Biogeochemistry of Isoprenoid Production and Anaerobic Hydrocarbon Degradation

144 Pages·2011·6.65 MB·English

by  Kat Dawson

The Pennsylvania State University

The Graduate School

Department of Geosciences

BIOGEOCHEMISTRY OF ISOPRENOID PRODUCTION AND ANAEROBIC
HYDROCARBON BIODEGRADATION

A Dissertation in
Geosciences

by

Katherine S. Dawson

© 2011 Katherine S. Dawson

Submitted in Partial Fulfillment
of the Requirements
for the Degree of

Doctor of Philosophy

August 2011
The dissertation of Katherine S. Dawson was reviewed and approved* by the following:

Jennifer L. Macalady
Assistant Professor of Geosciences
Dissertation Co-Advisor
Co-Chair of Committee

Katherine H. Freeman
Professor of Geosciences
Dissertation Co-Advisor
Co-Chair of Committee

Christopher H. House
Associate Professor of Geosciences

John M. Regan
Associate Professor of Environmental Engineering

Chris J. Marone
Professor of Geosciences
Associate Head for Graduate Programs and Research in Geosciences

*Signatures are on file in the Graduate School
iii
ABSTRACT

This dissertation is an exploration of microbial isoprenoid production and
destruction by anaerobic hydrocarbon biodegradation. Isoprenoids are methyl-branched
hydrocarbons, and include biomarkers from all three domains of life such as archaeal
lipids, hopanoids, and sterols. Isoprenoid production was examined through variation in
the molecular structure of archaeal lipids across a hypersaline gradient (Chapter 5). This
study identified unsaturated analogues of archaeol in four halophilic archaeal strains and
revealed an increase in the percentage of unsaturated lipids with increasing salinity.
Anaerobic isoprenoid biodegradation was examined through the enrichment of bacteria
under anaerobic conditions utilizing pristane as a carbon source (Chapter 2). Further
analysis of anaerobic degradation utilized 13C-labelled phytane as a stable isotope tracer
(Chapter 3). In both cases, a microbial community dominated by denitrifying Beta- and
Gammaproteobacteria was responsible for the degradation of pristane and phytane.
Environmental anaerobic hydrocarbon degradation was examined through the analysis of
in situ microbial communities associated with the transformation of coal to methane
(Chapter 4). Through FISH and 16S rRNA tag pyrosequencing a coal transformation
pathway ending in acetoclastic/methylotrophic methanogenesis was identified in the
Cook Inlet Basin, Alaska. These studies demonstrate the microbial impact on
hydrocarbon production and alteration, which influences the transition over geologic time
scales from biomolecules to biomarkers in the sedimentary record.
iv
TABLE OF CONTENTS

LIST OF FIGURES vii
LIST OF TABLES x
ACKNOWLEDGEMENTS xi

CHAPTER 1: INTRODUCTION
1.1 Isoprenoids 1
1.2 Microbial Hydrocarbon Degradation 2
1.3 Archaeal Membrane Lipids 5
1.4 Biogenic Coal Bed Methane 6
1.5 Organization Of The Thesis 8
1.6 Anticipated Publications From This Work 8
1.7 Figures 10
1.8 References 19

CHAPTER 2: ANAEROBIC BIODEGRADATION OF ISOPRENOID
BIOMARKERS BY A DENITRIFYING MICROCOSM
2.1 Abstract 26
2.2 Introduction 26
2.3 Materials and Methods 28
2.4 Results 32
2.5 Discussion 36
v
2.6 Acknowledgements 39
2.7 Figures and Tables 40
2.8 References 47

CHAPTER 3: STABLE ISOTOPE TRACING OF ANAEROBIC ISOPRENOID
BIODEGRADATION WITH 13C LABELLED PHYTANE
3.1 Abstract 52
3.2 Introduction 52
3.3 Experimental Procedures 54
3.4 Results 59
3.5 Discussion 62
3.6 Conclusions 65
3.7 Acknowledgements 66
3.8 Figures and Tables 67
3.9 References 78

CHAPTER 4: QUANTITATIVE FISH ANALYSIS OF MICROBIAL
CONSORTIA FROM A BIOGENIC GAS FIELD IN THE COOK INLET
BASIN, ALASKA
4.1 Abstract 83
4.2 Introduction 83
4.3 Materials and Methods 85
4.4 Results 88
vi
4.5 Discussion 92
4.6 Conclusions 94
4.7 Figures and Table 96
4.8 References 103

CHAPTER 5: MOLECULAR CHARACTERIZATION OF ARCHAEAL LIPIDS
ACROSS A HYPERSALINE GRADIENT
5.1 Abstract 107
5.2 Introduction 107
5.3 Methods 109
5.4 Results 111
5.5 Discussion 113
5.6 Conclusions 115
5.7 Acknowledgements 116
5.8 Figures and Tables 117
5.9 References 123

