sequential co-culture of anaerobic bacteria on switchgrass in a continuous flow-through reactor for

114 Pages·2017·5.21 MB·English

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Theses and Dissertations--Biosystems and
Biosystems and Agricultural Engineering
Agricultural Engineering
2014
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Noelia M. Elia
University of Kentucky, [email protected]
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Elia, Noelia M., "SEQUENTIAL CO-CULTURE OF ANAEROBIC BACTERIA ON SWITCHGRASS IN A
CONTINUOUS FLOW-THROUGH REACTOR FOR BIOFUEL PRODUCTION" (2014). Theses and
Dissertations--Biosystems and Agricultural Engineering. 22.
https://uknowledge.uky.edu/bae_etds/22
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Noelia M. Elia, Student
Dr. Sue E. Nokes, Major Professor
Dr. Donald Colliver, Director of Graduate Studies
SEQUENTIAL CO-CULTURE OF ANAEROBIC BACTERIA ON
SWITCHGRASS IN A CONTINUOUS FLOW-THROUGH REACTOR FOR
BIOFUEL PRODUCTION

THESIS

A thesis submitted in partial fulfillment of the requirements for the degree of Master of
Science in Biosystems and Agricultural Engineering the College of Engineering at the
University of Kentucky

By

Noelia M Elia

Lexington, Kentucky

Director: Dr. Sue E. Nokes, Professor of Biosystems and Agricultural Engineering

Lexington, Kentucky

2014

Copyright © Noelia M Elia 2014
ABSTRACT OF THESIS

SEQUENTIAL CO-CULTURE OF ANAEROBIC BACTERIA ON SWITCHGRASS IN
A CONTINUOUS FLOW-THROUGH REACTOR FOR BIOFUEL PRODUCTION

Solid substrate cultivation (SSC) using lignocellulosic non-food feedstock, such
as switchgrass, is an alternative for advanced biofuel production. Acetone-Butanol-
Ethanol (ABE) fermentation in two stages using a sequential culture of microorganisms
from the class Clostridia is an approach proposed to increase the butanol production.
The goal was to test the efficacy of a sequential culture on high solid substrate
cultivation in batch and continuous cultivation, and to evaluate conditions to optimize
butanol production using switchgrass as substrate.
Initial batch experiments were used to determine particle size effect, choice of
solvent producer and pretreatment evaluation: The effect of particle size on gas
production was surface area-dependent, 2 mm particle size of switchgrass was better
fermented by clostridia than the other particle sizes. C. thermocellum improved
switchgrass fermentation by C. beijerinckii. Moreover, C. saccharoperbutylacetonicum
produced the highest butanol yield on glucose as substrate. The Fenton reaction was
studied as a potential pretreatment for switchgrass. C. beijerinckii grew better on Fenton-
treated material, but solvent production was low.
The major conclusion of the continuous flow on SSC experiment was that there is
no statistical difference in the effect of flow rate within the flow range tested.

KEYWORDS: Switchgrass, Clostridium co-culture, particle size, Fenton pretreatment,
continuous fermentation

Noelia M Elia

January 17, 2014
SEQUENTIAL CO-CULTURE OF ANAEROBIC BACTERIA ON SWITCHGRASS IN
A CONTINUOUS FLOW-THROUGH REACTOR FOR BIOFUEL PRODUCTION.

By

Noelia M Elia

Sue E. Nokes

Donald Colliver

January 17, 2014
ACKNOWLEDGEMENTS

I would like to thank the many people who have helped me to not only to feel
comfortable in a culture different than mine but also for the encouragement to be part of
the “green generation” and to help the world being more sustainable.

I would like to thank Dr. Michael Flythe, who has provided me with tremendous
opportunities that have challenged me to learn more about the fantastic microbiological
world. Also, I am glad to have his professional point of view to guide me through my
Master’s degree.

Next, I would like to thank my advisor Dr. Sue Nokes who gave me the chance to
participate in this project and provide all the tools to be a leadership professional in the
area.

