Understanding Biofilms of Anaerobic, Thermophilic and Cellulolytic Bacteria

158 Pages·2014·3.93 MB·English

Understanding Biofilms of Anaerobic, Thermophilic and
Cellulolytic Bacteria: A Study towards the Advancement of
Consolidated Bioprocessing Strategies
by
Alexandru Dumitrache
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Graduate Department of Chemical Engineering and Applied Chemistry
University of Toronto
© Copyright by Alexandru Dumitrache (2014)
Understanding Biofilms of Anaerobic, Thermophilic and
Cellulolytic Bacteria: A Study towards the Advancement of
Consolidated Bioprocessing Strategies
Alexandru Dumitrache
Doctor of Philosophy
Chemical Engineering and Applied Chemistry
University of Toronto
2014
Abstract
The anaerobic, cellulolytic bacterium Clostridium thermocellum formed biofilms on cellulose
consisting of a single layer of cells which did not secrete an extracellular polymeric matrix.
Sporulation occurred under normal growth and was believed to assist with biofilm translocation
to new substrates. Although the cell-substrate distance was less than 210 nm, the biofilm layer
lost up to 29% of hydrolyzed oligomeric products when reactors were loaded with extreme
concentrations of cellulose (up to 200 g/litre). This effect was much less severe at lower cellulose
concentrations. Of the total cellulose carbon, 4% (gC/gC) was utilized for cell mass production
and up to 75% was converted into primary metabolites (ethanol, acetic acid, lactic acid, carbon
dioxide). Increasing the starting cellulose concentration shifted the ethanol-to-acetic acid ratio
from 0.91 g/g to 0.41 g/g. Such high substrate loadings and metabolite shifts have not been
previously reported and may be of interest for consolidated bioprocessing strategies. Cellulose
conversion was initially limited by microbial growth, with a biofilm development rate estimated
at 0.46h-1 to 0.33h-1 and where up to 20% of the substrate was consumed. Subsequently,
substrate-limited conditions determined the rate kinetics. Surface accessibility for microbial
colonization was the dominant rate limiting factor, while mass imposed constraints very late
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towards the end-point fermentation. CO was found to be an excellent reporter molecule for
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cellulose consumption and biofilm growth. Online CO tracking may also be used to assess the
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digestibility of substrates with unknown surface properties. A mathematical model that described
biofilm growth, substrate consumption and product formation was found to have an excellent fit
with experimental data of CO production which reinforced the previous findings on the
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cellulolytic biofilm form and function. Together, these results demonstrate that biofilms are
undeniably the key to understanding the effective microbial conversion of cellulosic substrates.
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Acknowledgments

I would like to graciously acknowledge my primary supervisors Dr. Gideon Wolfaardt and Dr.
Grant Allen for giving me the opportunity to complete this work and for their adamant
confidence in me; and my project’s committee members Dr. Emma Master and Dr. Elizabeth
Edwards for guiding me through this process.
I would like to further recognize all the collaborators in this quest of discovery, of whom I
mention Dr. Lee Lynd from Dartmouth College, Dr. Hermann Eberl from Guelph University and
Dr. Steven Liss from Queens University. You were integral to the publication of the work
presented here.
My deepest gratitude to all the good friends whom I had the pleasure to work with: Dr. Martina
Hausner, Dr. Otini Kroukamp (who was instrumental in the development of the CEMS devices
used in this study), Dr. Elanna Bester, Romeo Dumitrache, Maryam Goudarzi, Lindsay Jackson,
Evan and Patrick Ronan, David Jenkins and all the other members of the Wolfaardt, Allen and
Hausner labs.
Finally I thank my family and life-long friends for supporting me in every way possible.
This thesis is dedicated to my wife Anca Dumitrache for her patience and earnest
encouragements throughout this long academic journey.
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Table of Contents

