Microbial processes influencing the attenuation and impacts of fuel ethanol releases

243 Pages·2013·3.5 MB·English

by  Jie Ma

Copyright
Jie Ma
December 3, 2013

Abstract
Microbial processes influencing the attenuation and impacts of ethanol blend
fuel releases
By
Jie Ma
Fuel releases that impact groundwater are a common occurrence, and the growing use of
ethanol as a transportation biofuel is increasing the likelihood of encountering ethanol in such
releases. Therefore, it is important to understand how such releases behave and affect public
safety and environmental health, and how indigenous microorganisms respond and affect their
migration, fate, and overall impacts.
Vapor intrusion risk (i.e., methane explosion and enhanced fuel hydrocarbon vapor
intrusion) associated with ethanol blend releases is a potential concern. Using both experimental
measurements and mathematical model simulations, this thesis shows that methane is unlikely to
build up to pose an explosion hazard (5% v:v) if diffusion is the only mass transport pathway
through the unsaturated zone. However, if methanogenic activity near the source zone is
sufficiently high to cause advective gas transport, the methane indoor concentration may exceed
the flammable threshold. As a group of widely distributed microorganisms, methanotrophs can
significantly attenuate methane migration through the vadose zone, and thus alleviate the
associated explosion risk. However, methane biodegradation could consume soil oxygen that
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would otherwise be available to support biodegradation of volatile hydrocarbons, and increase
their vapor intrusion potential.
The release of an ethanol blend solution (10 % v:v ethanol solution mixed with 50 mg/L
benzene and 50 mg/L toluene) experiment into a pilot-scale (8 m3) aquifer tank produced a large
amount of volatile fatty acids (VFAs). The accumulation of VFAs (particularly butyric acid)
exceeded the secondary maximum contaminant level value for odor, which represents a
previously unreported aesthetic impact. After the release was shut off, ethanol anaerobic
degradation was temporarily stimulated when the dissolved ethanol concentration decreased
below its toxicity threshold (~2,000 mg/L for this system). Methane generation persisted for
more than 100 days after the disappearance of dissolved ethanol. The persistent methane was
likely generated from ethanol degradation byproducts (e.g., acetate) and solid organic carbon in
aquifer materials. Ethanol blend releases stimulate the microbial growth and increased the
organic carbon content in the aquifer.
Microorganisms play a critical role in the fate of ethanol-blended fuel releases, often
determining their region of influence and potential impacts. This thesis used advanced molecular
tools including 454 pyrosequencing and real-time PCR (qPCR) to characterize changes in
structure of indigenous microbial communities in response to 1) a pilot-scale ethanol blend
release and to 2) the shut-off of such release. This thesis shows that the ethanol blend release
stimulated microbial growth and significantly changed the microbial community structure by
enriching microbial groups involved in the fermentative degradation process. The growth of
putative hydrocarbon degraders and commensal anaerobes, and increases in degradation rates
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suggest an adaptive response that increases the potential for natural attenuation of ethanol blend
releases. After the release was shut off, the microbial community returned towards the pre-
contaminated state; however, restoration was relatively slow and far from complete even one
year later.
Overall, this thesis advanced current understanding of vapor intrusion risks and
groundwater quality impacts associated ethanol blend releases and microbial ecology in the
impacted aquifer. The integration of this knowledge with site-specific information on pertinent
hydrogeological processes will undoubtedly enhance engineering practices such as site
investigation, risk assessment, and bioremediation implementation and maintenance to deal with
releases of current and future biofuel blends.
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Acknowledgements
I would like to thank my advisor Dr. Pedro Alvarez for his unwavering support,
unconditional trust and outstanding example of how to become a good scientist. I wish to thank
Dr. Bill Rixey for his long-term collaborations and constant help in this study. My thanks also go
to other members of my dissertation committee, Dr. Qilin Li and Dr. George Bennett for their
valuable inputs and suggestions on the development of this dissertation. My research
collaborators Yi Zhang, Dr. Carlos W. Nossa, Dr. George DeVaull, Dr. Hong (Emma) Luo and
Dr. Brent Stafford provide a lot of time and effort on this study. I wish to thank Dr. Qiyou Jiang
for his help with the shared computing facilities at Rice University and Dr. .Jan Hewitt for her
help on the dissertation revisions. My office mate Dr. Zongming Xiu gave me a lot of guidance
in doing research and beyond. I am also grateful to all other labmates and colleagues who helped
make my time at Rice. Most importantly, I wish to thank my parents, who have always supported
and loved me.
This work was funded by the American Petroleum Institute. I also received a stipend
from the China Scholarship Council.

