Studies on microbial prospecting for exobiopolymeric flocculants A thesis submitted in fulfillment of the requirement for the award of the degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY Santosh Pathak (Regn No. 9051005) Department of Biotechnology and Environmental Sciences, Thapar University, Patiala –147004 Punjab (India) 2011 Acknowledgement I pay my reverence and gratitude to the Almighty for his everlasting blessings. This thesis arose out of years of research that has been done since I joined Thapar University. I have known a great number of people during these many years, whose contribution in assorted ways to the research and the making of the thesis deserved special mention. It is a pleasure to convey my gratitude to them all in my humble acknowledgment. Firstly, I offer my gratitude to my esteemed supervisor Dr. Moushumi Ghosh, Associate Professor, Thapar University, Patiala, for her supervision, advice, and guidance from the very early stage of this research as well as giving me extraordinary experiences throughout the work. Above all and the most needed, she provided me unflinching encouragement and support in various ways. Her truly scientific intuition has made her as a constant oasis of ideas and passions in Science, which exceptionally inspire and enrich my growth as a student, a researcher and a scientist want to be. I am indebted to her insightful comments, and for putting so much energy for this work. I take this opportunity to thank Dr. Abhijit Mukherjee, Director, Thapar University, for all the facilities which have been immensely helpful for the completion of my work. I express my gratitude to Dr. P. K. Bajpai, Dean (Research and Sponsored Projects), and Dr. Susheel Mittal, former Dean, Thapar University, Patiala, for their encouragement and support during my research work in the University. I am extremely thankful to Dr. M Sudhakara Reddy, Professor and Head Department of Biotechnology and Environmental Sciences, Thapar University, Patiala, for providing me all the help and support during the progress of my work at the Institute. I would also like to thank Dr. Niranjan Das, former Head and Associate Professor, for his encouragement and suggestions during the course of my work. I am highly thankful to the members of my doctoral committee Dr. N. Tejo Prakash, Associate Professor, Department of Biotechnology and Environmental Sciences, Thapar University and Dr. Haripada Bhunia, Associate Professor, Department of Chemical Engineering, for their comments and suggestions during the course of my PhD. It is my privilege to thank Dr. Abhijit Ganguli, Asssociate Professor, Department of Biotechnology and Environmental Sciences for providing necessary guidance, help and suggestions, for my work and at personal level. 1 List of Publications Publications in peer reviewed journals 1. Ghosh, M., Ganguli, A. and Pathak, S., 2009. Application of a novel biopolymer for removal of Salmonella from poultry wastewater. Environ. Technol., 30: 337–344. 2. Ghosh, M., Pathak, S. and Ganguli, A., 2009. Effective removal of Cryptosporidium by a novel bioflocculant. Water Environ. Res., 81: 160-164. 3. Ghosh, M. and Pathak, S., 2009. Biopolymer based intervention for Salmonella in water. Ind. J. Biotechnol., 8: 298-303. 4. Ghosh, M., and Pathak, S., 2010. Application of microbial exopolymers for removal of protozoan parasites from potable water. Res. J. Biotechnol., 5: 58-62. Conference Proceedings Oral Presentations 1. Pathak, S., Ghosh, M., and Ganguli. A. Physicochemical properties of a novel polymeric flocculant material from microbial source at National Conference on Emerging Trends in Engineering Materials, Thapar Institute of Engineering & Technology (February 1-3, 2006). 2. Ghosh, M., Pathak, S., and Ganguli, A. Biotechnological Evaluation of a novel Exopolymeric Flocculant produced by Klebsiella terrigena for removal of Cryptosporidium parvum – an Emerging Water Borne Pathogen at ICBioE‟07 in Kuala Lumpur , Malaysia (May 8-10, 2007). 3. Pathak, S. and Ghosh, M. Targeted removal of Salmonella spp. by a novel biopolymer – a futuristic biodefense tool at XIth Punjab Science Congress, Thapar University (7-10 February, 2008). 4. Pathak, S., Ghosh, M. and Ganguli, A. Application of microbial exopolymers for removal of pathogens from potable water. First International Biotechnology Conference, SMIT, Sikkim (28-30 December, 2008). 1Related to present thesis work Poster presentations 1. Pathak, S. and Ghosh, M. Removal of high risk pathogens in waste water by novel biopolymeric flocculants. 49th Annual conference of Association of Microbiologists of India, University of Delhi, Delhi, November 18-20, 2008. 