Characterization of C. debilis GB1 a Thermophilic Facultative Anaerobe Capable of Lending

438 Pages·2017·6.67 MB·English

by  Scott Wushke

Characterization of C. debilis GB1
a Thermophilic Facultative
Anaerobe Capable of Lending
Aerotolerance in Co-culture with
C. thermocellum

by
Scott Wushke

A Thesis Submitted to the Faculty of Graduate Studies of
The University of Manitoba
in partial fulfilment of the requirments of degree of

DOCTOR OF PHILOSOPHY

Faculty of Science/Department of Microbiology
University of Manitoba
Winnipeg

Copyright © 2017 by Scott Wushke
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Characterization of C. debilis GB1
a Thermophilic Facultative
Anaerobe Capable of Lending
Aerotolerance in Co-culture with
C. thermocellum

by
Scott Wushke
Ph.D., University of Manitoba, 2016

Supervisory Committee

Dr. Richard Sparling (Department of Microbiology, University of Manitoba)
Supervisor

Dr. Nazim Cicek (Department of Biosystems Engineering, University of Manitoba)
Committee member

Dr. Karen Brassinga (Department of Microbiology, University of Manitoba)
Committee member

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Abstract

Cellulosic ethanol production is mainly described as an anaerobic process.
Through use of aerobic selective pressure, using inoculate from samples taken from a
farm in South-Eastern Saskatchewan, Canada, we were able to create highly cellulolytic
aerotolerant enrichments which produced both hydrogen and ethanol. We were able to
recreate this highly cellulolytic aerotolerant phenotype using C. thermocellum DSM
1237 and one of the Caldibacillus debilis strains isolated from the aerotolerant
enrichments, strain GB1.
Caldibacillus debilis GB1 is a thermophilic facultative anaerobe. C. debilis GB1
displayed a physiology distinct from that of the type strain Tf which is an obligate
aerobe. Under oxygen limiting conditions, both GB1 and Tf produce end-products
lactate, acetate, formate and CO ;however, GB1 alone produces ethanol.
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A key feature necessary for aerotolerant growth of the co-culture with C.
thermocellum was the capacity of C. debilis GB1 to grow both aerobically and
anaerobically. In order to characterize GB1, we sequenced and annotated the genome
then used high throughput proteomics to characterise the protein expression changes
between aerobic and anaerobic metabolism. We found C. debilis GB1 has typical mixed
fermentation pathways and aerobic/anaerobic regulation similar to those found in E.
coli K-12 and B. cereus.
In order to explain the lack of ethanol production in the type strain, C. debilis Tf,
we did a genomic comparison of both strains Tf and GB1, focusing on the genes
concerning pyruvate metabolism. In particular, there were amino acid changes in the
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aldehyde-alcohol dehydrogenase ADHE that could lead to inactivity in strain Tf. This
genomic evidence, plus Tf having physiology consistent with an adhE deletion mutant
in the closely related Geobacillus thermoglucosidasius, leads us to propose that the
adhE gene is either differently regulated or inactive in C. debilis Tf.
From our work, we showed that C. debilis GB1 allowed us to further define and
characterize the physiology of the genus Caldibacillus, find a new aerotolerant mode
of mode of Consolidated bioprocessing using C. thermocellum, and further
understanding of aerobic/anaerobic regulation in thermophilic facultative anaerobes.

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Acknowledgements

Thank you to my advisor and mentor Dr. Sparling for giving me guidance, support,
and the opportunity to do this research.
Thank you to my supervisory committee members: Dr. Brassinga and Dr. Cicek
Thank you to collaborators who offered guidance and support: Dr. Levin, Dr. Stott,
and Dr. Fristensky.
Thank you to those guided and helped me with the Omics work: Peyman Ezzati,
Xiang Li Zhang, Vic Spicer and Dr. Krokhin
Thank you to my friends and lab mates: Tobin Verbeke, Tom Rydzak, Marcel
Taillefer, Alan Froese, Charushi Panditharatne Carlo Carere, Umesh Ramachandran,
Rumana Islam, Nathan Wrana, Valery Agbor, Jilagamazhi Fu, Ryan Sestric, Riffat
Munir, Tatiana Kozlova, Parveen Sharma, Maryam Mirzaie, Chris Dartiailh, Warren
Blunt and Elsie Jordaan, as well as many others that are too numerous to mention.
Thank you to Ariadne Valadares who suffered through so much proof reading for me.

