69 white Marine 10 10 15-06-15 to 23-06-15 70 white Marine 10 10 15-06-15 to 23-06-15 2A 1 dark yellow Marine 10 10 15-06-15 to 23-06-15 2 dark yellow Marine 10 10 15-06-15 to 23-06-15 3 dark yellow Marine 10 10 15-06-15 to 23-06-15 4 dark yellow Marine 10 10 15-06-15 to 23-06-15 5 dark yellow Marine 10 10 15-06-15 to 23-06-15 6 light yellow Marine 10 10 15-06-15 to 23-06-15 7 light yellow Marine 10 10 15-06-15 to 23-06-15 8 light yellow Marine 10 10 15-06-15 to 23-06-15 9 light yellow Marine 10 10 15-06-15 to 23-06-15 10 light yellow Marine 10 10 15-06-15 to 23-06-15 11 light yellow Marine 10 10 15-06-15 to 23-06-15 12 light yellow Marine 10 10 15-06-15 to 23-06-15
xix
13 light yellow Marine 10 10 15-06-15 to 23-06-15 14 light yellow Marine 10 10 15-06-15 to 23-06-15 15 light yellow Marine 10 10 15-06-15 to 23-06-15 16 light yellow Marine 10 10 15-06-15 to 23-06-15 17 light yellow Marine 10 10 15-06-15 to 23-06-15 18 light yellow Marine 10 10 15-06-15 to 23-06-15 19 light yellow Marine 10 10 15-06-15 to 23-06-15 20 light yellow Marine 10 10 15-06-15 to 23-06-15 21 light yellow Marine 10 10 15-06-15 to 23-06-15 22 orange Marine 10 10 15-06-15 to 23-06-15 23 orange Marine 10 10 15-06-15 to 23-06-15 24 orange Marine 10 10 15-06-15 to 23-06-15 25 orange Marine 10 10 15-06-15 to 23-06-15 26 pink Marine 10 10 15-06-15 to 23-06-15 27 white Marine 10 10 15-06-15 to 23-06-15 28 white Marine 10 10 15-06-15 to 23-06-15 29 white Marine 10 10 15-06-15 to 23-06-15 30 white Marine 10 10 15-06-15 to 23-06-15 31 white Marine 10 10 15-06-15 to 23-06-15 32 white Marine 10 10 15-06-15 to 23-06-15 33 white Marine 10 10 15-06-15 to 23-06-15 34 white Marine 10 10 15-06-15 to 23-06-15 35 white Marine 10 10 15-06-15 to 23-06-15 36 white Marine 10 10 15-06-15 to 23-06-15 37 white Marine 10 10 15-06-15 to 23-06-15 38 white Marine 10 10 15-06-15 to 23-06-15 39 white Marine 10 10 15-06-15 to 23-06-15 40 white Marine 10 10 15-06-15 to 23-06-15
xx
3A 1 dark yellow Marine 10 10 15-06-15 to 23-06-15 2 dark yellow Marine 10 10 15-06-15 to 23-06-15 3 dark yellow Marine 10 10 15-06-15 to 23-06-15 4 dark yellow Marine 10 10 15-06-15 to 23-06-15 5 dark yellow Marine 10 10 15-06-15 to 23-06-15 6 dark yellow Marine 10 10 15-06-15 to 23-06-15 7 dark yellow Marine 10 10 15-06-15 to 23-06-15 8 dark yellow Marine 10 10 15-06-15 to 23-06-15 9 dark yellow Marine 10 10 15-06-15 to 23-06-15 10 dark yellow Marine 10 10 15-06-15 to 23-06-15 11 dark yellow Marine 10 10 15-06-15 to 23-06-15 12 dark yellow Marine 10 10 15-06-15 to 23-06-15 13 dark yellow Marine 10 10 15-06-15 to 23-06-15 14 dark yellow Marine 10 10 15-06-15 to 23-06-15 15 dark yellow Marine 10 10 15-06-15 to 23-06-15 16 dark yellow Marine 10 10 15-06-15 to 23-06-15 17 dark yellow Marine 10 10 15-06-15 to 23-06-15 18 dark yellow Marine 10 10 15-06-15 to 23-06-15 19 dark yellow Marine 10 10 15-06-15 to 23-06-15 20 dark yellow Marine 10 10 15-06-15 to 23-06-15 21 dark yellow Marine 10 10 15-06-15 to 23-06-15 22 dark yellow Marine 10 10 15-06-15 to 23-06-15 23 dark yellow Marine 10 10 15-06-15 to 23-06-15 24 dark yellow Marine 10 10 15-06-15 to 23-06-15 25 dark yellow Marine 10 10 15-06-15 to 23-06-15 26 dark yellow Marine 10 10 15-06-15 to 23-06-15 27 dark yellow Marine 10 10 15-06-15 to 23-06-15 28 dark yellow Marine 10 10 15-06-15 to 23-06-15
xxi
29 dark yellow Marine 10 10 15-06-15 to 23-06-15 30 dark yellow Marine 10 10 15-06-15 to 23-06-15 31 dark yellow Marine 10 10 15-06-15 to 23-06-15 32 dark yellow Marine 10 10 15-06-15 to 23-06-15 33 dark yellow Marine 10 10 15-06-15 to 23-06-15 34 dark yellow Marine 10 10 15-06-15 to 23-06-15 35 dark yellow Marine 10 10 15-06-15 to 23-06-15 36 dark yellow Marine 10 10 15-06-15 to 23-06-15 37 dark yellow Marine 10 10 15-06-15 to 23-06-15 38 dark yellow Marine 10 10 15-06-15 to 23-06-15 39 dark yellow Marine 10 10 15-06-15 to 23-06-15 40 dark yellow Marine 10 10 15-06-15 to 23-06-15 41 dark yellow Marine 10 10 15-06-15 to 23-06-15 42 dark yellow Marine 10 10 15-06-15 to 23-06-15 43 dark yellow Marine 10 10 15-06-15 to 23-06-15 44 dark yellow Marine 10 10 15-06-15 to 23-06-15 45 dark yellow Marine 10 10 15-06-15 to 23-06-15 46 dark yellow Marine 10 10 15-06-15 to 23-06-15 47 dark yellow Marine 10 10 15-06-15 to 23-06-15 48 dark yellow Marine 10 10 15-06-15 to 23-06-15 49 dark yellow Marine 10 10 15-06-15 to 23-06-15 50 dark yellow Marine 10 10 15-06-15 to 23-06-15 51 light yellow Marine 10 10 15-06-15 to 23-06-15 52 light yellow Marine 10 10 15-06-15 to 23-06-15 53 light yellow Marine 10 10 15-06-15 to 23-06-15 54 light yellow Marine 10 10 15-06-15 to 23-06-15 55 light yellow Marine 10 10 15-06-15 to 23-06-15 56 light yellow Marine 10 10 15-06-15 to 23-06-15
xxii
57 orange Marine 10 10 15-06-15 to 23-06-15 58 orange Marine 10 10 15-06-15 to 23-06-15 59 orange Marine 10 10 15-06-15 to 23-06-15 60 orange Marine 10 10 15-06-15 to 23-06-15 61 orange Marine 10 10 15-06-15 to 23-06-15 62 orange Marine 10 10 15-06-15 to 23-06-15 63 orange Marine 10 10 15-06-15 to 23-06-15 64 orange Marine 10 10 15-06-15 to 23-06-15 65 white Marine 10 10 15-06-15 to 23-06-15 66 white Marine 10 10 15-06-15 to 23-06-15 67 white Marine 10 10 15-06-15 to 23-06-15 68 white Marine 10 10 15-06-15 to 23-06-15 69 white Marine 10 10 15-06-15 to 23-06-15 70 white Marine 10 10 15-06-15 to 23-06-15 71 white Marine 10 10 15-06-15 to 23-06-15 72 white Marine 10 10 15-06-15 to 23-06-15 73 white Marine 10 10 15-06-15 to 23-06-15 74 white Marine 10 10 15-06-15 to 23-06-15 75 white Marine 10 10 15-06-15 to 23-06-15 4A 1 orange Marine 10 10 15-06-15 to 23-06-15 2 orange Marine 10 10 15-06-15 to 23-06-15 3 white Marine 10 10 15-06-15 to 23-06-15 4 white Marine 10 10 15-06-15 to 23-06-15 5 white Marine 10 10 15-06-15 to 23-06-15 5A 1 dark yellow Marine 10 10 15-06-15 to 23-06-15 2 dark yellow Marine 10 10 15-06-15 to 23-06-15 3 dark yellow Marine 10 10 15-06-15 to 23-06-15 4 dark yellow Marine 10 10 15-06-15 to 23-06-15
xxiii
