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Potential of Marine-Derived Fungi and Their Enzymes in Bioremediation of Industrial Pollutants

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Potential of Marine-Derived Fungi and Their Enzymes in Bioremediation of Industrial Pollutants Potential of marine-derived fungi and their enzymes in bioremediation of industrial pollutants Thesis submitted for the degree of Doctor of Philosophy in Marine Sciences to the Goa University by Ashutosh Kumar Verma Work carried out at National Institute of Oceanography, Dona Paula, Goa403004, India March 2011 STATEMENT As per requirement, under the University Ordinance 0.19.8 (vi), I state that the present thesis titled "Potential of marine-derived fungi and their enzymes in bioremediation of industrial pollutants" is my original contribution and the same has not been submitted on any previous occasion. To the best of my knowledge, the present study is the first comprehensive work of its kind from the area mentioned. The literature related to the problem investigated has been cited. Due acknowledgements have been made whenever facilities or suggestions have been availed of. Ashutosh Kumar Verma CERTIFICATE This is to certify that the thesis titled "Potential of marine-derived fungi and their enzymes in bioremediation of industrial pollutants" submitted for the award of the degree of Doctor of Philosophy in the Department of Marine Sciences, Goa University, is the bona fide work of Mr Ashutosh Kumar Verma. The work has been carried out under my supervision and the thesis or any part thereof has not been previously submitted for any degree or diploma in any university or institution. Place: 0 \„c__ cL co, Date: RA \ (2,1_ \ Dr. Chandralata Raghukumar Ph.D. Supervisor, Marine Biotechnology Laboratory, National Institute of Oceanography, Dona Paula, Goa, India. Ail RvtcA vrtc..1& s u.net-l-cJ Q.cue. i-L, L.3.-k— A-1 Acknowledgments This is the best opportunity for me to convey my sincere regards for all those people who enabled me to accomplish thesis work successfully. It is difficult to overstate my gratitude to my Ph. D. supervisor, Dr. Chandralata Raghukumar. Her enthusiasm and great efforts to explain things clearly and patiently, made my work even more interesting. She encouraged me to choose this topic for my thesis and I am very grateful to her for valuable guidance and constant encouragement throughout this study. She always supported and taught me experimental and writing skills. Throughout my thesis-writing period, she provided encouragement, sound advice, good teaching, good company and lots of good ideas. She as a person is always ready to hear and solve the problems of her students in whatever way possible whilst allowing the room to work in their own way. I feel proud and very fortunate of getting opportunity to work in her guidance, as one simply could not wish for a better or friendlier supervisor. Working with her was the most memorable achievement for me and I attribute the present work to her encouragement and constant effort and without her this thesis, too, would not have been completed. I also thank Dr. S. Raghukumar, for his valuable and thoughtful suggestions regarding my work. At several instances, he provided me with insights and guidance to recognize my mistakes. Both of them have influenced me a lot with their enthusiastic and positive approach. Their confidence imbibing attitude and endless endeavors through which I have gained and learned a lot building up my future and personality. I wish to thank Dr. S. R. Shetye, Director, NIO for giving me opportunity to work and providing the infrastructure facilities in the institute. I am thankful to my Co-guide Dr. S. Ghadi, Department of Biotechnology, Goa University for all his support throughout my study. I would also like to extend my appreciation to all my FRC members, Dr. G. N. Nayak, Dean, Department of Marine Science, Goa University, Dr. H. Menon, Head, Department of Marine Sciences, Goa University and VC's nominee Dr. N. Ramaiah, Scientist, NIO for all their critical comments and for their periodic assessment of my work. I would also thank Dr. D. J. Bhat, Head, Department of Botany, Goa University for helping me in the identification of fungi. I humbly acknowledge Dr. N. Ramaiah, DU leader, Microbial Ecology Laboratory who provided me the facilities for completion of the work as and when required. I am thankful to Dr. M.P. Tapaswi, Head Librarian, NIO, for providing some important references whenever required. I am also grateful to Mr. Arun Mahale, and other staff specially Mr. Javali of DTP section to help me in modification of some of the figures in this thesis. I express my sincere thanks to Dr Chandrakant Govind Naik for his efforts in spectrometric analysis. His help and contribution is notable in the completion of this thesis. I am thankful to Dr. Yogesh Shouche, Scientist, NCCS, Pune for extending the sequencing facility in his lab. I am indebted to my dear friend Pankaj Verma for his great help and useful suggestions throughout my