THE ANAEROBIC MICROBIAL METABOLISM OF ENERGY SUBSTRATES: AN EXPLORATION INTO COALBED METHANOGENESIS, THE BIODETERIORATION OF DIESEL FUEL, AND BIOCORROSION

Under anaerobic conditions the complete mineralization of energy substrate can be achieved by specialized individual microorganisms or through syntrophic partnerships involving bacteria and archaea that ultimately convert parent compounds to methane and carbon dioxide. Such biotransformations generally result in the production of several fatty acids (i.e. acetate, propionate, butyrate, benzoate) that are particularly diagnostic for in situ microbial activity as well as postulated intermediates for anaerobic hydrocarbon degradation. The metabolic fate of these organic acid intermediates has been the subject of multiple investigations. However, what seems clear is that fatty acid-oxidizing bacteria generally catalyze the subsequent conversion of these intermediate compounds under a thermodynamically-based microbial syntrophism in co-culture with hydrogen/formate-utilizing microorganisms. Thus, C1-C5 volatile fatty acid (VFA) compounds are used throughout these chapters to represent metabolic intermediates of coal or hydrocarbon degradation. The ecological consequences of these anaerobic bioconversions are highly dependent on the prevailing geochemical conditions as well as the interrelationships between microorganisms and carbon sources. This dissertation focuses on the degradation of hydrocarbons or proposed fatty acid intermediates either relating to the production of coal bed methane (CBM) or the biodeterioration of fuels and how the latter subsequently impacts the biocorrosion of carbon steel. Coal is extremely difficult to chemically characterize; the organic fraction varies based on the starting plant material, the conditions of decomposition, and the physical and chemical changes that occur during the process of coalification. Thus, coal does not make for amenable methanogenic substrate, and requires a diverse microbial assemblage along with a thermodynamically based microbial syntrophism for the bioconversion of coal to methane. Considering the vast worldwide reserves of coal, the ever-expanding need for energy across the globe, and the environmental benefits of methane utilization as an energy source, there is considerable interest in stimulating the modern bioconversion of coal to methane. It has been previously proposed that ecological factors such as substrate bioavailability, coal recalcitrance, the absence of the requisite microorganisms, and chemical/nutrient limitations might contribute to the inhibition of biogenic CBM production. To assess coalfield methanogenesis, formation water from 17 sites collected from the Illinois and Powder River Basins as well as the Cook Inlet gas field was used as inocula for nutrient-replete incubations amended with C1-C5 fatty acids. Methanogenic rates for these incubations were extremely slow with long lag times and expected stoichiometric values of methane were typically not produced. Additionally, a functional gene microarray indicated that the genetic potential associated with a variety of microbial functions was present in all samples. Out of the three coalfields, the Cook Inlet incubations produced the most methane, particularly when amended with butyrate or valerate, a result that correlated with the number of unique mcr gene sequences and is consistent with the in situ detection of C4-C5 alkanoic acids. Collectively, these results suggested that the soft lignite coal in the Cook Inlet is easier to degrade than the sub-bituminous and bituminous coals of the Illinois and Powder River Basins. The degradation of the lignite coal in turn leads to the production of intermediate polar organic compounds (i.e. butanoic, pentanoic acids) within production waters that are then syntrophically converted to methane. These findings highlight the role of syntrophy in CBM production, and we concluded that coal methanogenesis is probably not limited by the inherent lack of metabolic potential, the presence of alternate electron acceptors, or the lack of available nutrients, but more likely restricted by the recalcitrant nature of the coal itself. Stringent regulations have been imposed worldwide mandating that diesel fuels contain ≤15 ppm sulfur; as a consequence ultra-low sulfur diesel (ULSD) has been fully integrated into the worldwide infrastructure to reduce chemical and particulate emissions. The process of desulfurization results in several changes to the physical properties of diesel fuel, which might differentially impact the biodeterioration of ULSD, compared to traditional diesel fuel formulations. We hypothesized that the removal of potentially