5 dark yellow Marine 10 10 15-06-15 to 23-06-15 6 dark yellow Marine 10 10 15-06-15 to 23-06-15 7 dark yellow Marine 10 10 15-06-15 to 23-06-15 8 dark yellow Marine 10 10 15-06-15 to 23-06-15 9 dark yellow Marine 10 10 15-06-15 to 23-06-15 10 dark yellow Marine 10 10 15-06-15 to 23-06-15 11 dark yellow Marine 10 10 15-06-15 to 23-06-15 12 dark yellow Marine 10 10 15-06-15 to 23-06-15 13 dark yellow Marine 10 10 15-06-15 to 23-06-15 14 dark yellow Marine 10 10 15-06-15 to 23-06-15 15 dark yellow Marine 10 10 15-06-15 to 23-06-15 16 dark yellow Marine 10 10 15-06-15 to 23-06-15 17 dark yellow Marine 10 10 15-06-15 to 23-06-15 18 dark yellow Marine 10 10 15-06-15 to 23-06-15 19 dark yellow Marine 10 10 15-06-15 to 23-06-15 20 dark yellow Marine 10 10 15-06-15 to 23-06-15 21 dark yellow Marine 10 10 15-06-15 to 23-06-15 22 dark yellow Marine 10 10 15-06-15 to 23-06-15 23 dark yellow Marine 10 10 15-06-15 to 23-06-15 24 dark yellow Marine 10 10 15-06-15 to 23-06-15 25 dark yellow Marine 10 10 15-06-15 to 23-06-15 26 dark yellow Marine 10 10 15-06-15 to 23-06-15 27 dark yellow Marine 10 10 15-06-15 to 23-06-15 28 dark yellow Marine 10 10 15-06-15 to 23-06-15 29 dark yellow Marine 10 10 15-06-15 to 23-06-15 30 dark yellow Marine 10 10 15-06-15 to 23-06-15 31 dark yellow Marine 10 10 15-06-15 to 23-06-15 32 dark yellow Marine 10 10 15-06-15 to 23-06-15
xxiv
33 light yellow Marine 10 10 15-06-15 to 23-06-15 34 light yellow Marine 10 10 15-06-15 to 23-06-15 35 light yellow Marine 10 10 15-06-15 to 23-06-15 36 light yellow Marine 10 10 15-06-15 to 23-06-15 37 light yellow Marine 10 10 15-06-15 to 23-06-15 38 light yellow Marine 10 10 15-06-15 to 23-06-15 39 light yellow Marine 10 10 15-06-15 to 23-06-15 40 light yellow Marine 10 10 15-06-15 to 23-06-15 41 light yellow Marine 10 10 15-06-15 to 23-06-15 42 light yellow Marine 10 10 15-06-15 to 23-06-15 43 light yellow Marine 10 10 15-06-15 to 23-06-15 44 light yellow Marine 10 10 15-06-15 to 23-06-15 45 orange Marine 10 10 15-06-15 to 23-06-15
xxv
ANNEXURE TABLE 5
Sample Name Strain No. Color with X-gal Media pH Temperature ◦C Incubation Time 1A 1 blue Marine 10 10 23-06-15 to 01-07-15 2 blue Marine 10 10 23-06-15 to 01-07-15 3 blue Marine 10 10 23-06-15 to 01-07-15 4 blue Marine 10 10 23-06-15 to 01-07-15 5 blue Marine 10 10 23-06-15 to 01-07-15 6 blue Marine 10 10 23-06-15 to 01-07-15 7 blue Marine 10 10 23-06-15 to 01-07-15 8 blue Marine 10 10 23-06-15 to 01-07-15 9 blue Marine 10 10 23-06-15 to 01-07-15 10 blue Marine 10 10 23-06-15 to 01-07-15 11 blue Marine 10 10 23-06-15 to 01-07-15 12 blue Marine 10 10 23-06-15 to 01-07-15 13 blue Marine 10 10 23-06-15 to 01-07-15 14 blue Marine 10 10 23-06-15 to 01-07-15 15 blue Marine 10 10 23-06-15 to 01-07-15 16 blue Marine 10 10 23-06-15 to 01-07-15 17 blue Marine 10 10 23-06-15 to 01-07-15 18 blue Marine 10 10 23-06-15 to 01-07-15 19 blue Marine 10 10 23-06-15 to 01-07-15 20 blue Marine 10 10 23-06-15 to 01-07-15 21 blue Marine 10 10 23-06-15 to 01-07-15 22 blue Marine 10 10 23-06-15 to 01-07-15 23 blue Marine 10 10 23-06-15 to 01-07-15 24 blue Marine 10 10 23-06-15 to 01-07-15 25 blue Marine 10 10 23-06-15 to 01-07-15
xxvi
26 blue Marine 10 10 23-06-15 to 01-07-15 27 blue Marine 10 10 23-06-15 to 01-07-15 28 blue Marine 10 10 23-06-15 to 01-07-15 29 blue Marine 10 10 23-06-15 to 01-07-15 30 blue Marine 10 10 23-06-15 to 01-07-15 31 blue Marine 10 10 23-06-15 to 01-07-15 32 blue Marine 10 10 23-06-15 to 01-07-15 33 blue Marine 10 10 23-06-15 to 01-07-15 34 blue Marine 10 10 23-06-15 to 01-07-15 35 blue Marine 10 10 23-06-15 to 01-07-15 36 blue Marine 10 10 23-06-15 to 01-07-15 37 blue Marine 10 10 23-06-15 to 01-07-15 38 blue Marine 10 10 23-06-15 to 01-07-15 39 blue Marine 10 10 23-06-15 to 01-07-15 40 blue Marine 10 10 23-06-15 to 01-07-15 41 blue Marine 10 10 23-06-15 to 01-07-15 42 blue Marine 10 10 23-06-15 to 01-07-15 43 brown Marine 10 10 23-06-15 to 01-07-15 44 dark yellow Marine 10 10 23-06-15 to 01-07-15 45 dark yellow Marine 10 10 23-06-15 to 01-07-15 46 light yellow Marine 10 10 23-06-15 to 01-07-15 47 light yellow Marine 10 10 23-06-15 to 01-07-15 48 pink Marine 10 10 23-06-15 to 01-07-15 49 pink Marine 10 10 23-06-15 to 01-07-15 50 pink Marine 10 10 23-06-15 to 01-07-15 51 white Marine 10 10 23-06-15 to 01-07-15 52 white Marine 10 10 23-06-15 to 01-07-15 53 white Marine 10 10 23-06-15 to 01-07-15
xxvii
54 white Marine 10 10 23-06-15 to 01-07-15 55 white Marine 10 10 23-06-15 to 01-07-15 56 white Marine 10 10 23-06-15 to 01-07-15 57 white Marine 10 10 23-06-15 to 01-07-15 58 white Marine 10 10 23-06-15 to 01-07-15 59 white Marine 10 10 23-06-15 to 01-07-15 60 white Marine 10 10 23-06-15 to 01-07-15 61 white Marine 10 10 23-06-15 to 01-07-15 62 white Marine 10 10 23-06-15 to 01-07-15 63 white Marine 10 10 23-06-15 to 01-07-15 64 white Marine 10 10 23-06-15 to 01-07-15 65 white Marine 10 10 23-06-15 to 01-07-15 66 white Marine 10 10 23-06-15 to 01-07-15 67 white Marine 10 10 23-06-15 to 01-07-15 68 white Marine 10 10 23-06-15 to 01-07-15 69 white Marine 10 10 23-06-15 to 01-07-15 70 white Marine 10 10 23-06-15 to 01-07-15 2A 1 blue Marine 10 10 23-06-15 to 01-07-15 2 blue Marine 10 10 23-06-15 to 01-07-15 3 blue Marine 10 10 23-06-15 to 01-07-15 4 blue Marine 10 10 23-06-15 to 01-07-15 5 blue Marine 10 10 23-06-15 to 01-07-15 6 blue Marine 10 10 23-06-15 to 01-07-15 7 blue Marine 10 10 23-06-15 to 01-07-15 8 blue Marine 10 10 23-06-15 to 01-07-15 9 blue Marine 10 10 23-06-15 to 01-07-15 10 blue Marine 10 10 23-06-15 to 01-07-15 11 blue Marine 10 10 23-06-15 to 01-07-15
xxviii