Acknowledgments .......................................................................................................................... iv
Table of Contents ............................................................................................................................ v
List of Tables ................................................................................................................................. ix
List of Figures ................................................................................................................................. x
List of Appendices ...................................................................................................................... xvii
Chapter 1: Introduction ............................................................................................................. 1
1.1 Rationale of this thesis ........................................................................................................ 1
1.2 Hypothesis ........................................................................................................................... 2
1.3 Specific research objectives ................................................................................................ 3
1.4 Thesis Outline and Statement of Authorship ...................................................................... 4
1.4.1 Thesis Outline ......................................................................................................... 4
1.4.2 Statement of Authorship ......................................................................................... 7
Chapter 2: Literature review ..................................................................................................... 9
2.1 Sustainable biofuels from bacterial conversion of cellulosic materials .............................. 9
2.1.1 Advances in energy biotechnology and cellulosic feedstock production ............... 9
2.1.2 Microbial conversion of biomass through consolidated bioprocessing (CBP) ..... 11
2.2 The biofilm mode of bacterial existence ........................................................................... 14
2.2.1 Sessile and planktonic cell populations ................................................................ 14
2.2.2 Biofilm attachment: inert versus reactive substrates ............................................ 15
2.3 Cell-cellulose interactions in nature .................................................................................. 17
2.4 Tools for discovery and current state of cellulolytic biofilm research ............................. 19
2.5 Clostridium thermocellum as a model cellulolytic organisms .......................................... 23
2.6 The context of this study in the current literature ............................................................. 28
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Chapter 3: Form and function of Clostridium thermocellum biofilms ................................... 29
3.1 Abstract ............................................................................................................................. 29
3.2 Introduction ....................................................................................................................... 30
3.3 Materials and methods ...................................................................................................... 31
3.3.1 Bacterial strains, chemicals and culture media ..................................................... 31
3.3.2 Imaging of in-situ and in-vivo biofilms ................................................................ 32
3.3.3 Cell counts in effluent stream ............................................................................... 33
3.3.4 Continuous-flow reactors for carbon mass balance analysis ................................ 34
3.3.5 Solid-phase carbon ................................................................................................ 34
3.3.6 Gas-phase carbon .................................................................................................. 35
3.3.7 Liquid-phase carbon .............................................................................................. 36
3.3.8 Carbon balance calculations ................................................................................. 36
3.3.9 Statistical analysis ................................................................................................. 37
3.4 Results ............................................................................................................................... 37
3.4.1 Biofilm structural characterization ....................................................................... 37
3.4.2 Substrate colonization and cell detachment .......................................................... 39
3.4.3 Carbon flow in C. thermocellum biofilms ............................................................ 41
3.5 Discussion ......................................................................................................................... 43
3.5.1 Biofilm structure and growth ................................................................................ 43
3.5.2 Cell adhesion and detachment .............................................................................. 44
3.5.3 Sporulation ............................................................................................................ 45
3.5.4 Biofilm activity and carbon flow .......................................................................... 46
3.6 Conclusions ....................................................................................................................... 49
Chapter 4: Tracking the cellulolytic activity of Clostridium thermocellum biofilms ............ 50
4.1 Abstract ............................................................................................................................. 50
4.2 Introduction ....................................................................................................................... 51
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4.3 Materials and methods ...................................................................................................... 53
4.3.1 Bacterial strains, chemicals and culture media ..................................................... 53
4.3.2 Continuous-flow reactors and substrate preparation ............................................. 54
4.3.3 Imaging of biofilm substrate-penetration at different growth stages .................... 55
4.3.4 Cell counts in the continuous-flow reactor effluent stream .................................. 55
4.3.5 Online carbon dioxide measurements ................................................................... 56
4.3.6 Carbon dioxide data analysis ................................................................................ 57
4.3.7 Statistical analysis ................................................................................................. 57
4.4 Results ............................................................................................................................... 58
4.4.1 Microscopic structure of cellulosic sheets ............................................................ 58
4.4.2 Correlation of CO production with cellulose consumption ................................. 58
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4.4.3 Conversion limitation regions described by CO production profiles .................. 59
2
4.4.4 Biofilm invasion and cell yield during the identified cellulose conversion
regions ................................................................................................................... 61
4.4.5 Correlation between biofilm activity and the accessible cellulosic surface ......... 64
4.4.6 Comparison of substrate digestibility from analysis of CO data ......................... 65
2
4.5 Discussion ......................................................................................................................... 70
4.5.1 Tracking biofilm activity and surface colonization through CO production
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from substrate consumption .................................................................................. 70
4.5.2 Online CO measurements: a tool for comparative analysis of substrate
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digestibility and conversion kinetics ..................................................................... 72
4.6 Conclusions ....................................................................................................................... 74
Chapter 5: Mathematical modeling to validate online CO2 measurements as a metric for
cellulolytic biofilm activity in continuous-flow bioreactors .................................................... 75
5.1 Abstract ............................................................................................................................. 75
5.2 Introduction ....................................................................................................................... 75
5.3 Materials and methods ...................................................................................................... 77
5.3.1 Experimental work ................................................................................................ 77
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5.3.2 Computational methods ........................................................................................ 79
5.4 Theory and calculations .................................................................................................... 80
5.5 Results and Discussion ..................................................................................................... 89
5.5.1 Parameter identification ........................................................................................ 89
5.5.2 Alternative model variants tested .......................................................................... 96
5.6 Conclusions ....................................................................................................................... 99
Chapter 6: Significance, conclusions and future research recommendations ....................... 100
6.1 Significance for applied research methods ..................................................................... 100
6.2 Significance for the fundamental microbiology and ecology of cellulolytic biofilms ... 101
6.3 Significance for engineered microbial conversion strategies ......................................... 103
6.4 Final conclusions ............................................................................................................ 106
6.5 Future work recommendations ....................................................................................... 108
References ................................................................................................................................... 111
Appendix 1 – Nomenclature: symbols and units ........................................................................ 122
Appendix 2 - Abbreviations ........................................................................................................ 127
Appendix 3 - Additional information and data ........................................................................... 130

