Marquette University e-Publications@Marquette Dissertations, Theses, and Professional Dissertations (1934 -) Projects Preservation of Methanogenic Cultures to Enhance Anaerobic Digestion Ujwal H. Bhattad Marquette University Follow this and additional works at: https://epublications.marquette.edu/dissertations_mu Part of the Environmental Engineering Commons Recommended Citation Bhattad, Ujwal H., "Preservation of Methanogenic Cultures to Enhance Anaerobic Digestion" (2012). Dissertations (1934 -). 209. https://epublications.marquette.edu/dissertations_mu/209 PRESERVATION OF METHANOGENIC CULTURES TO ENHANCE ANAEROBIC DIGESTION by Ujwal H. Bhattad, M.E. A Dissertation submitted to the Faculty of the Graduate School, Marquette University, in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy Milwaukee, Wisconsin August 2012 ABSTRACT PRESERVATION OF METHANOGENIC CULTURES TO ENHANCE ANAEROBIC DIGESTION Ujwal H. Bhattad, M.E. Marquette University, 2012 The use of anaerobic biotechnology is increasing as a sustainable process to treat various organic wastes. Methanogens convert organic COD into CH4 and play the key role to drive thermodynamically unfavorable biochemical fermentation reactions and keep the digestion process steady and efficient. Progressive understanding of anaerobic microbiology with digester functionality may help to develop efficient, customized methanogenic cultures to enhance anaerobic bioprocesses. Preservation of methanogenic cultures via drying would be a cost-effective option for research and practical applications. However, preservation of methanogenic cultures is challenging due to methanogen sensitivity to O2 toxicity and drying, and very limited work is reported on their preservation. The work described herein involves preservation and subsequent storage of various methanogenic cultures in oxic conditions as well as applications to improve performance of anaerobic digesters and standardize laboratory testing. Five methanogenic cultures were customized under different growth conditions. The cultures were preserved using freeze- and heat-drying, and subsequently stored for short and long periods in the presence of air. Their activity was then assayed by measuring specific methanogenic activity. The influences of growth conditions and protective agent addition were investigated to improve methanogenic activity after preservation. Clone library and qPCR techniques were used to identify and quantify methanogenic communities before and after drying. The usefulness of preserved cultures was examined to bioaugment transiently upset anaerobic digesters and as seed inocula for a standard laboratory test, the biochemical methane potential (BMP) assay. The effect of bioaugmentation was correlated with methanogenic community structure using the DGGE molecular fingerprinting technique. All customized methanogenic cultures were significantly active even after handling, drying and subsequent storage in the presence of air, suggesting methanogenic culture preservation and storage in air is feasible. Freeze-dried cultures maintained higher methanogenic activity than heat-dried cultures. The culture developed in the presence of limited O2 exhibited higher methanogenic activity than cultures developed in strict anaerobic conditions regardless of the drying method employed. Glucose as a protective agent resulted in higher methanogenic activity, more so in freeze drying than heat drying. Some methanogenic community members were found to be more tolerant to drying stress than others. Dried methanogenic cultures were found to be viable options to use as a bioaugment to improve treatment efficiency of anaerobic digesters after toxic upset and for the BMP assay. i ACKNOWLEDGEMENTS Ujwal H. Bhattad, M.E. I am sincerely and heartily thankful to all the people who directly or indirectly supported me in completing my research. I strongly feel that without their contribution it would have been impossible to reach this level. Formost, I owe my deepest gratitude to my research advisor Dr. Danial Zitomer who made this dissertation possible and because of him, my graduate experience has been one that I will cherish forever. I am greatly thankful to his excellent guidance, caring, patience, and providing me with an excellent atmosphere for doing research. He provided me a full-time research assistantship through out my study which helped me a lot to focus on my research and not to worry on my finance. His mentorship was a key in providing a well rounded experience consistent to my long-term career goals. He encouraged me to not only grow as a scientist but also an independent thinker. I am confident that these professional qualities will help me in my future success. I am also grateful to my research committee members for their continuous support, encouragement and suggestions to improve the quality of my research. I am thankful to Dr. Jim Maki for helping me to develop my background in molecular biology and providing his lab facility to do some advance molecular work. It was a wonderful experience working under his guidance, also a great relief talking to him during many stressful situations in dissertation writing. It was also a great pleasure for having Dr. Michael Switzenbaum and Dr. Michael King in the research committee. Their remarkable suggestions were undoubtly helpful for exploring the depth of my research. I am delighted to thank Mike Dollhopf, (Lab Manager, Water Quality Center, Marquette University) for assisting me in lab experimental set-up, and keeping track of lab supplies which helped a lot in completing the research tasks on time without any interreptions. I found him a great person on professional and personal level. I am very thankful to Keerthi Cherukuri, (Graduate student, Department of Biological Sciences, Marquette University) for teaching, and working collaboratively on ii few of my research objectives with her qPCR molecular skills. Her contribution to my research was significant and helped me a lot in strenthening my research findings. I would also like to extend my appericiation to Prince Mathai, (Graduate Student, Department of Biological Sciences, Marquette University) for enriching my knowledge on molecular biology, and teaching and helping me in cloning and sequencing of DGGE bands. I am also greatly thankful to Dr. Naveen Bansal (Department of Maths, Statistics, and Computer Sciences, Marquette University), and Snehal Patil (Graduate student) for teaching me statistical and bioinformatics tools, and helping me several times when ever I needed. I am obliged to thank Eslami Mehdi, Emery Nelson and Parwinder Virk (Undergraduates at Marquette University) for their great contribution in assisting routing monitoring and operation of my research experiments. Without them, it was challenging for me to collect huge amount of data and do the quality analysis. I feel so fortunate to associate with all my past and present lab-mates; Dr. Vaibhav Tale, Dr. Anne Schauer-Gimenez, Dr. Rachel Morris, Dr. Navaneethan Navaratnam, Ben Bocher, Kaushik Venkateshweren, Steve Gaziano and Matt Saib for being a great charm in every day of my work. They were a great help, motivation and fun. I am thankful to all of them for keeping me easy during hard journey of my research. I want to further extend my thanks to Dr. Tale for introducing me a doctoral research opportunity in this lab, and doing needful during my beginning at Marquatte University. Last but no least, I am so grateful to all donors of Arthur J.Schmitt Fellowship, Richard W. Jobling Fellowship, and Albert and Cecil Lue-Hing Engineering Graduate Scholarship, as well as State of Wisconsin, Department of Agriculture, Trade and Consumer Protection for supporting my education and research. I would like to express my special thanks to Marquette Univesity for giving me a great platform for excellence, faith, leadership and service. iii TABLE OF CONTENTS Title Page Acknowledgements……………………………………………………………………………………. i List of Tables…………………………………………………………………………...................... vii List of Figures………………………………………………………………………………………... viii Chapter 1 Introduction......................................................................................................1 1.1 Preservation of microorganisms by drying ...................................................2 1.1.1 Type of microorganisms ................................................................................4 1.1.2 Cell concentration ..........................................................................................5 1.1.3 Growth phase .................................................................................................5 1.1.4 Growth media ................................................................................................5 1.1.5 Stress-induced growth conditions .................................................................7 1.1.6 Drying methodology ......................................................................................7 1.1.6.1 Freeze drying ..........................................................................................8 Freezing ...............................................................................................................10 Drying..................................................................................................................11 Cryo-injury ..........................................................................................................14 1.1.6.2 Liquid drying ........................................................................................15