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Dedication

To my parents, who never stopped believing in me.

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Table of Contents
Abstract ........................................................................................................................ iii
Acknowledgements ........................................................................................................ v
Dedication ..................................................................................................................... vi
Table of Figures ...........................................................................................................xii
Table of Tables ............................................................................................................ xiv
Table of Appendix Figures........................................................................................... xv
Table of Appendix Tables ........................................................................................... xvi
List of Materials for Which Copyright Permission was Obtained .............................xvii
Chapter 2: ...................................................................................................................xvii
Chapter 1: Developing Consortia for Ethanol Production from Consolidated
Bioprocessing ................................................................................................................. 1
1.1. Introduction and Justification for Use of Consolidated Bioprocessing ........... 1
1.2. The Need for Biofuels in 2016 ........................................................................ 2
1.3. Ethanol and Hydrogen ..................................................................................... 5
1.4. First, Second, and Third Generation Biofuels ................................................. 7
1.4.1. Feedstock for Consolidated Bioprocessing .............................................. 8
1.4.2. Lignocellulosic Composition ................................................................... 9
1.5. Typical Mixed Acid Fermentation of Bacteria and Ethanol Production ....... 11
1.6. Organisms for Potential Use in Consolidated Bioprocessing ....................... 13
1.6.1. C. thermocellum Role in Consolidated Bioprocessing .......................... 13
1.6.2. Caldicellulosiruptor Role in Consolidated Bioprocessing .................... 16
1.6.3. C. phytofermentans Role in Consolidated Bioprocessing ...................... 17
1.6.4. C. cellulolyticum Role in Consolidated Bioprocessing .......................... 18
1.7. Designed Co-cultures for CBP ...................................................................... 20
1.7.1. Co-cultures of C. thermocellum with Thermoanaerobacter sp. or
Thermoanaerobacterium sp. ................................................................................. 20
1.7.2. C. besci and T. maritma Co-Cultures ..................................................... 22
1.7.3. Co-cultures under Aerobic Conditions................................................... 22
1.8. Bioprospecting for Consolidated Bioprocessing ........................................... 23
1.9. Oxygen Removal/Oxygen Tolerance ............................................................ 24
1.10. Geobacillus and Caldibacillus in CBP ...................................................... 26
1.11. Omics for Microbial Characterization and Predictive Leverage ............... 27
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1.12. Conclusion ................................................................................................. 30
1.13. Thesis Objectives: ...................................................................................... 31
1.14. Work not Included in the Thesis Chapters................................................. 33
Chapter 2: Characterization of Enriched Aero-tolerant Cellulose Degrading
Communities for Biofuels Production Using Differing Selection Pressures and
Inoculum Sources ......................................................................................................... 34
2.1. Abstract ......................................................................................................... 34
2.2. Introduction ................................................................................................... 35
2.3. Materials and Methods .................................................................................. 37
2.3.1. Inocula and Enrichment Conditions ....................................................... 37
2.3.2. Variation in Growth Medium and Selection Conditions........................ 37
2.3.3. Experimental Setup ................................................................................ 38
2.3.4. Sugar and End-Product Analyses ........................................................... 40
2.3.5. DNA extraction and PCR Amplification ............................................... 40
2.3.6. Denaturing Gradient Gel Electrophoresis (DGGE) ............................... 41
2.3.7. DNA Sequencing and Phylogenetic Analyses ....................................... 41
2.4. Results and Discussion .................................................................................. 42
2.4.1. Culture Enrichment ................................................................................ 42
2.4.2. Analysis of Microbial Community......................................................... 42
2.5. Conclusion ..................................................................................................... 51
Chapter 3: Characterization of the Facultative Anaerobe Caldibacillus debilis GB1
and its use in a Designed Aerotolerant, Cellulose Degrading, Co-Culture with
Clostridium thermocellum ............................................................................................ 53
3.1. Abstract ......................................................................................................... 53
3.2. Introduction ................................................................................................... 54
3.3. Materials and Methods .................................................................................. 56
3.3.1. Culturing Method ................................................................................... 56
3.3.2. Cell Growth ............................................................................................ 57
3.3.3. Physiological Characterization............................................................... 57
3.3.4. Sugar and End-Product Analysis ........................................................... 58
3.3.5. Bioinformatic Analyses .......................................................................... 59
3.3.6. Culture Purity Confirmation .................................................................. 59
3.4. Results ........................................................................................................... 60
3.4.1. Caldibacillus Strain Comparison ........................................................... 60