12 blue Marine 10 10 23-06-15 to 01-07-15 13 blue Marine 10 10 23-06-15 to 01-07-15 14 blue Marine 10 10 23-06-15 to 01-07-15 15 blue Marine 10 10 23-06-15 to 01-07-15 16 blue Marine 10 10 23-06-15 to 01-07-15 17 blue Marine 10 10 23-06-15 to 01-07-15 18 dark yellow Marine 10 10 23-06-15 to 01-07-15 19 dark yellow Marine 10 10 23-06-15 to 01-07-15 20 light yellow Marine 10 10 23-06-15 to 01-07-15 21 light yellow Marine 10 10 23-06-15 to 01-07-15 22 light yellow Marine 10 10 23-06-15 to 01-07-15 23 light yellow Marine 10 10 23-06-15 to 01-07-15 24 white Marine 10 10 23-06-15 to 01-07-15 25 white Marine 10 10 23-06-15 to 01-07-15 26 white Marine 10 10 23-06-15 to 01-07-15 27 white Marine 10 10 23-06-15 to 01-07-15 28 white Marine 10 10 23-06-15 to 01-07-15 29 white Marine 10 10 23-06-15 to 01-07-15 30 white Marine 10 10 23-06-15 to 01-07-15 31 white Marine 10 10 23-06-15 to 01-07-15 32 white Marine 10 10 23-06-15 to 01-07-15 33 white Marine 10 10 23-06-15 to 01-07-15 34 white Marine 10 10 23-06-15 to 01-07-15 35 white Marine 10 10 23-06-15 to 01-07-15 36 white Marine 10 10 23-06-15 to 01-07-15 37 white Marine 10 10 23-06-15 to 01-07-15 38 white Marine 10 10 23-06-15 to 01-07-15 39 white Marine 10 10 23-06-15 to 01-07-15
xxix
40 white Marine 10 10 23-06-15 to 01-07-15 3A 1 blue Marine 10 10 23-06-15 to 01-07-15 2 blue Marine 10 10 23-06-15 to 01-07-15 3 blue Marine 10 10 23-06-15 to 01-07-15 4 blue Marine 10 10 23-06-15 to 01-07-15 5 blue Marine 10 10 23-06-15 to 01-07-15 6 blue Marine 10 10 23-06-15 to 01-07-15 7 dark yellow Marine 10 10 23-06-15 to 01-07-15 8 dark yellow Marine 10 10 23-06-15 to 01-07-15 9 dark yellow Marine 10 10 23-06-15 to 01-07-15 10 dark yellow Marine 10 10 23-06-15 to 01-07-15 11 dark yellow Marine 10 10 23-06-15 to 01-07-15 12 dark yellow Marine 10 10 23-06-15 to 01-07-15 13 dark yellow Marine 10 10 23-06-15 to 01-07-15 14 dark yellow Marine 10 10 23-06-15 to 01-07-15 15 dark yellow Marine 10 10 23-06-15 to 01-07-15 16 dark yellow Marine 10 10 23-06-15 to 01-07-15 17 dark yellow Marine 10 10 23-06-15 to 01-07-15 18 dark yellow Marine 10 10 23-06-15 to 01-07-15 19 dark yellow Marine 10 10 23-06-15 to 01-07-15 20 dark yellow Marine 10 10 23-06-15 to 01-07-15 21 dark yellow Marine 10 10 23-06-15 to 01-07-15 22 dark yellow Marine 10 10 23-06-15 to 01-07-15 23 dark yellow Marine 10 10 23-06-15 to 01-07-15 24 dark yellow Marine 10 10 23-06-15 to 01-07-15 25 dark yellow Marine 10 10 23-06-15 to 01-07-15 26 dark yellow Marine 10 10 23-06-15 to 01-07-15 27 dark yellow Marine 10 10 23-06-15 to 01-07-15
xxx
28 dark yellow Marine 10 10 23-06-15 to 01-07-15 29 dark yellow Marine 10 10 23-06-15 to 01-07-15 30 dark yellow Marine 10 10 23-06-15 to 01-07-15 31 dark yellow Marine 10 10 23-06-15 to 01-07-15 32 dark yellow Marine 10 10 23-06-15 to 01-07-15 33 dark yellow Marine 10 10 23-06-15 to 01-07-15 34 dark yellow Marine 10 10 23-06-15 to 01-07-15 35 dark yellow Marine 10 10 23-06-15 to 01-07-15 36 dark yellow Marine 10 10 23-06-15 to 01-07-15 37 dark yellow Marine 10 10 23-06-15 to 01-07-15 38 dark yellow Marine 10 10 23-06-15 to 01-07-15 39 dark yellow Marine 10 10 23-06-15 to 01-07-15 40 dark yellow Marine 10 10 23-06-15 to 01-07-15 41 dark yellow Marine 10 10 23-06-15 to 01-07-15 42 dark yellow Marine 10 10 23-06-15 to 01-07-15 43 dark yellow Marine 10 10 23-06-15 to 01-07-15 44 dark yellow Marine 10 10 23-06-15 to 01-07-15 45 dark yellow Marine 10 10 23-06-15 to 01-07-15 46 dark yellow Marine 10 10 23-06-15 to 01-07-15 47 dark yellow Marine 10 10 23-06-15 to 01-07-15 48 light yellow Marine 10 10 23-06-15 to 01-07-15 49 light yellow Marine 10 10 23-06-15 to 01-07-15 50 light yellow Marine 10 10 23-06-15 to 01-07-15 51 orange Marine 10 10 23-06-15 to 01-07-15 52 white Marine 10 10 23-06-15 to 01-07-15 53 white Marine 10 10 23-06-15 to 01-07-15 54 white Marine 10 10 23-06-15 to 01-07-15 55 white Marine 10 10 23-06-15 to 01-07-15
xxxi
56 white Marine 10 10 23-06-15 to 01-07-15 57 white Marine 10 10 23-06-15 to 01-07-15 58 white Marine 10 10 23-06-15 to 01-07-15 59 white Marine 10 10 23-06-15 to 01-07-15 60 white Marine 10 10 23-06-15 to 01-07-15 61 white Marine 10 10 23-06-15 to 01-07-15 62 white Marine 10 10 23-06-15 to 01-07-15 63 white Marine 10 10 23-06-15 to 01-07-15 64 white Marine 10 10 23-06-15 to 01-07-15 65 white Marine 10 10 23-06-15 to 01-07-15 66 white Marine 10 10 23-06-15 to 01-07-15 67 white Marine 10 10 23-06-15 to 01-07-15 68 white Marine 10 10 23-06-15 to 01-07-15 69 white Marine 10 10 23-06-15 to 01-07-15 70 white Marine 10 10 23-06-15 to 01-07-15 71 white Marine 10 10 23-06-15 to 01-07-15 72 white Marine 10 10 23-06-15 to 01-07-15 73 white Marine 10 10 23-06-15 to 01-07-15 74 white Marine 10 10 23-06-15 to 01-07-15 75 white Marine 10 10 23-06-15 to 01-07-15 4A 1 light yellow Marine 10 10 23-06-15 to 01-07-15 2 light yellow Marine 10 10 23-06-15 to 01-07-15 3 white Marine 10 10 23-06-15 to 01-07-15 4 white Marine 10 10 23-06-15 to 01-07-15 5 white Marine 10 10 23-06-15 to 01-07-15 5A 1 dark yellow Marine 10 10 23-06-15 to 01-07-15 2 dark yellow Marine 10 10 23-06-15 to 01-07-15 3 dark yellow Marine 10 10 23-06-15 to 01-07-15
xxxii
4 dark yellow Marine 10 10 23-06-15 to 01-07-15 5 dark yellow Marine 10 10 23-06-15 to 01-07-15 6 dark yellow Marine 10 10 23-06-15 to 01-07-15 7 dark yellow Marine 10 10 23-06-15 to 01-07-15 8 dark yellow Marine 10 10 23-06-15 to 01-07-15 9 dark yellow Marine 10 10 23-06-15 to 01-07-15 10 dark yellow Marine 10 10 23-06-15 to 01-07-15 11 dark yellow Marine 10 10 23-06-15 to 01-07-15 12 dark yellow Marine 10 10 23-06-15 to 01-07-15 13 dark yellow Marine 10 10 23-06-15 to 01-07-15 14 dark yellow Marine 10 10 23-06-15 to 01-07-15 15 dark yellow Marine 10 10 23-06-15 to 01-07-15 16 dark yellow Marine 10 10 23-06-15 to 01-07-15 17 dark yellow Marine 10 10 23-06-15 to 01-07-15 18 dark yellow Marine 10 10 23-06-15 to 01-07-15 19 dark yellow Marine 10 10 23-06-15 to 01-07-15 20 dark yellow Marine 10 10 23-06-15 to 01-07-15 21 dark yellow Marine 10 10 23-06-15 to 01-07-15 22 dark yellow Marine 10 10 23-06-15 to 01-07-15 23 dark yellow Marine 10 10 23-06-15 to 01-07-15 24 dark yellow Marine 10 10 23-06-15 to 01-07-15 25 dark yellow Marine 10 10 23-06-15 to 01-07-15 26 dark yellow Marine 10 10 23-06-15 to 01-07-15 27 dark yellow Marine 10 10 23-06-15 to 01-07-15 28 dark yellow Marine 10 10 23-06-15 to 01-07-15 29 light yellow Marine 10 10 23-06-15 to 01-07-15 30 light yellow Marine 10 10 23-06-15 to 01-07-15 31 light yellow Marine 10 10 23-06-15 to 01-07-15
