Preservation of Methanogenic Cultures to Enhance Anaerobic Digestion

171 Pages·2016·2.87 MB·English

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Dissertations, Theses, and Professional
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Ujwal H. Bhattad
Marquette University
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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 CH and play the key role to
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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 O toxicity and drying, and very limited work is reported on
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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 O exhibited higher methanogenic activity than cultures developed in strict
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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.
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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
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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.
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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
1.1.6.3 Thermal drying .....................................................................................16
Thermal injury .....................................................................................................18
1.1.7 Protective agent ...........................................................................................19
1.1.8 Storage .........................................................................................................24
1.1.9 Rehydration .................................................................................................27
1.2 Methanogen tolerance to drying, O and heat .............................................29
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1.3 Bioaugmentation in anaerobic biotechnology .............................................32
1.3.1 Bioaugmentation for difficult-to-degrade organic compounds ...................32
1.3.2 Bioaugmentation for odor reduction ...........................................................33
1.3.3 Bioaugmentation for accelerating the reactor start-up ................................34
1.3.4 Bioaugmentation for shock load upset ........................................................34
1.3.5 Bioaugmentation for toxic upset .................................................................35
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1.3.6 Bioaugmentation for psychrophilic digester ...............................................36
1.4 References ...................................................................................................36
Chapter 2 Methanogens Endurance to Drying in Presence of Air under Extreme
Temperature ................................................................................................44
2.1 Introduction .................................................................................................44
2.2 Methods .......................................................................................................48
2.2.1 Culture and medium ....................................................................................48
2.2.2 Culture thickening and drying .....................................................................48
2.2.3 Specific methanogenic activity (SMA) testing ...........................................49
2.2.4 Microbial community identification and quantification ..............................50
2.2.4.1 Methanogenic community identification using mcrA gene-specific
primer ...................................................................................................50
2.2.4.2 Methanogenic community quantification using mcrA gene-specific
and 16S rRNA genus-specific primers .................................................52
2.2.5 Analysis .......................................................................................................54
2.3 Results .........................................................................................................55
2.3.1 Methanogenic activity .................................................................................55
2.3.2 mcrA transcript and gene copies quantification ..........................................57
2.3.3 Relationship between SMA, activity lag and mcrA transcript copy
numbers .......................................................................................................58
2.3.4 mcrA clone library .......................................................................................58
2.3.5 16S rRNA transcript and gene copy quantification .....................................61
2.4 Discussion ...................................................................................................64
2.5 Conclusion ...................................................................................................69
2.6 References ...................................................................................................69
2.7 Appendix I ...................................................................................................76
Chapter 3 Methanogenic Culture Preservation and Long-Term Storage in
Presence of Air ............................................................................................79
3.1 Introduction .................................................................................................79
3.2 Methods .......................................................................................................82
3.2.1 Culture development ...................................................................................82
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3.2.2 Preservation methods ..................................................................................83
3.2.3 Short-term and simulated long-term storage ...............................................84
3.2.4 Actual Storage over five months .................................................................84
3.2.5 Methanogenic activity testing .....................................................................84
3.2.5.1 SMA for H ...........................................................................................85
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3.2.5.2 SMA for acetate ....................................................................................86
3.2.6 Analysis .......................................................................................................86
3.3 Results .........................................................................................................87
3.3.1 RMC and drying time of dried cultures .......................................................89
3.3.2 Methanogenic activity without glucose addition .........................................90
3.3.3 Methanogenic activity with glucose addition ..............................................92
3.3.4 Activity lag ..................................................................................................92
3.3.5 Methanogenic activity over five months of storage ....................................95
3.3.6 Influence of culture growth conditions on methanogenic activity
preservation .................................................................................................96
3.4 Discussion ...................................................................................................97
3.5 Conclusion .................................................................................................103
3.6 References .................................................................................................105
3.7 Appendix II ................................................................................................110
Chapter 4 Micro-Oxic Growth Condition Enhanced Preservation of H -utilizing
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Methanogenic Culture in Presence of Air ..............................................111
4.1 Introduction ...............................................................................................111
4.2 Methods .....................................................................................................113
4.2.1 Culture development .................................................................................113
4.2.2 Culture preservation and simulated long-term storage ..............................114
4.2.3 Methanogenic activity of preserved culture ..............................................114
4.2.4 Quantification of methanogenic community .............................................114
4.2.5 Analysis .....................................................................................................116
4.3 Results and Discussion ..............................................................................117
4.4 Conclusion .................................................................................................122
4.5 References .................................................................................................122
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Chapter 5 Preserved, Dried Methanogenic Biomass for Bioaugmentation and
Standard Laboratory Tests: A Novel Approach ....................................126
5.1 Introduction ...............................................................................................126
5.2 Methods .....................................................................................................128
5.2.1 Culture development and freeze drying ....................................................128
5.2.2 Methanogenic activity testing for freeze-dried biomass ...........................129
5.2.3 Bioaugmentation effect of freeze-dried biomass .......................................130
5.2.3.1 Digester monitoring ............................................................................131
5.2.3.2 Microbial community analysis ...........................................................131
5.2.4 BMP testing using freeze-dried inocula ....................................................134
5.2.5 Statistical analysis .....................................................................................135
5.3 Results and Discussion ..............................................................................135
5.3.1 Methanogenic activity of freeze-dried biomass ........................................135
5.3.2 Bioaugmentation effect of freeze-dried biomass .......................................136
5.3.2.1 Digester Monitoring ...........................................................................138
5.3.2.2 Microbial community analysis ...........................................................140
5.3.3 BMP test using freeze-dried inoculum ......................................................147
5.4 Conclusion .................................................................................................149
5.5 References .................................................................................................151
Chapter 6 Overall Conclusions .....................................................................................156
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LIST OF TABLES
Title Page

Table 1.1. Freeze drying parameters ..................................................................................13

Table 2.1. Methanogenic gene and genus specific primers ...............................................55

Table 3.1. Methanogenic activity of hydrogen-utilizing methanogenic cultures before
and after preservation and after simulated long-term storage ...........................88
Table 3.2. Percent residual moisture content (RMC) of hydrogen-utilizing
methanogenic cultures after preservation ..........................................................88