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List of Tables

Table 3-1: Characteristics of continuous-flow cultures of C. thermocellum biofilms operated at a
dilution rate of 2 h-1a ..................................................................................................................... 41
Table 3-2: Summary of carbon mass balance analysis for continuous-flow cultures of C.
thermocellum grown on cellulose filter papera.............................................................................. 42
Table 5-1: Parameters of the model (17)-(20) with population maintenance terms, as identified
by the Levenberg-Marquart algorithm using the R-package FME and function ModFit ............. 94

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List of Figures

Figure 2-1: Microbial cellulose conversion strategies. Evolution of biomass-processing schemes
featuring dedicated cellulase production towards a single-step consolidated bioprocessing.
Adapted from (Lynd et al., 2002). ................................................................................................ 12
Figure 2-2: The comparative cost of ethanol production by consolidated bioprocessing (CBP)
and by simultaneous saccharification and co-fermentation (SSCF) featuring dedicated cellulase
production. Adapted from (Lynd et al., 2005). Although price variations may have occurred for
both technologies, here we emphasize the motivation behind the current thesis study when the
project was conceived. .................................................................................................................. 13
Figure 2-3: Simplified diagram of the primary pathway of C. thermocellum carbohydrate
catabolism. Carbon dioxide production (X ) is equal to the average acetate (X ) and ethanol
CO2 A
(X ) molar yields. [X = (X + X )/2] ........................................................................................ 26
E CO2 A E
Figure 2-4: Schematic representation of the cellulosome organization and attachment to the C.
thermocellum cell surface. Adapted from (Bayer et al., 1998). (physical size estimated at less
than 10 nm) ................................................................................................................................... 27
Figure 3-1: (A to C) Confocal laser scanning micrographs of Clostridium thermocellum 27405
biofilms on cellulose cotton fibers show substrate colonization with various degrees of cell
density after approximately 48 h of incubation; before total substrate colonization occurred, the
density on the fibers decreased by imaging further from the inoculation region. (D) A monolayer
of cells was the defining characteristic of fresh and mature biofilms, while typical terminal
endospores attached at the nonsporulating end (red circles), cell division occurred at the interface
(white circles), and highly elongated sporangia (red arrows) and detaching cells (white arrows)
were frequently observed. Cells were stained with Syto 9; cellulose fibers were stained with
wheat germ agglutinin-TRITC. ..................................................................................................... 38
Figure 3-2: (A) Clostridium thermocellum biofilm on cotton fiber shown in the x-y plane of a
reconstituted volume render. (B) Optical sectioning along the cellulose in the x-y plane, and
x