xxxiii
32 light yellow Marine 10 10 23-06-15 to 01-07-15 33 light yellow Marine 10 10 23-06-15 to 01-07-15 34 light yellow Marine 10 10 23-06-15 to 01-07-15 35 light yellow Marine 10 10 23-06-15 to 01-07-15 36 light yellow Marine 10 10 23-06-15 to 01-07-15 37 light yellow Marine 10 10 23-06-15 to 01-07-15 38 light yellow Marine 10 10 23-06-15 to 01-07-15 39 light yellow Marine 10 10 23-06-15 to 01-07-15 40 light yellow Marine 10 10 23-06-15 to 01-07-15 41 light yellow Marine 10 10 23-06-15 to 01-07-15 42 light yellow Marine 10 10 23-06-15 to 01-07-15 43 light yellow Marine 10 10 23-06-15 to 01-07-15 44 light yellow Marine 10 10 23-06-15 to 01-07-15 45 white Marine 10 10 23-06-15 to 01-07-15
xxxiv
ANNEXURE TABLE 6
Sample Plate Strain Temperatu Name Name No. Colour with X-gal Media pH re ◦C Incubation Time Medium Light blue blue Dark blue Other colour 1A (i) 3 yes Marine 10 10 01-07-15 to 27-07-15 4 yes Marine 10 10 01-07-15 to 27-07-15 5 yes Marine 10 10 01-07-15 to 27-07-15 6 white Marine 10 10 01-07-15 to 27-07-15 9 yes Marine 10 10 01-07-15 to 27-07-15 12 yes Marine 10 10 01-07-15 to 27-07-15 13 yes Marine 10 10 01-07-15 to 27-07-15 15 yes Marine 10 10 01-07-15 to 27-07-15 1A (ii) 16 yes Marine 10 10 01-07-15 to 27-07-15 17 yes Marine 10 10 01-07-15 to 27-07-15 20 yes Marine 10 10 01-07-15 to 27-07-15 21 yes Marine 10 10 01-07-15 to 27-07-15 22 white Marine 10 10 01-07-15 to 27-07-15 23 yes Marine 10 10 01-07-15 to 27-07-15 24 yes Marine 10 10 01-07-15 to 27-07-15 25 yes Marine 10 10 01-07-15 to 27-07-15 1A (iii) 26 yes Marine 10 10 01-07-15 to 27-07-15 27 yes Marine 10 10 01-07-15 to 27-07-15 29 yes Marine 10 10 01-07-15 to 27-07-15 32 yes Marine 10 10 01-07-15 to 27-07-15 34 pink Marine 10 10 01-07-15 to 27-07-15 38 yes Marine 10 10 01-07-15 to 27-07-15 39 yes Marine 10 10 01-07-15 to 27-07-15
xxxv
40 yes Marine 10 10 01-07-15 to 27-07-15 1A (iv) 41 yes Marine 10 10 01-07-15 to 27-07-15 42 white Marine 10 10 01-07-15 to 27-07-15 44 blue Marine 10 10 01-07-15 to 27-07-15 45 white Marine 10 10 01-07-15 to 27-07-15 46 blue Marine 10 10 01-07-15 to 27-07-15 47 blue Marine 10 10 01-07-15 to 27-07-15 48 blue Marine 10 10 01-07-15 to 27-07-15 50 blue Marine 10 10 01-07-15 to 27-07-15 1A (v) 52 yes Marine 10 10 01-07-15 to 27-07-15 53 white Marine 10 10 01-07-15 to 27-07-15 54 yes Marine 10 10 01-07-15 to 27-07-15 55 yes Marine 10 10 01-07-15 to 27-07-15 56 yes Marine 10 10 01-07-15 to 27-07-15 57 yes Marine 10 10 01-07-15 to 27-07-15 58 yes Marine 10 10 01-07-15 to 27-07-15 59 yes Marine 10 10 01-07-15 to 27-07-15 60 white Marine 10 10 01-07-15 to 27-07-15 1A (vi) 61 yes Marine 10 10 01-07-15 to 27-07-15 63 white Marine 10 10 01-07-15 to 27-07-15 66 yes Marine 10 10 01-07-15 to 27-07-15 68 yes Marine 10 10 01-07-15 to 27-07-15 69 yes Marine 10 10 01-07-15 to 27-07-15 2A (i) 1 yes Marine 10 10 01-07-15 to 27-07-15 4 yes Marine 10 10 01-07-15 to 27-07-15 7 yes Marine 10 10 01-07-15 to 27-07-15 2A (ii) 10 yes Marine 10 10 01-07-15 to 27-07-15 11 yes Marine 10 10 01-07-15 to 27-07-15
xxxvi
17 yes Marine 10 10 01-07-15 to 27-07-15 18 yes Marine 10 10 01-07-15 to 27-07-15 21 yes Marine 10 10 01-07-15 to 27-07-15 22 yes Marine 10 10 01-07-15 to 27-07-15 24 yes Marine 10 10 01-07-15 to 27-07-15 26 yes Marine 10 10 01-07-15 to 27-07-15 2A (iii) 27 yes Marine 10 10 01-07-15 to 27-07-15 28 yes Marine 10 10 01-07-15 to 27-07-15 30 yes Marine 10 10 01-07-15 to 27-07-15 31 yes Marine 10 10 01-07-15 to 27-07-15 32 yes Marine 10 10 01-07-15 to 27-07-15 37 yes Marine 10 10 01-07-15 to 27-07-15 38 yes Marine 10 10 01-07-15 to 27-07-15 19 yes Marine 10 10 01-07-15 to 27-07-15 3A (i) 6 yes Marine 10 10 01-07-15 to 27-07-15 8 yes Marine 10 10 01-07-15 to 27-07-15 9 yes Marine 10 10 01-07-15 to 27-07-15 32 yes Marine 10 10 01-07-15 to 27-07-15 75 yes Marine 10 10 01-07-15 to 27-07-15
xxxvii
ANNEXURE TABLE 7
Sample Name Strain No. Size of Colony (with X-gal) Media pH Temperature ◦C Incubation Time Small Medium Large 1A 1 yes R2 Agar 10 10 24-06-15 to 30-07-15 2 yes R2 Agar 10 10 24-06-15 to 30-07-15 3 yes R2 Agar 10 10 24-06-15 to 30-07-15 4 yes R2 Agar 10 10 24-06-15 to 30-07-15 5 yes R2 Agar 10 10 24-06-15 to 30-07-15 6 yes R2 Agar 10 10 24-06-15 to 30-07-15 7 yes R2 Agar 10 10 24-06-15 to 30-07-15 8 yes R2 Agar 10 10 24-06-15 to 30-07-15 9 yes R2 Agar 10 10 24-06-15 to 30-07-15 10 yes R2 Agar 10 10 24-06-15 to 30-07-15 11 yes R2 Agar 10 10 24-06-15 to 30-07-15 12 yes R2 Agar 10 10 24-06-15 to 30-07-15 13 yes R2 Agar 10 10 24-06-15 to 30-07-15 14 yes R2 Agar 10 10 24-06-15 to 30-07-15 15 yes R2 Agar 10 10 24-06-15 to 30-07-15 16 yes R2 Agar 10 10 24-06-15 to 30-07-15 17 yes R2 Agar 10 10 24-06-15 to 30-07-15 18 yes R2 Agar 10 10 24-06-15 to 30-07-15 19 yes R2 Agar 10 10 24-06-15 to 30-07-15 20 yes R2 Agar 10 10 24-06-15 to 30-07-15 2A 1 yes R2 Agar 10 10 24-06-15 to 30-07-15 2 yes R2 Agar 10 10 24-06-15 to 30-07-15 3 yes R2 Agar 10 10 24-06-15 to 30-07-15 4 yes R2 Agar 10 10 24-06-15 to 30-07-15 xxxviii
5 yes R2 Agar 10 10 24-06-15 to 30-07-15 6 yes R2 Agar 10 10 24-06-15 to 30-07-15 7 yes R2 Agar 10 10 24-06-15 to 30-07-15 8 yes R2 Agar 10 10 24-06-15 to 30-07-15 9 yes R2 Agar 10 10 24-06-15 to 30-07-15 10 yes R2 Agar 10 10 24-06-15 to 30-07-15 11 yes R2 Agar 10 10 24-06-15 to 30-07-15 12 yes R2 Agar 10 10 24-06-15 to 30-07-15 13 yes R2 Agar 10 10 24-06-15 to 30-07-15 14 yes R2 Agar 10 10 24-06-15 to 30-07-15 15 yes R2 Agar 10 10 24-06-15 to 30-07-15 16 yes yes R2 Agar 10 10 24-06-15 to 30-07-15 17 R2 Agar 10 10 24-06-15 to 30-07-15 18 yes R2 Agar 10 10 24-06-15 to 30-07-15 19 yes R2 Agar 10 10 24-06-15 to 30-07-15 20 yes R2 Agar 10 10 24-06-15 to 30-07-15 3A 1 yes R2 Agar 10 10 24-06-15 to 30-07-15 2 yes R2 Agar 10 10 24-06-15 to 30-07-15 3 yes R2 Agar 10 10 24-06-15 to 30-07-15 4 yes R2 Agar 10 10 24-06-15 to 30-07-15 5 yes R2 Agar 10 10 24-06-15 to 30-07-15 6 yes R2 Agar 10 10 24-06-15 to 30-07-15 7 yes R2 Agar 10 10 24-06-15 to 30-07-15 8 yes R2 Agar 10 10 24-06-15 to 30-07-15 9 yes R2 Agar 10 10 24-06-15 to 30-07-15 10 yes R2 Agar 10 10 24-06-15 to 30-07-15 11 yes R2 Agar 10 10 24-06-15 to 30-07-15 12 yes R2 Agar 10 10 24-06-15 to 30-07-15 xxxix