thesis. I acknowledge the financial assistance provided by CSIR (Council for Scientific and Industrial Research) during my research work. I am thankful to all my friends in the Marine Biotechnology and Microbial Ecology Lab, NIO for all their timely help. Special mention is required of Dasu and I owe everlasting gratefulness to him, who was always there to help me in all the possible ways. I thank Cathrine, Samir, Varada, Donna and Purnima, for making the lab a wonderful place to work. They provided a stimulating and fun environment. I thank all my friends from BOD, COD, GOD and POD for the help provided during the study. I am thankful to Atul Ltd., Gujrat for the supply of textile mill effluents, Jeypore Sugar Co. Ltd., Andhra Pradesh and Emmellen Biotech Pharmaceuticals Ltd., Maharshtra for molasses-based effluents. I am deeply indebted to all my family members for their support and constant encouragement and showing trust in me. And last but not the least; I am thankful to almighty for giving me all the strength to achieve this goal. Ashutosh Kumar Verma Table of Contents Page No. Chapter 1 Review of Literature 1-32 Chapter 2 Isolation of fungi from various marine habitats, specifically for lignin degrading enzymes and screening them for decolorization of pollutants 33-72 Chapter 3 Influence of various parameters on decolorization and detoxification of colored effluents by selected marine fungi producing these enzymes 73-157 Chapter 4 Homology studies of laccase produced by different fungi. Isolation of laccase gene from a fungal isolate and its expression in the compatible yeast species. 158-171 Chapter 5 Summary & Future Prospect's 172-181 Appendix 182-189 Bibliography 190-258 Publications 259-260 Chapter 1 44Pview of Literature Review of Literature 1.1 Industrial pollutants/effluents Color is the first contaminant to be recognized in wastewater. These effluents dumped into the water bodies prohibit usage and affects its aesthetic and sentimental value. Paper and pulp mills, molasses based-alcohol distilleries, tanneries, dye-making units, and textile industries are some of the major industries that produce and discharge highly colored effluents. Each of these industrial effluents creates some specific problem besides producing aesthetically unacceptable intense coloring of soil and water bodies. Growing public awareness of the environment is forcing several industrial units to practice stringent pollution treatment on a top priority. 1.1.1 Paper and pulp mills The pulp and paper industry is quite old. In India, more than 150 paper and board mills with an installed capacity of nearly 3 million tones year-1 are in operation (Subramanyam, 1990) of which 36 are the large mills with a production capacity >55 tones day-1 , and the rest are small mills with production capacity <30 tones day-1(Sastri, 1986). The large pulp-paper mills equipped with soda recovery discharge about 270 to 450 1 effluent ke of paper containing 40 to 50 g lignin kg-1 bleached paper produced. Contrary to that, the small paper mills without soda recovery discharge nearly 300 to 400 1 of black liquor effluent containing 200 to 250 g lignin kal of paper manufactured (Garg and Modi, 1999). More than 150x106 tons of pulp is produced annually and about 50x106 tons of lignin together with the chemicals used is released from the P&P industry indicating that a lot of efforts have to be undertaken to handle the enormous amounts of hazardous potential (Call and Miicke, 1997). One of the major problems of effluent discharge from the pulp and paper industry is its brown/black color, generally known as black liquor. The color of these wastewaters is primarily due to lignin and its derivatives, which are discharged in such effluents mainly from the pulping, bleaching, and chemical recovery stages of the plant. High 1 Review of Literature molecular-weight chlorinated lignins are generally not removed from the effluents. These products include chlorolignins, chlorophenols, and chloroaliphatics (Ali and Sreekrishnan, 2001). Besides, these paper mill effluents are highly alkaline and alter the pH of the soil and water bodies into which they are discharged. 1.1.2 Textile dyes and textile mill effluent The total annual world textile dye production is estimated at about 800 kt. In 1999 the value of the global dyestuff market was estimated at 6.6 billion US$, North America accounting for 1.2 billion US$, Central and South America for 0.7 billion US$, Western Europe for 1.2 billion US$ and Asia for 2.7 billion US$. India, the former USSR, Eastern Europe, China, South Korea and Taiwan consume approximately 600 thousand tons (kt) of dyes per annum (Ishikawa et al., 2000). The distribution of global dyestuff market has changed during the last decade, with Asia being the largest dyestuff market today (about 42%). Even though the dye industry is characterized by a large number of producers (about 2000 worldwide), just four Western companies accounted for nearly half of the market in 2000 (Wesenberg et al., 2003).
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