CHAPTER 6: CONCULSIONS AND FUTURE DIRECTIONS
6.1 Anaerobic isoprenoid degradation 128
6.2 Coal bed methane biogeochemistry 129
6.3 Halophilic archaeal lipids 130
6.4 References 131
vii
LIST OF FIGURES
Figure 1-1: Examples of isoprenoid biopolymers that may serve as biomarkers 10
Figure 1-2: Proposed mechanisms for anaerobic hydrocarbon degradation 11
Figure 1-3: Membrane glycerolipid structures in the three domains of life 12
Figure 1-4: Structures of archaeal glycerol diphytanyl glycerol tetraethers (GDGT’s) and
diphytanyl glycerol diethers (DGD’s) 13
Figure 1-5: Isotopic values of methane showing the differences between thermogenic and
biogenic methane generated from different pathways 14
Figure 1-6: Phylogenetic tree of 16s rDNA sequences of Firmicutes, Bacteroidetes,
Planctomycetes, Spirochaetes, and other bacterial lineages associated with coal bed
methane 15
Figure 1-7: Phylogenetic tree of 16s rDNA sequences of Betaproteobacteria and
Gammaproteobacteria associated with CBM 16
Figure 1-8: Phylogenetic tree of 16s rDNA sequences of Alphaproteobacteria,
Deltaproteobacteria and Epsilonproteobacteria associated with CBM 17
Figure 1-9: Phylogenetic tree of 16s rDNA sequences of methanogenic archaea
associated with CBM 18
Figure 2-1: FISH micrographs of cells in the denitrifying, pristane-degrading enrichment
culture 40
Figure 2-2: Neighbor-joining phylogenetic tree showing betaproteobacteria from pristane
degrading enrichments 41
Figure 2-3: Neighbor-joining phylogenetic tree showing gammaproteobacteria from
pristane degrading enrichments 42
viii
Figure 2-4: Fraction of bacterial cells hybridizing to specific oligonucleotide probes in
pristane degrading cultures 43
Figure 2-5: Anaerobic consumption of nitrate, and the production of nitrite and
bicarbonate in a denitrifying, pristane degrading enrichment culture 44
Figure 2-6: Anaerobic consumption of nitrate in denitrifying enrichments with pristane,
DGD and GDGT core lipids 45
Figure 3-1: Preparation scheme for 13C-labeled phytane 67
Figure 3-2: Gas chromatogram and mass spectra of saponified 13C-labeled diphytanyl
glycerol diether from Haloferax sulfurifontis SD1 68
Figure 3-3: Gas chromatograms and mass spectra of 13C-phytane before and after SPE
purification 69
Figure 3-4: Loss of nitrate and production of nitrite and bicarbonate in incubation grown
with 13C-labeled phytane as a carbon source 70
Figure 3-5: Values of δ13C for the CO produced during incubations on 13C-labeled
2
phytane, unlabeled phytane and with no added carbon 71
Figure 3-6: Keeling plot of 13C for CO produced during incubation on 13C-labeled
2
phytane versus 1/[HCO -] 72
3
Figure 3-7: Percentage of cells hybridizing to specific oligonucleotide probes in phytane
degrading incubations 73
Figure 3-8: FISH micrographs of cells in the denitrifying, phytane-degrading enrichment
culture 74
Figure 3-9: Neighbor-joining phylogenetic tree showing gammaproteobacteria clones in
13C-phytane and pristane degrading enrichments 75
ix
Figure 3-9: Neighbor-joining phylogenetic tree showing betaproteobacteria clones in 13C-
labeled phytane and pristane degrading enrichments 76
Figure 4-1: FISH micrographs depicting major bacterial and archaeal lineages observed
in production water samples from the Cook Inlet Basin 97
Figure 4-2: Microbial communities of the Cook Inlet gas field expressed as a percentage
of DAPI stained cells 98
Figure 4-3: Comparison of the microbial communities of the Cook Inlet gas field
determined by FISH and 16S rRNA tag pyrosequencing 99
Figure 4-4: Carbon and hydrogen isotopic classification of methane of the Cook Inlet
sample sites 100
Figure 4-5: PCA of Cook Inlet basin microbial population data with geochemical data
projected onto the ordination using the ‘envfit’ function 101
Figure 4-6: PCA of microbial population data from five sedimentary basins with
geochemical data projected onto the ordination using the ‘envfit’ function 102
Figure 5-1: Structures of archaeal diphytanyl glycerol diether (DGD) with various chain
lengths, cyclization, unsaturation and hydroxyl substitutions 118
Figure 5-2: Total ion chromatograms of saturated C and C DGD 119
20-20 25-20
Figure 5-3: Partial GC-MS chromatograms of polar lipid extracts of halophilic archaea
showing C and C DGDs 120
20-20 25-20
Figure 5-4: Plot of total unsaturated archaeal C and C DGD lipids for four
20-20 25-20
halophilic archaeal strains 121
Figure 5-5: Average fraction of unsaturated DGDs versus optimal % NaCl (w/v) for four
halophilic archaeal strains 122
x
LIST OF TABLES
Table 2-1: Ratios of isoprenoid peak areas in GC-MS chromatograms 46
Table 2-2: Oligonucleotide probes used in the study of denitrifying, pristane degrading
enrichments 46
Table 3-1: Oligonucleotide probes used in the study of denitrifying, phytane degrading
incubations 77
Table 4-1: Oligonucleotide probes used in the study of coal bed methane production
water 96
Table 5-1: Presence of absence of C and C DGDs in halophilic archaea from
20-20 25-20
various genera 117