13 yes R2 Agar 10 10 24-06-15 to 30-07-15 14 yes R2 Agar 10 10 24-06-15 to 30-07-15 15 yes R2 Agar 10 10 24-06-15 to 30-07-15 16 yes R2 Agar 10 10 24-06-15 to 30-07-15 17 yes R2 Agar 10 10 24-06-15 to 30-07-15 18 yes R2 Agar 10 10 24-06-15 to 30-07-15 19 yes R2 Agar 10 10 24-06-15 to 30-07-15 20 yes R2 Agar 10 10 24-06-15 to 30-07-15 4A no blue colony R2 Agar 10 10 24-06-15 to 30-07-15 5A no blue colony R2 Agar 10 10 24-06-15 to 30-07-15
xl
ANNEXURE TABLE 8
Sample Name Strain No. Size of colony (with X-gal) Media pH Temperature ◦C Incubation Time Small Medium Large 1A no growth R2 Agar 7 10 26-06-15 to 30-07-15 2A no growth R2 Agar 7 10 26-06-15 to 30-07-16 3A 1 yes R2 Agar 7 10 26-06-15 to 30-07-17 2 yes R2 Agar 7 10 26-06-15 to 30-07-18 3 yes R2 Agar 7 10 26-06-15 to 30-07-19 4 yes R2 Agar 7 10 26-06-15 to 30-07-20 5 yes R2 Agar 7 10 26-06-15 to 30-07-21 6 yes R2 Agar 7 10 26-06-15 to 30-07-22 7 yes R2 Agar 7 10 26-06-15 to 30-07-23 8 yes R2 Agar 7 10 26-06-15 to 30-07-24 9 yes R2 Agar 7 10 26-06-15 to 30-07-25 10 yes R2 Agar 7 10 26-06-15 to 30-07-26 11 yes R2 Agar 7 10 26-06-15 to 30-07-27 12 yes R2 Agar 7 10 26-06-15 to 30-07-28 4A no growth R2 Agar 7 10 26-06-15 to 30-07-29 5A no growth R2 Agar 7 10 26-06-15 to 30-07-30
xli
ANNEXURE TABLE 9
Sample Name Strain No. Size of colony (with X-gal) Media pH Temperature ◦C Incubation Time Small Medium Large 1A 1 yes Marine 7 10 26-06-15 to 30-07-15 2 yes Marine 7 10 26-06-15 to 30-07-15 3 yes Marine 7 10 26-06-15 to 30-07-15 4 yes Marine 7 10 26-06-15 to 30-07-15 5 yes Marine 7 10 26-06-15 to 30-07-15 6 yes Marine 7 10 26-06-15 to 30-07-15 7 yes Marine 7 10 26-06-15 to 30-07-15 8 yes Marine 7 10 26-06-15 to 30-07-15 9 yes Marine 7 10 26-06-15 to 30-07-15 10 yes Marine 7 10 26-06-15 to 30-07-15 11 yes Marine 7 10 26-06-15 to 30-07-15 12 yes Marine 7 10 26-06-15 to 30-07-15 13 yes Marine 7 10 26-06-15 to 30-07-15 14 yes Marine 7 10 26-06-15 to 30-07-15 15 yes Marine 7 10 26-06-15 to 30-07-15 16 yes Marine 7 10 26-06-15 to 30-07-15 17 yes Marine 7 10 26-06-15 to 30-07-15 18 yes Marine 7 10 26-06-15 to 30-07-15 19 yes Marine 7 10 26-06-15 to 30-07-15 20 yes Marine 7 10 26-06-15 to 30-07-15 2A 1 yes Marine 7 10 26-06-15 to 30-07-15 2 yes Marine 7 10 26-06-15 to 30-07-15 3 yes Marine 7 10 26-06-15 to 30-07-15 4 yes Marine 7 10 26-06-15 to 30-07-15
xlii
5 yes Marine 7 10 26-06-15 to 30-07-15 6 yes Marine 7 10 26-06-15 to 30-07-15 7 yes Marine 7 10 26-06-15 to 30-07-15 8 yes Marine 7 10 26-06-15 to 30-07-15 9 yes Marine 7 10 26-06-15 to 30-07-15 10 yes Marine 7 10 26-06-15 to 30-07-15 11 yes Marine 7 10 26-06-15 to 30-07-15 12 yes Marine 7 10 26-06-15 to 30-07-15 13 yes Marine 7 10 26-06-15 to 30-07-15 14 yes Marine 7 10 26-06-15 to 30-07-15 15 yes Marine 7 10 26-06-15 to 30-07-15 16 yes Marine 7 10 26-06-15 to 30-07-15 17 yes Marine 7 10 26-06-15 to 30-07-15 18 yes Marine 7 10 26-06-15 to 30-07-15 19 yes Marine 7 10 26-06-15 to 30-07-15 20 yes Marine 7 10 26-06-15 to 30-07-15 3A 1 yes Marine 7 10 26-06-15 to 30-07-15 2 yes Marine 7 10 26-06-15 to 30-07-15 3 yes Marine 7 10 26-06-15 to 30-07-15 4 yes Marine 7 10 26-06-15 to 30-07-15 5 yes Marine 7 10 26-06-15 to 30-07-15 6 yes Marine 7 10 26-06-15 to 30-07-15 7 yes Marine 7 10 26-06-15 to 30-07-15 8 yes Marine 7 10 26-06-15 to 30-07-15 9 yes Marine 7 10 26-06-15 to 30-07-15 10 yes Marine 7 10 26-06-15 to 30-07-15 4A 1 yes Marine 7 10 26-06-15 to 30-07-15 2 yes Marine 7 10 26-06-15 to 30-07-15
xliii
3 yes Marine 7 10 26-06-15 to 30-07-15 4 yes Marine 7 10 26-06-15 to 30-07-15 5 yes Marine 7 10 26-06-15 to 30-07-15 6 yes Marine 7 10 26-06-15 to 30-07-15 7 yes Marine 7 10 26-06-15 to 30-07-15 8 yes Marine 7 10 26-06-15 to 30-07-15 9 yes Marine 7 10 26-06-15 to 30-07-15 10 yes Marine 7 10 26-06-15 to 30-07-15 11 yes Marine 7 10 26-06-15 to 30-07-15 12 yes Marine 7 10 26-06-15 to 30-07-15 13 yes Marine 7 10 26-06-15 to 30-07-15 14 yes Marine 7 10 26-06-15 to 30-07-15 15 yes Marine 7 10 26-06-15 to 30-07-15 5A no blue colony Marine 7 10 26-06-15 to 30-07-15
xliv
ANNEXURE TABLE 10
Sample Name Media pH Temperature ◦C Growth with x-gal Incubation Time I (1) R2 Agar 10 10 no 24-06-15 to 30-07-15 I (2) R2 Agar 10 10 no 24-06-15 to 30-07-15 I (3) R2 Agar 10 10 no 24-06-15 to 30-07-15 I (4) R2 Agar 10 10 no 24-06-15 to 30-07-15 I (5) R2 Agar 10 10 no 24-06-15 to 30-07-15 I (6) R2 Agar 10 10 no 24-06-15 to 30-07-15 I (7) R2 Agar 10 10 no blue colony 24-06-15 to 30-07-15 I (8) R2 Agar 10 10 no 24-06-15 to 30-07-15 I (9) R2 Agar 10 10 no 24-06-15 to 30-07-15 I (10) R2 Agar 10 10 no 24-06-15 to 30-07-15 I (1) R2 Agar 7 10 no 24-06-15 to 30-07-15 I (2) R2 Agar 7 10 no 24-06-15 to 30-07-15 I (3) R2 Agar 7 10 no 24-06-15 to 30-07-15 I (4) R2 Agar 7 10 no 24-06-15 to 30-07-15 I (5) R2 Agar 7 10 no 24-06-15 to 30-07-15 I (6) R2 Agar 7 10 no 24-06-15 to 30-07-15 I (7) R2 Agar 7 10 no 24-06-15 to 30-07-15 I (8) R2 Agar 7 10 no 24-06-15 to 30-07-15 I (9) R2 Agar 7 10 no 24-06-15 to 30-07-15 I (10) R2 Agar 7 10 no 24-06-15 to 30-07-15 I (1) Marine 7 10 no 24-06-15 to 30-07-15 I (2) Marine 7 10 no 24-06-15 to 30-07-15 I (3) Marine 7 10 one blue colony 24-06-15 to 30-07-15 I (4) Marine 7 10 no 24-06-15 to 30-07-15 I (5) Marine 7 10 no 24-06-15 to 30-07-15
xlv
I (6) Marine 7 10 no 24-06-15 to 30-07-15 I (7) Marine 7 10 no 24-06-15 to 30-07-15 I (8) Marine 7 10 no 24-06-15 to 30-07-15 I (9) Marine 7 10 no 24-06-15 to 30-07-15 I (10) Marine 7 10 no 24-06-15 to 30-07-15
xlvi
ANNEXURE TABLE 11
GROWTH RATE Sample Temperature R2 Agar pH R2 Agar Marine Marine Name (◦C) (10) pH (7) pH (10) pH (7) V.good good no V.good good no V.good good no V.good good no 1 (A) 10 yes yes yes yes 2 (A) 10 yes yes yes yes 3 (A) 10 yes yes yes yes 4 (A) 10 no growth no yes yes no blue 5 (A) 10 colony no yes yes one blue 6 (A) 10 yes no yes colony one blue 7 (A) 10 colony no yes yes