I would like to thank Gloria Gellin and the other graduate students for all of their
kindness and help in the lab.

Also, I would like to thank my committee members, Dr. Flythe and Dr. Nokes (whom I
already mentioned) and Dr. Montross for their review of my thesis and great suggestions
and questions that encouraged me to look at science from many perspectives.

I would like to thank Juan for his love and encouragement and help in all sense of the
word; my family for their love and continued support of my decision to pursue this
degree and my friends for be always there.

Finally, I appreciate of all people that crossed my path and made my Master’s degree
years an experience to never forget.

iii
TABLE OF CONTENTS
Acknowledgements……………………………………………………………………..iii
List of Tables………………………………………………….……….………….…......vi
List of Figures…………………………………………....................................................vii

1. Chapter One: Review of Literature………………………………...………..………1
Introduction………………………………………………………………...……………...1
Biofuels……………………………………………………………………...…………….2
Biorefineries…………………………………………………………………...…………..4
Biomass as feedstock: lignocellulose structure….……………………………...………....5
Biomass as feedstock: biomass to biofuels………………………………………...……...6
Switchgrass as a dedicated energy crop…………………………………………….…....10
Microbiology of biofuel production………………………………………………….......11

2. Chapter Two: ABE Fermentation in Batch System……………………………….14
2.1 Introduction…………………………………………………………………………..14
2.2 Objectives………………………………………………………………………...….21
2.3 Materials and Methods…………………………………………………………….....22
Strains and Culture conditions………………………………………….……………….22
Biomass preparation……………………………………………………………………..23
Short term fermentation: solvent producer strains on glucose and switchgrass…...........24
Effect of the particle size: Gas production………………………………………............25
Effect of the particle size: product formation……………………………………….…..25
2.4 Results………………………………………………………………………………..26
Short term fermentation: solvent producer strains on glucose and switchgrass…….…..26
Effect of the particle size: Gas production……………………………………………….30
Effect of the particle size: product formation……………………………………………30
2.5 Discussion…………………………………………………………………………....33

3. Chapter Three: Fenton reaction as a pretreatment on Switchgrass……………...36
3.1 Introduction…………………………………………………………………………..36
3.2 Objectives………………………………………………………….……………...…37
3.3 Materials and Methods……………………………………………………………….37
Strains and Culture conditions……………………………………….………………….37
Short term fermentation………………………………………………………………….37
Fenton-treated vs untreated switchgrass: Gas production……………..………………..37
Fenton-treated vs untreated switchgrass: Survival experiment (Viable cell number)…..38
Fenton-treated vs untreated switchgrass: Product quantification of C. thermocellum….39
Toxicity of C. thermocellum inoculum…………………………………………………...39
3.4 Results………………………………………………………………………………..41
iv
Short term fermentation……………………………..…………………………………...41
Fenton-treated vs untreated switchgrass: Gas production ……...……………………….41
Fenton-treated vs untreated switchgrass: Survival experiment (Viable cell number)…...44
Fenton-treated vs untreated switchgrass: Product quantification of C. thermocellum….46
Toxicity of C. thermocellum inoculum………………….………………………………..47
3.5 Discussion……………………………………………………………………………50

4. Chapter Four: ABE Fermentation in a Solid Substrate Cultivation with
Continuous Product Removal………………………………………………………….52
4.1 Introduction……………………………………………………………………….….52
4.2 Objectives……………………………………………………………………………54
4.3 Materials and Methods……………………………………………………………….55
Strains and Culture Conditions.…………………………………………………………55
Preliminary Batch Fermentation..……………………………………………………….55
Continuous Fermentation………………………………………………………………..57
4.4 Results………………………………………………………………………………..60
Preliminary Batch Fermentation………………………………………………………...60
Continuous Fermentation………………………………………………………………..61
4.5 Discussion……………………………………………………………………………66