2. Pathak, S., Ghosh, M., and Ganguli. A. Removal of Cryptosporidium parvum from water samples by a novel exopolymeric bioflocculant produced by Klebsiella terrigena at 47th Annual Conference of Association of Microbiologists of India (AMI), Barkatullah University, Bhopal (MP), during December 6-8, 2006. Contents Page no. List of Publications v Abstract vii List of Abbreviations xiv List of Symbols xv List of Tables xvi List of Figures xvii Chapter 1. INTRODUCTION 1-6 Chapter 2. REVIEW OF LITERATURE 7-36 2.1 Flocculation dynamics 7 2.2 Types of flocculation 2.2.1 Charge neutralization 9 2.2.2 Bridging 10 2.2.3 Colloid Entrapment 10 2.3 Mechanisms involved in flocculation 2.3.1 Balancing Opposing Forces 11 2.3.2 The Energy Barrier 12 2.4 Types of flocculants 13 2.5 Limitation of synthetic flocculants 16 2.6 Microbial flocculants 17 2.7 Production and characterization of bacterial bioflocculants 19 2.8 Flocculant attributes 23 2.9 Factors affecting flocculant activity 2.9.1 Molecular Structure 24 2.9.2 Molecular Weight 25 2.9.3 Temperature 25 2.9.4 pH 26 2.9.5 Inorganic metal ions 27 2.9.6 Dose 27 2.10 Application of bioflocculants 28 2.11 Constraints of bioflocculant application 29 2.12 Heavy-metal Contamination and Toxicity 30 2.13 Water contamination by pathogens 33 Chapter 3. MATERIALS AND METHODS 37-62 3.1 Isolation and characterization of flocculant producing microorganisms 3.1.1 Collection of sludge samples 37 3.1.2 Chemicals and Media 37 3.1.3 Screening of bioflocculant producing bacteria 38 3.1.4 Bacteria and Culture conditions 38 3.1.5 DNA Isolation and PCR amplification 39 3.1.6 DNA sequencing and analysis of sequence data 39 3.1.7 Amplified Ribosomal DNA Restriction Analysis (ARDRA) 40 3.2 Growth kinetics and flocculating activity of Klebsiella terrigena and purification of its bioflocculant 3.2.1 Growth kinetics of Klebsiella terrigena RD4 strain 40 3.2.2 Determination of flocculating activity 41 3.2.3 Extraction and partial purification of bioflocculant (KBF) 42 3.2.4 Homogeneity of bioflocculant 42 3.2.5 Stability and storage of bioflocculant 43 3.2.6 Determination of alcian blue binding activity 43 3.3 Analysis of structure, composition and properties of bioflocculant 3.3.1 Enzymatic and EDTA treatment of Bioflocculant 43 3.3.2 Molecular mass determination of bioflocculant 44 3.3.3 Scanning electron microscopy 44 3.3.4 Compositional Analysis 47 3.3.5 Thin Layer Chromatography 47 3.3.6 High Performance Liquid Chromatography 48 3.3.7 Fourier Transform Infrared Spectroscopy 48 3.3.8 Thermal stability 48 3.3.9 Rheological measurements of the bioflocculant 49 3.4 Optimization of reaction system conditions 3.4.1 Bioflocculant Dosage 49 3.4.2 Medium pH 49 3.4.3 Cations 50 3.5 Evaluation of bioflocculant efficiency 3.5.1 Comparison of bioflocculant (KBF) with other flocculants 50 3.5.2 Flocculation of various suspended solids in aqueous solution 50 3.6 Effect of nutritional factors and culture conditions 3.6.1 Carbon-source(s) 51 3.6.2 Nitrogen-source(s) 52 3.6.3 Metal ion 52 3.6.4 Culture pH 53 3.6.5 Incubation temperature 53 3.6.6 Inoculum size 53 3.6.7 Agitation 54 3.7 Cellular response of K. terrigena RD4 to environmental modulators 3.7.1 Mutagenesis 54 3.7.2 Screening and selection of mutants 55 3.7.3 Oxidative stress 56 3.7.4 Water activity 56 3.8 Simulated and real time evaluation of pathogen removal efficacy 3.8.1 Salmonella removal assays 56 3.8.2 Fluorescent in situ Hybridization (FISH) experiments with pathogen and Salmonella removal 57 3.8.3 Microscopy and digital image analysis 58 3.8.4 Cryptosporidium parvum removal assays 59 3.9 Optimization studies on heavy metal removal 60 3.10 Statistical Evaluation 61 Chapter 4. RESULTS 63-115 4.1 Screening of exopolymeric flocculant producing potential strain 63 4.2 Characterization and Identification of the strain RD4 4.2.1 Morphological and biochemical characterization 65 4.2.2 Molecular characterization and identification 65 4.2.3 Genetic fingerprinting of Klebsiella terrigena RD4 strain by ARDRA 69 4.3 Kinetics of growth and biofloculant production 69 4.4 Nature of K. terrigena bioflocculant (KBF) 71 4.5 Optimization of factors effecting flocculant ativity 4.5.1 Effect of Bioflocculant dosage 72 4.5.2 Optimization of Cations 72 4.5.3 Effect of medium pH and bioflocculant stablity in solutions 73 4.5.4 Incubation temperature 73 4.6 Evaluation of bioflocculant efficiency 4.6.1 Comparative study with some natural and synthetic flocculants 75 4.6.2 Flocculation of various suspended solids 75 4.7 Partial Characterization of the purified polymeric flocculant (KBF) of Klebsiella terrigena RD4 strain 4.7.1 Surface properties of K. terrigena bioflocculant 78 4.7.2 Biochemical composition and physical properties 79 4.7.3 Thermal properties of KBF 83 4.7.4 Rheological studies of the bioflocculant 84 4.8 Effect of nutritional factors and culture conditions 4.8.1 Carbon source(s) 88 4.8.2 Nitrogen source(s) 92 4.8.3 Carbon/Nitrogen (C/N) ratio 94 4.8.4 pH of culture medium and cultivation temperature 95 4.8.5 Agitation speed and inoculum size 95 4.8.6 Effect of metal ion(s) 98 4.9 Cellular response of K.