xlvii
ANNEXURE TABLE 12
Sample Name Plate Name Strain No. Media with x-gal) pH Temperature ◦C Incubation Time 1A (i) 3 Marine 10 10 04-08-15 to 16-09-15 5 Marine 10 10 04-08-15 to 16-09-15 12 Marine 10 10 04-08-15 to 16-09-15 13 Marine 10 10 04-08-15 to 16-09-15 1A (ii) 16 Marine 10 10 04-08-15 to 16-09-15 17 Marine 10 10 04-08-15 to 16-09-15 20 Marine 10 10 04-08-15 to 16-09-15 23 Marine 10 10 04-08-15 to 16-09-15 25 Marine 10 10 04-08-15 to 16-09-15 1A (iii) 26 Marine 10 10 04-08-15 to 16-09-15 27 Marine 10 10 04-08-15 to 16-09-15 29 Marine 10 10 04-08-15 to 16-09-15 39 Marine 10 10 04-08-15 to 16-09-15 1A (iv) 41 Marine 10 10 04-08-15 to 16-09-15 47 Marine 10 10 04-08-15 to 16-09-15 48 Marine 10 10 04-08-15 to 16-09-15 1A (v) 52 Marine 10 10 04-08-15 to 16-09-15 54 Marine 10 10 04-08-15 to 16-09-15 55 Marine 10 10 04-08-15 to 16-09-15 57 Marine 10 10 04-08-15 to 16-09-15 1A (vi) 61 Marine 10 10 04-08-15 to 16-09-15 68 Marine 10 10 04-08-15 to 16-09-15 69 Marine 10 10 04-08-15 to 16-09-15 2A (i) 1 Marine 10 10 04-08-15 to 16-09-15 7 Marine 10 10 04-08-15 to 16-09-15
xlviii
2A (ii) 11 Marine 10 10 04-08-15 to 16-09-15 17 Marine 10 10 04-08-15 to 16-09-15 22 Marine 10 10 04-08-15 to 16-09-15 26 Marine 10 10 04-08-15 to 16-09-15 2A (iii) 27 Marine 10 10 04-08-15 to 16-09-15 30 Marine 10 10 04-08-15 to 16-09-15 32 Marine 10 10 04-08-15 to 16-09-15 37 Marine 10 10 04-08-15 to 16-09-15 3A 6 Marine 10 10 04-08-15 to 16-09-15 32 Marine 10 10 04-08-15 to 16-09-15 75 Marine 10 10 04-08-15 to 16-09-15
xlix
ANNEXURE TABLE 13
Growth Rate Size of Colonies pH Temperature (◦C) Incubation Time Color with x-gal Slow Medium Good Small Medium Large 10 10 05-08-15 to 16-09-15 yellow yes yes 10 10 05-08-15 to 16-09-15 yellow yes yes 10 10 05-08-15 to 16-09-15 yellow + light blue yes yes 10 10 05-08-15 to 16-09-15 orangish yellow + blue yes yes 10 10 05-08-15 to 16-09-15 yellow + blue yes yes 10 10 05-08-15 to 16-09-15 green + blue yes yes 10 10 05-08-15 to 16-09-15 yellow + blue yes yes 10 10 05-08-15 to 16-09-15 yellow + blue yes yes 10 10 05-08-15 to 16-09-15 yellow + blue yes yes 10 10 05-08-15 to 16-09-15 yellow + blue yes yes 10 10 05-08-15 to 16-09-15 green + yellow + blue yes yes 10 10 05-08-15 to 16-09-15 yellow + blue yes yes 10 10 05-08-15 to 16-09-15 yellow + blue yes yes 10 10 05-08-15 to 16-09-15 blue yes yes 10 10 05-08-15 to 16-09-15 greenish yellow + blue yes yes 10 10 05-08-15 to 16-09-15 blue yes yes 10 10 05-08-15 to 16-09-15 blue + white yes yes 10 10 05-08-15 to 16-09-15 blue yes yes 10 10 05-08-15 to 16-09-15 blue yes yes 10 10 05-08-15 to 16-09-15 blue yes yes 10 10 05-08-15 to 16-09-15 white + blue yes yes 10 10 05-08-15 to 16-09-15 dark blue yes yes 10 10 05-08-15 to 16-09-15 white + blue + dark blue yes yes 10 10 05-08-15 to 16-09-15 dark blue yes yes
l
10 10 05-08-15 to 16-09-15 yellow + blue + orange yes yes 10 10 05-08-15 to 16-09-15 yellow + blue yes yes 10 10 05-08-15 to 16-09-15 dark blue yes yes 10 10 05-08-15 to 16-09-15 white + blue + dark blue yes yes 10 10 05-08-15 to 16-09-15 blue yes yes 10 10 05-08-15 to 16-09-15 blue yes yes 10 10 05-08-15 to 16-09-15 white + blue yes yes 10 10 05-08-15 to 16-09-15 light blue yes yes 10 10 05-08-15 to 16-09-15 light blue yes yes 10 10 05-08-15 to 16-09-15 yellow + green + blue yes yes 10 10 05-08-15 to 16-09-15 light blue yes 10 10 05-08-15 to 16-09-15 dark blue yes yes 10 10 05-08-15 to 16-09-15 white + blue yes yes 10 10 05-08-15 to 16-09-15 white + blue yes yes 10 10 05-08-15 to 16-09-15 white + blue yes yes 10 10 05-08-15 to 16-09-15 white + blue yes yes 10 10 05-08-15 to 16-09-15 light yellow + blue yes yes 10 10 05-08-15 to 16-09-15 blue yes yes 10 10 05-08-15 to 16-09-15 blue yes yes 10 10 05-08-15 to 16-09-15 v. light yellow + blue yes yes 10 10 05-08-15 to 16-09-15 v. light yellow + blue yes yes 10 10 05-08-15 to 16-09-15 blue yes yes 10 10 05-08-15 to 16-09-15 blue yes yes 10 10 05-08-15 to 16-09-15 v. light blue yes yes 10 10 05-08-15 to 16-09-15 no colonies 10 10 05-08-15 to 16-09-15 blue yes yes 10 10 05-08-15 to 16-09-15 v. small blue colonies yes yes 10 10 05-08-15 to 16-09-15 v. small blue colonies yes yes
li
10 10 05-08-15 to 16-09-15 v. small blue colonies yes yes 10 10 05-08-15 to 16-09-15 v. small blue colonies yes yes 10 10 05-08-15 to 16-09-15 v. small blue colonies yes yes 10 10 05-08-15 to 16-09-15 v. small blue colonies yes yes 10 10 05-08-15 to 16-09-15 v. small blue colonies yes yes 10 10 05-08-15 to 16-09-15 v. small blue colonies yes yes 10 10 05-08-15 to 16-09-15 v. small blue colonies yes yes 10 10 05-08-15 to 16-09-15 v. small blue colonies yes yes 10 10 05-08-15 to 16-09-15 blue yes yes 7 10 05-08-15 to 16-09-15 v. light blue yes yes 7 10 05-08-15 to 16-09-15 v. light blue yes yes 7 10 05-08-15 to 16-09-15 v. light blue yes yes 7 10 05-08-15 to 16-09-15 v. light blue yes yes 7 10 05-08-15 to 16-09-15 no growth 7 10 05-08-15 to 16-09-15 no growth 7 10 05-08-15 to 16-09-15 v. light blue yes yes 7 10 05-08-15 to 16-09-15 v. light blue yes yes 7 10 05-08-15 to 16-09-15 yellow + blue yes yes 7 10 05-08-15 to 16-09-15 yellow + blue + green yes yes 7 10 05-08-15 to 16-09-15 yellow yes yes 7 10 05-08-15 to 16-09-15 no growth 7 10 05-08-15 to 16-09-15 no growth 10 10 04-08-15 to 16-09-15 blue + white yes yes 10 10 04-08-15 to 16-09-16 blue yes yes 10 10 04-08-15 to 16-09-17 dark blue + white yes yes 10 10 04-08-15 to 16-09-18 blue + white yes yes 10 10 04-08-15 to 16-09-19 blue yes yes 10 10 04-08-15 to 16-09-20 blue yes yes
lii