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Based on this research, the following papers are published or submitted or in preparation:

Ma, J., Xiu, Z., Monier, A., Mamonkina, I., Zhang, Y., He, Y., Stafford, B., Rixey, W. and
Alvarez, P. (2011) Aesthetic Groundwater Quality Impacts from a Continuous Pilot-Scale
Release of an Ethanol Blend. Ground Water Monitoring & Remediation 31(3), 47–54.
Ma, J.,Rixey, W.G., DeVaull, G.E., Stafford, B.P. and Alvarez, P.J.J. (2012) Methane
bioattenuation and implications for explosion risk reduction along the groundwater to soil
surface pathway above a plume of dissolved ethanol. Environmental Science &
Technology 46(11), 6013–6019.
Ma, J., Rixey, W.G., and Alvarez, P.J.J. Microbial processes influencing the transport, fate
and groundwater impacts of fuel ethanol releases. Current Opinion in Biotechnology
24(3): 457-466.
Ma, J., Nossa, C.W., Xiu, Z., Rixey, W.G. and Alvarez, P.J.J. Adaptive changes in microbial
community structure in response to a continuous pilot-scale release of an ethanol blend.
Environmental Pollution 178(0): 419-425.
Ma, J., Luo, H., DeVaull, G.E., Rixey, W.G.,Alvarez, P.J.J.A numerical model investigation
for potential methane explosion and benzene vapor intrusion associated with high-ethanol
blend releases. Environmental Science & Technology (minor revision)
Ma, J., et al., Response to the shut-off of a pilot-scale ethanol blended release: increased
ethanol degradation activities and persistent methanogenesis. (in preparation)
Ma, J., et al., Pyrosequencing-based investigation for microbial response to a 2-years ethanol
blended release and the shut-off of such release. (in preparation)