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3.5. Caldibacillus debilis Plus Clostridium thermocellum Co-Culture ................ 66
3.6. Discussion ..................................................................................................... 67
3.6.1. Caldibacillus Strain Comparison ........................................................... 67
3.6.2. Caldibacillus debilis Plus Clostridium thermocellum Co-Culture ........ 69
3.7. Conclusion ..................................................................................................... 75
Chapter 4: Genome and proteome characterization of Caldibacillus debilis GB1 core
metabolism under aerobic and anaerobic conditions ................................................... 76
4.1. Abstract ......................................................................................................... 76
4.2. Introduction ................................................................................................... 77
4.3. Materials and Methods .................................................................................. 80
4.3.1. Cell Culturing and Harvesting ............................................................... 80
4.3.2. DNA Isolation and Processing ............................................................... 81
4.3.3. Preparation of Cells for Protein Extraction ............................................ 82
4.3.4. Mass Spectrometry ................................................................................. 84
4.3.5. Database Search, Protein Identification, and Statistical Analysis .......... 84
4.3.6. Comparison of C. debilis GB1 Proteome with E. coli K-12
Transcriptome ....................................................................................................... 87
4.4. Results and Discussion .................................................................................. 88
4.4.1. General Genome Features ...................................................................... 88
4.4.2. Growth Characteristics at the Proteomic Sampling Point ...................... 89
4.4.3. General Features of the Proteomes ........................................................ 91
4.4.4. Tricarboxylic Acid Cycle............................................................................ 99
4.4.5. Glycolysis ............................................................................................. 103
4.4.6. Pyruvate Metabolism and End-Product Synthesis ............................... 103
4.4.7. Oxygen Respiration .............................................................................. 105
4.4.8. Reactive Oxygen Species Protection ................................................... 106
4.4.9. Pyruvate Dehydrogenase ...................................................................... 107
4.4.10. Other Highly Differentially-Expressed Pathways ............................ 108
4.4.11. Core Metabolism Overview ............................................................. 116
4.4.12. Comparison against the E. coli K-12 data set................................... 119
4.5. Conclusion ................................................................................................... 125
Chapter 5: Genomic Comparison of a Facultatively Anaerobic and Obligately Aerobic
Caldibacillus debilis Strains GB1 and Tf Helps Explain Physiological Differences 127
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5.1. Abstract ....................................................................................................... 127
5.2. Introduction ................................................................................................. 128
5.3. Materials and Methods ................................................................................ 129
5.3.1. Cell Culturing ....................................................................................... 129
5.3.2. Genomic Comparison........................................................................... 130
5.3.3. Protein Extraction................................................................................. 130
5.3.4. Mass Spectrometry Methods ................................................................ 131
5.3.5. Proteogenomics .................................................................................... 132
5.4. Results and Discussion ................................................................................ 133
5.4.1. GB1 and Tf Genome Comparison........................................................ 133
5.4.2. Proteogenomics .................................................................................... 145
5.4.3. Core Metabolic Genes Relative to Observed Phenotype ..................... 147
5.4.4. Pyruvate Fermentation ......................................................................... 152
5.4.5. Lactate Dehydrogenase ........................................................................ 168
5.4.6. Pyruvate Dehydrogenase ...................................................................... 168
5.4.7. Pyruvate Formate Lyase ....................................................................... 169
5.4.8. Alcohol Aldehyde Dehydrogenase ...................................................... 170
5.4.9. Cellobiose Phosphotransferase Systems .............................................. 179
5.4.10. Respirofermentative Metabolism ..................................................... 181
5.5. Conclusion ................................................................................................... 183
Chapter 6: General Discussion and Conclusions ....................................................... 185
6.1. Core Objectives ........................................................................................... 185
6.2. Thesis Work in the Context of 2016 ........................................................... 188
6.3. Future Work ................................................................................................ 190
6.4. Conclusion ................................................................................................... 191
6.5. Future Perspectives ...................................................................................... 193
Appendices ................................................................................................................. 195
Appendix A: Physiological and Genomic Characterization of Fervidobacterium
pennivorans strain DYC in Relation to the Type Strain ............................................ 195
A.1. Abstract ....................................................................................................... 195
A.2. Introduction ................................................................................................. 196
A.3. Materials and Methods ................................................................................ 198
A.3.1. Isolation of DYC and Cell Culturing ................................................... 198
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