10 10 04-08-15 to 16-09-21 blue + white yes yes 10 10 04-08-15 to 16-09-22 blue + white yes yes 10 10 04-08-15 to 16-09-23 white yes yes 10 10 04-08-15 to 16-09-24 dark blue yes yes 10 10 04-08-15 to 16-09-25 blue + white yes yes 10 10 04-08-15 to 16-09-26 blue + white yes yes 10 10 04-08-15 to 16-09-27 blue + white yes yes 10 10 04-08-15 to 16-09-28 blue + yellow yes yes 10 10 04-08-15 to 16-09-29 blue + white yes yes 10 10 04-08-15 to 16-09-30 blue yes yes 10 10 04-08-15 to 16-09-31 blue + white yes yes 10 10 04-08-15 to 16-09-32 dark blue yes yes 10 10 04-08-15 to 16-09-33 blue + white + yellow yes yes 10 10 04-08-15 to 16-09-34 blue + white yes yes 10 10 04-08-15 to 16-09-35 blue + white yes yes 10 10 04-08-15 to 16-09-36 dark blue yes yes 10 10 04-08-15 to 16-09-37 blue + white yes yes 10 10 04-08-15 to 16-09-38 dark blue yes yes 10 10 04-08-15 to 16-09-39 blue + white yes yes 10 10 04-08-15 to 16-09-40 dark blue yes yes 10 10 04-08-15 to 16-09-41 dark blue yes yes 10 10 04-08-15 to 16-09-42 dark blue yes yes 10 10 04-08-15 to 16-09-43 dark blue yes yes 10 10 04-08-15 to 16-09-44 white + blue yes yes 10 10 04-08-15 to 16-09-45 white + blue yes yes 10 10 04-08-15 to 16-09-46 v. light blue + white yes yes 10 10 04-08-15 to 16-09-47 dark blue yes yes 10 10 04-08-15 to 16-09-48 dark blue yes yes
liii
10 10 04-08-15 to 16-09-49 dark blue yes yes 10 10 04-08-15 to 16-09-50 dark blue yes yes 7 10 05-08-15 to 16-09-15 yellow + blue + orange yes yes 7 10 05-08-15 to 16-09-15 white yes yes 7 10 05-08-15 to 16-09-15 yellow + blue + orange yes yes 7 10 05-08-15 to 16-09-15 white + blue yes yes 7 10 05-08-15 to 16-09-15 yellow + blue + orange yes yes 7 10 05-08-15 to 16-09-15 white + blue yes yes 7 10 05-08-15 to 16-09-15 orange yes yes 7 10 05-08-15 to 16-09-15 yellow + blue yes yes 7 10 05-08-15 to 16-09-15 orange + blue+ white yes yes 7 10 05-08-15 to 16-09-15 green yes yes 7 10 05-08-15 to 16-09-15 light blue + yellow yes yes 7 10 05-08-15 to 16-09-15 white + blue yes yes 7 10 05-08-15 to 16-09-15 white + blue yes yes 7 10 05-08-15 to 16-09-15 blue yes yes 7 10 05-08-15 to 16-09-15 blue yes yes 7 10 05-08-15 to 16-09-15 blue yes yes 7 10 05-08-15 to 16-09-15 dark blue yes yes 7 10 05-08-15 to 16-09-15 dark blue yes yes 7 10 05-08-15 to 16-09-15 white + blue yes yes 7 10 05-08-15 to 16-09-15 blue yes yes 7 10 05-08-15 to 16-09-15 blue + white yes yes 7 10 05-08-15 to 16-09-15 white + blue yes yes 7 10 05-08-15 to 16-09-15 yellow + blue yes yes 7 10 05-08-15 to 16-09-15 yellow + blue yes yes 7 10 05-08-15 to 16-09-15 yellow + blue yes yes 7 10 05-08-15 to 16-09-15 white yes yes
liv
7 10 05-08-15 to 16-09-15 white + light blue yes yes 7 10 05-08-15 to 16-09-15 white + light blue yes yes 7 10 05-08-15 to 16-09-15 white + light blue yes yes 7 10 05-08-15 to 16-09-15 white + blue yes yes 7 10 05-08-15 to 16-09-15 white + blue yes yes 7 10 05-08-15 to 16-09-15 white + blue yes yes 7 10 05-08-15 to 16-09-15 white + light blue yes yes 7 10 05-08-15 to 16-09-15 white + light blue yes yes 7 10 05-08-15 to 16-09-15 white + blue yes yes 7 10 05-08-15 to 16-09-15 white + light blue yes yes 7 10 05-08-15 to 16-09-15 white + light blue yes yes 7 10 05-08-15 to 16-09-15 white + blue yes yes 7 10 05-08-15 to 16-09-15 white + blue yes yes 7 10 05-08-15 to 16-09-15 white + light blue yes yes 7 10 05-08-15 to 16-09-15 light blue + yellow yes yes 7 10 05-08-15 to 16-09-15 dark blue yes yes 7 10 05-08-15 to 16-09-15 white + light blue yes yes 7 10 05-08-15 to 16-09-15 no growth 7 10 05-08-15 to 16-09-15 white + blue yes yes 7 10 05-08-15 to 16-09-15 white + ligt blue yes yes 7 10 05-08-15 to 16-09-15 white + ligt blue yes yes 7 10 05-08-15 to 16-09-15 white + ligt blue yes yes 7 10 05-08-15 to 16-09-15 white yes yes 7 10 05-08-15 to 16-09-15 yellow + blue yes yes 7 10 05-08-15 to 16-09-15 green yes yes 7 10 05-08-15 to 16-09-15 orange + blue yes yes 7 10 05-08-15 to 16-09-15 orange + blue yes yes 7 10 05-08-15 to 16-09-15 orange + blue yes yes
lv
7 10 05-08-15 to 16-09-15 orange + blue yes yes 7 10 05-08-15 to 16-09-15 yellow + blue yes yes 7 10 05-08-15 to 16-09-15 white + blue yes yes 7 10 05-08-15 to 16-09-15 yellow + blue yes yes 7 10 05-08-15 to 16-09-15 yellow + blue yes yes 7 10 05-08-15 to 16-09-15 orange + light blue yes yes 7 10 05-08-15 to 16-09-15 orange + blue yes yes 7 10 05-08-15 to 16-09-15 orange + blue yes yes 7 10 05-08-15 to 16-09-15 orange yes yes 7 10 05-08-15 to 16-09-15 orange + blue yes yes 7 10 05-08-15 to 16-09-15 orange + blue yes yes 7 10 05-08-15 to 16-09-15 blue yes yes
lvi
ANNEXURE TABLE 14
Colour with X-gal Size of Colonies Sample No. Strain No. Media pH Temperature (◦C) Incubation Time Light blue Dark blue V. dark blue Small Medium Large 1 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 2 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 3 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 4 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 5 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 6 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 7 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 8 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 9 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 10 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 11 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 12 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 13 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 14 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 15 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 16 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 17 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 18 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 19 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 20 Marine 7 10 16-09-15 to 28-10-15 yes yes 21 Marine 7 10 16-09-15 to 28-10-15 yes yes 22 Marine 7 10 16-09-15 to 28-10-15 yes yes 23 Marine 7 10 16-09-15 to 28-10-15 yes yes 24 Marine 7 10 16-09-15 to 28-10-15 yes yes
lvii