5. Chapter Five: Future work………………………………………………………….70

Appendices………………………………………………………………………………71

References……………………………………………………………………………. ...91

Vita……………………………………………………………………………………..101

v
LIST OF TABLES
Table 1.1. Properties of different fuels……………………………………………………4
Table 1.2. Energy consumption comparison between hammer mill and knife mill………8
Table 2.1. Analysis results of switchgrass by “DairyOne” Forage Testing Laboratory...23
Table 2.4.1. Products formation by solventogenic strains on RCM…………………….27
Table 2.4.2. Product formation by solventogenic strains on switchgrass ……………....28
Table 2.4.3. Product formation by C. beijerinckii on different particle size of switchgrass
during 10 days……………………………………………………………………………32
Table 2.4.4. Product formation by the sequential culture of C. thermocellum followed by
C. beijerinckii on different particle sizes of switchgrass during 10 days………………..32
Table 3.4.1 .pH of cultures of C. beijerinckii grown on different concentrations of C.
thermocellum………………………………………………………………………….….49
Table 4.4.1. Chemical characterization of switchgrass by Forage Testing Laboratory…65
Table 4.4.2. . Lignocellulose composition of switchgrass………………………………65

vi
LIST OF FIGURES
Figure 1.1.Schematic of lignocellulose composition………………………………….....7
Figure 2.1.1.Simplified figure of glycolysis leading acetone, butanol and ethanol
production from Clostridium species…………………………………………………….15
Figure 2.1.2.Simplified figure of metabolic pathway of C. thermocellum. …………….16
Figure 2.1.3.Schematic representation of the cellulosome from C. thermocellum. …….17
Figure 2.1.4. Gas production from switchgrass by Clostridium thermocellum...……….18
Figure 2.2.1. Experimental design to test different solvent producer on glucose and
switchgrass as substrate…………………………………………………………….……24
Figure 2.4.1. Total Product formation by solventogenic strains on RCM (a) and
switchgrass (b)……………………………………………………………………….…..29
Figure 2.4.2. Butanol production along time by solventogenic strains.………….……..30
Figure 2.4.3. Gas production by C. beijerinckii on switchgrass………………….….….31
Figure 2.4.4. Gas production by sequential culture on switchgrass..…………….….…..31
Figure 3.3.1. Experimental design to count the viable cell number …………………….40
Figure 3.3.2. Experimental design to evaluate the toxicity of the inoculum of C.
thermocellum on C. beijerinckii………………………………………………………….41
Figure 3.4.1. Gas production by the sequential culture of C. thermocellum followed by C.
beijerinckii on untreated and Fenton-treated switchgrass....……………………………..42
Figure 3.4.2. Gas production by C. beijerinckii on untreated and Fenton-treated
switchgrass……………………………………………………………………………….43
Figure 3.4.3. Comparison of gas produced by C. beijerinckii 51743 on Fenton-treated
and untreated switchgrass………………………………………………………………..43
Figure 3.4.4. Viable cell number of C. beijerinckii grown on untreated switchgrass…...45
Figure 3.4.5. Viable cell number of C. beijerinckii grown on Fenton-treated
switchgrass……………………………………………………………………………….45
Figure 3.4.6. pH variation along the growth of C. thermocellum on Fenton-treated and
untreated switchgrass………………………………………………………………….…46
Figure 3.4.7. Product formation by C. thermocellum ATCC 27405 on switchgrass……47
Figure 3.4.8. Product formation by C. thermocellum ATCC 27405 on Fenton-treated
switchgrass…………………………………………………………………………….…47
Figure 3.4.9. ∆ Optical density (600nm) of cultures of C. beijerinckii ……………..….48
Figure 3.4.10. ∆ Optical density (600nm) of cultures of C. beijerinckii……………......49
Figure 4.3.1. Experimental design to count the viable cell number...…………………...57
Figure 4.3.2. Scheme of the Solid-substrate cultivation system with continuous product
removal…………………………………………………………………………………..59
Figure 4.4.1. Viable cell number of C. saccharoperbutylacetonicum grown on
switchgrass in sequential culture with C. thermocellum and pure culture of C.
saccharoperbutylacetonicum. ………………………………………………..………….61
vii