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Table of Contents
Abstract ..................................................................................................................................... i
Acknowledgements ...................................................................................................................... iv
List of Figures ............................................................................................................................... xi
List of Tables ............................................................................................................................... xv
Chapter 1 Introduction................................................................................................................... 1
1.1 Problem statement ................................................................................................................. 1
1.2 Objectives and hypothesis ..................................................................................................... 4
1.3 Dissertation outline ............................................................................................................... 6
1.4 Significance and potential benefits of this study ................................................................... 9
Chapter 2 Literature Review ....................................................................................................... 10
2.1 Physical behavior of ethanol-blended fuel releases ............................................................ 10
2.2 Biodegradation of ethanol-blended fuel .............................................................................. 12
2.3 How would ethanol affect BTEX degradation? .................................................................. 14
2.3.1 Gene expression ............................................................................................................ 16
2.3.2 Metabolic flux dilution ................................................................................................. 17
2.3.3 Thermodynamic inhibition ........................................................................................... 17
2.3.4 Cell physiology ............................................................................................................. 18
2.3.5 Community structure .................................................................................................... 19
2.3.6 Overall effect of ethanol on BTEX plume dynamics ................................................... 22
2.4 Existing knowledge gaps ..................................................................................................... 25
2.4.1 Methane explosion risk ................................................................................................. 25
2.4.2 Enhanced potential for benzene vapor intrusion .......................................................... 26
2.4.3 VFAs accumulation ...................................................................................................... 26
2.4.4 Microbial ecology ......................................................................................................... 26
Chapter 3 Methodology ............................................................................................................... 28
3.1 Pilot-scale aquifer tank ........................................................................................................ 28
3.2 Releases experiment ............................................................................................................ 34
3.3 Chemical analytical methods .............................................................................................. 35
3.3.1 Methane ........................................................................................................................ 36
3.3.2 Ethanol degradation byproducts ................................................................................... 37
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3.3.3 Ethanol .......................................................................................................................... 38
3.3.4 Benzene and toluene ..................................................................................................... 38
3.3.5 Bromide tracer .............................................................................................................. 38
3.4 Groundwater geochemical parameters monitoring ............................................................. 39
3.5 Quantitative real-time PCR analysis ................................................................................... 39
3.6 Pyrosequencing ................................................................................................................... 41
3.7 Geochip ............................................................................................................................... 45
3.7 Biovapor 1-D analytic vapor intrusion model ..................................................................... 47
3.8 Abreu and Johson 3-D numerical vapor intrusion model ................................................... 49
Chapter 4 Experimental and 1-D analytic model investigation on CH4 explosion and benzene
vapor intrusion .......................................................................................................... 51
4.1 Introduction ......................................................................................................................... 51
4.2 Materials and Methods ........................................................................................................ 53
4.2.1 Pilot-scale aquifer system ............................................................................................. 53
4.2.2 Flux chamber description ............................................................................................. 54
4.2.3 Sampling and analysis methods for CH and O .......................................................... 55
4 2
4.2.4 Assessment of CH oxidation activity at different depths ............................................ 56
4
4.2.5 qPCR assays for pmoA gene ......................................................................................... 56
4.2.6 Biovapor model simulation .......................................................................................... 58
4.3 Results and Discussion ........................................................................................................ 61
4.3.1 CH accumulation in the flux chamber......................................................................... 61
4
4.3.2 Aerobic biodegradation of CH in the pilot-scale aquifer ............................................ 63
4
4.3.3 CH accumulation simulation ....................................................................................... 67
4
4.3.4 Impacts of CH oxidation on benzene vapor intrusion ................................................. 69
4
4.4 Conclusion ........................................................................................................................... 71
Chapter 5 A 3-D numerical model investigation for methane explosion and benzene vapor
intrusion potential associated with ethanol-blended fuel releases ........................... 73
5.1 Introduction ......................................................................................................................... 73
5.2 Materials and Methods ........................................................................................................ 76
5.2.1 3-D numerical model .................................................................................................... 76
5.2.2 Simulated scenarios and model input parameters......................................................... 77
5.2.3 Assumptions and limitations of this modeling study.................................................... 84
5.3 Results and Discussion ........................................................................................................ 86
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5.3.1 The explosion risk for diffusion-driven CH migration is negligible ........................... 86
4
5.3.2 The explosion risk increases significantly for advection-driven CH migration .......... 89
4
5.3.3 Oxygen consumption during CH4 biodegradation in the vadose zone increases
benzene vapor intrusion potential .......................................................................................... 95
5.3.4 Implications for site assessment and remedial action ................................................. 103
Chapter 6 Seasonal variation of ethanol fermentative degradation and aesthetic impact of
volatile fatty acids generation ................................................................................ 106
6. 1 Introduction ...................................................................................................................... 106
6.2 Materials and Methods ...................................................................................................... 107
6.2.1 Pilot-scale aquifer system ........................................................................................... 107
6.2.2 CH and VFAs analysis .............................................................................................. 108
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6.3 Results and Discussion ...................................................................................................... 109
6.3.1 Effect of groundwater temperature on VFAs production ........................................... 109
6.3.2 VFAs odor generation ................................................................................................ 111
6.3.3 Effect of temperature on CH production ................................................................... 115
4
6.3.4 Ethanol, benzene, and toluene attenuation ................................................................. 117
6.4 Conclusion ......................................................................................................................... 118
Chapter 7 Response to the shut-off of a pilot-scale ethanol blended release: increased ethanol
degradation activities and persistent methanogenesis ............................................ 119
7. 1 Introduction ...................................................................................................................... 119
7.2 Materials and Methods ...................................................................................................... 121
7.2.1 Pilot-scale aquifer system ........................................................................................... 121
7.2.2 Chemical analysis methods ........................................................................................ 122
7.2.3 Microcosm experiment ............................................................................................... 122
7.2.4 Sand organic carbon content ....................................................................................... 123
7.2.5 Sand sample collection and DNA extraction .............................................................. 123
7.2.6 qPCR analysis ............................................................................................................. 123
7.2.7 Microarray .................................................................................................................. 124
7.2.8 Microarray Data processing ........................................................................................ 125
7.3 Results ............................................................................................................................... 126
7.3.1 Microcosm experiments for ethanol toxicity .............................................................. 126
7.3.2 Higher ethanol degradation activity following source removal ................................. 129
7.3.3 Persistence of dissolved methane and methane metabolism genes ............................ 131
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