25 Marine 7 10 16-09-15 to 28-10-15 yes yes 26 Marine 7 10 16-09-15 to 28-10-15 yes yes 27 Marine 7 10 16-09-15 to 28-10-15 yes yes 28 Marine 7 10 16-09-15 to 28-10-15 yes yes 29 Marine 7 10 16-09-15 to 28-10-15 yes yes 30 Marine 7 10 16-09-15 to 28-10-15 yes yes 31 Marine 7 10 16-09-15 to 28-10-15 yes yes 32 Marine 7 10 16-09-15 to 28-10-15 yes yes 33 Marine 7 10 16-09-15 to 28-10-15 yes yes 34 Marine 7 10 16-09-15 to 28-10-15 yes yes 35 Marine 7 10 16-09-15 to 28-10-15 yes yes 36 Marine 7 10 16-09-15 to 28-10-15 yes yes 37 Marine 7 10 16-09-15 to 28-10-15 yes yes 38 Marine 7 10 16-09-15 to 28-10-15 yes yes 39 Marine 10 10 16-09-15 to 28-10-15 yes yes 40 Marine 10 10 16-09-15 to 28-10-15 yes yes 41 Marine 10 10 16-09-15 to 28-10-15 yes yes 42 Marine 10 10 16-09-15 to 28-10-15 yes yes 43 Marine 10 10 16-09-15 to 28-10-15 yes yes 44 Marine 10 10 16-09-15 to 28-10-15 yes yes 45 Marine 10 10 16-09-15 to 28-10-15 yes yes 46 Marine 10 10 16-09-15 to 28-10-15 yes yes 47 Marine 10 10 16-09-15 to 28-10-15 yes yes 48 Marine 10 10 16-09-15 to 28-10-15 yes yes 49 Marine 10 10 16-09-15 to 28-10-15 yes yes 50 Marine 10 10 16-09-15 to 28-10-15 yes yes 51 Marine 10 10 16-09-15 to 28-10-15 yes yes 52 Marine 10 10 16-09-15 to 28-10-15 yes yes
lviii
53 Marine 10 10 16-09-15 to 28-10-15 yes yes 54 Marine 10 10 16-09-15 to 28-10-15 yes yes 55 Marine 10 10 16-09-15 to 28-10-15 yes yes 56 Marine 10 10 16-09-15 to 28-10-15 yes yes 57 Marine 10 10 16-09-15 to 28-10-15 yes yes 58 R2 Agar 7 10 16-09-15 to 28-10-15 yes yes 59 R2 Agar 7 10 16-09-15 to 28-10-15 yes yes 60 R2 Agar 10 10 16-09-15 to 28-10-15 yes yes 61 Marine 7 10 16-09-15 to 28-10-15 yes yes
lix
ANNEXURE TABLE 15
Strain No. UP-PCR (Bands) Undiluted Diluted (10 Times) No band 1 yes no 2 yes no 3 yes no 4 yes 5 yes 6 yes 7 yes 8 yes no 9 yes no 10 yes no 11 yes 12 yes 13 yes 14 yes 15 yes no 16 weak band no 17 yes no 18 yes no 19 yes no 20 yes no 21 yes no 22 yes no 23 weak band no 24 weak band no lx
25 weak band no 26 yes 27 yes 28 yes 29 yes 30 weak band no 31 yes 32 yes 33 yes 34 yes 35 weak band no 36 yes 37 yes 38 yes yes 39 yes yes 40 no 41 weak band 42 weak band 43 44 yes no 45 yes yes 46 Weak band no 47 no 48 no yes 49 yes 50 yes 51 yes 52 yes lxi
53 yes no 54 yes 55 yes yes 56 yes yes 57 yes 58 yes yes 59 yes yes 60 yes no 61 yes no
lxii
ANNEXURE TABLE 16
Strain No. UP-PCR No Bands Bands Similar Different 1 yes yes 8 yes yes 10 yes 15 yes 18 yes 19 yes 20 yes 21 yes 22 yes 30 yes 38 yes 39 yes 43 yes 53 yes 55 yes 56 yes 58 yes 59 yes 60 yes 61 yes
lxiii
ANNEXURE TABLE 17
16s Bands Sample No. Weak Strong No band 1 yes 8 yes 9 yes 16 yes 19 yes 20 yes 21 yes 22 yes 38 yes 53 yes 55 yes 56 yes 58 yes 59 yes
lxiv
ANNEXURE TABLE 18
Sample 16s No. Sequencing Worked Not worked A yes B yes 1 yes 9 yes 16 yes 8 yes 20 yes 21 yes 22 yes 38 yes 53 yes 56 yes 58 yes 59 yes
lxv
ANNEXURE TABLE 19
16s Sequencing
Sample No. Origion Media pH Temperature (◦C) Closest Match Similarity (%)
A Ikka Column R2 Agar 10 10 Pseudomonas pelagia CL-AP6(T) 97.15
B Ikka Column R2 Agar 10 10 Pseudomonas pelagia CL-AP6(T) 73.32
1 Mc Murdo R2 Agar 10 10 Pseudomonas pelagia CL-AP6(T) 98.50
16 Mc Murdo R2 Agar 10 10 Marinilactibacilus piezotolerans LT20(T) 94.89
20 Canary Pond Marine 7 10 Alkalibacterium subtropicum O24-2(T) 94.66
21 Canary Pond Marine 7 10 Alkalibacterium putridalgicola T129-2-1(T) 92.00
22 Canary Pond Marine 7 10 Marinobacter psychrophilus 20041(T) 96.11
58 Kingfisher Pond R2 Agar 7 10 Rhizobium herbae CCBAU83011(T) 95.22
59 Kingfisher Pond R2 Agar 7 10 Rhizobium herbae CCBAU83011(T) 96.66
lxvi
ANNEXURE TABLE 20
Whole Genome Sequencing
Sample No. Origion Media pH Temperature (◦C) Gram Staining DNA Conc. (ng/ul) Closest Match Similarity (%)
B Ikka Column R2 agar 10 10 negative 30 Pseudomonas pelagia CL-AP6(T) 99.66
20 Canary Pond Marine 7 10 positive 30 Alkalibacterium subtropicum O24-2(T) 97.83
22 Canary Pond Marine 7 10 negative 83.8 Marinobacter psychrophilus 20041(T) 99.32
58 Kingfisher Pond R2 agar 7 10 negative 33 Rhizobium giardinii H152(T) 98.2
59 Kingfisher Pond R2 agar 7 10 negative 48.9 Rhizobium giardinii H152(T) 98.2
lxvii
ANNEXURE TABLE 21
Sample No. Species Name Whole genome sequencing Similarity (%) Media Temperature (◦C) pH Origion
58 NAQVI-58 Rhizobium giardinii H152(T) 98.2% R2 Agar 10 7 Kingfisher Pond
59 NAQVI-59 Rhizobium giardinii H152(T) 98.2% R2 Agar 10 7 Kingfisher Pond
lxviii lxix
ANNEXURE 1
lxx
lxxi
ANNEXURE II
lxxii
lxxiii
lxxiv
lxxv
ANNEXURE III
lxxvi
ANNEXURE IV
Whole Genome Sequencing Report of Strain NAQVI 58
All statistics are based on contigs of size >= 500 bp, unless otherwise noted (e.g., "# contigs (>= 0 bp)" and "Total length (>= 0 bp)" include all contigs).
lxxvii
lxxviii
lxxix lxxx
Whole Genome Sequencing Report of Strain NAQVI 59
All statistics are based on contigs of size >= 500 bp, unless otherwise noted (e.g., "# contigs (>= 0 bp)" and "Total length (>= 0 bp)" include all contigs).
lxxxi
lxxxii
lxxxiii lxxxiv
ANNEXURE V
lxxxv
ANNEXURE VI
lxxxvi
lxxxvii
ANNEXURE VII
lxxxviii
ANNEXURE VIII
lxxxix
xc
xci
xcii
ANNEXURE IX
SEM for Bio-Samples
1. Sample stocks on liquid media. 2. Wash the sample cells with PBS, centrifuge for 5min at 10000 g, then carefully discard the supernatant. 3. Different dilution should be made for samples, usually I diluted at 5×10-1, 5×10-2,5×10-3,5×10-4. 4. Add 1ml 2.5% Gluteraldehyde to each sample, Allow to 4 ℃ for 4.5 hour. 5. After 4.5 hour, add 500 µl 50% ethanol to 500 µl each sample, allow to dry 5 min, centrifuge for 2 min at 10000 g. 6. Carefully discard the supernatant, add 700µl 70% ethanol to each sample,allow to dry 5 min, centrifuge for 2min at 10000 g. 7. Carefully discard the supernatant, add 800 µl 80% ethanol to each sample, allow to dry 5 min, centrifuge for 2min at 10000 g. 8. Carefully discard the supernatant, add 900 µl 90% ethanol to each sample, allow to dry 5 min, centrifuge for 2min at 10000 g. 9. Carefully discard the supernatant, add 960 µl 96% ethanol to each sample, allow to dry 5 min. 10. 10µlsuspension for each sample should be drop on the slide.
xciii
ANNEXURE X
xciv
ANNEXURE XI
xcv
xcvi
ANNEXURE XII
xcvii
ANNEXURE XIII
xcviii
xcix
ANNEXURE XIV
c
ANNEXURE XV
ci
PLAGIARISM REPORT
cii
ciii
LIST OF PUBLICATIONS