inhibitory organosulfur compounds could alleviate the negative impact of these substances on the microbes responsible for diesel fuel biodegradation and that the intense process of desulfurization leads to the production of residual lower-molecular weight by products, which might increase the proliferation of problematic microbes relative to the fuel hydrocarbons. To test these hypotheses, an inoculum from a seawater-compensated ballast tank, along with two other known hydrocarbon- degrading inocula, were amended with fuel from the same ship or with refinery fractions of ULSD, low- (LSD), and high sulfur diesel (HSD) and monitored for sulfate depletion. The rates of sulfate removal in incubations amended with the refinery fuels were elevated relative to the fuel-unamended controls, but indistinguishable from one another. Thus, anaerobic hydrocarbon metabolism likely occurred in these incubations regardless of fuel sulfur content. The microbial community structure from each incubation was also largely independent of the fuel amendment type, based on molecular analysis of 16S rRNA gene sequences. Our results suggest that removal of organosulfur compounds and the production of easily amenable low molecular weight hydrocarbons via desulfurization do not significantly influence sulfate reduction rates or the structure of microbial communities. Thus, the process of desulfurization cannot explain the propensity of ULSD to decay faster than traditional diesel fuel formulations. The major implications of this work are that the biodegradation of diesel hydrocarbons or, by inference, the degree of biocorrosion is not influenced by the concentration of organosulfur species in the fuel. There is no doubt that the anaerobic degradation of hydrocarbons can be accomplished axenically by microbial pure cultures under a variety of electron accepting conditions as well as syntrophically by nutritionally diverse microbial consortia. However, the environmental consequences of anaerobic hydrocarbon transformation depend on the geochemical setting. Under sulfate-reducing conditions the production of sulfide can lead to health and safety concerns, reservoir souring, and metal biocorrosion; whereas the consequences under methanogenic conditions include the overall diminishment of petroleum quality and the production of methane, a greenhouse gas. Given that syntrophic bacteria are common in oil production facilities, we focused our efforts to understanding the impact of syntrophic hydrocarbon and fatty acid intermediates on the deleterious process of biocorrosion under both methanogenic and sulfate reducing conditions. Thus, defined microbial incubations using the hydrocarbon-degrading, sulfate-reducing bacterium Desulfoglaeba alkanexedens strain ALDC, was cultured with either sulfate or Methanospirillum hungatei strain JF-1 as electron acceptors and tested for the ability to corrode carbon steel coupons. The anaerobic biodegradation of hydrocarbons can also lead to the formation of a suite of fatty acid intermediates that can then be syntrophically metabolized, thus the corrosive potential of a model fatty acid-oxidizing bacteria S. aciditrophicus strain SB was assessed both in axenic culture, as well as syntrophically in co-culture with both Desulfovibrio sp. strain G11 and M. hungatei strain JF-1. Corrosion was measured using both electrochemical and profilometry techniques. Our results suggested that the corrosion of carbon steel decreased when D. alkanexedens strain ALDC was in co-culture with M. hungatei strain JF-1 and increased when S. aciditrophicus strain SB was co-cultured with Desulfovibrio sp. strain G11. The results highlight the role of acetate and sulfide production on the corrosion of carbon steel, and suggest that the metabolic interactions between syntrophs and hydrogen/formate utilizing microorganisms can substantially influence both instantaneous corrosion rates (1/Rp) and localized corrosion of the metal coupon. Despite these trends, corrosion was highly variable between replicate incubations. Differences in the amount of biomass, initial substrate concentrations, metabolic activity, end product production, and experimental conditions within the defined incubations did not account for this variation. Thus, the variability was ascribed to differences in the elemental composition of the metal coupons

Elucidation of the methanogenic potential from coalbed microbial communities amended with volatile fatty acids.

The potential for modern coalfield methanogenesis was assessed using formation water from the Illinois Basin, Powder River Basin and Cook Inlet gas field as inocula for nutrient-replete incubations amended with C1-C5 fatty acids as presumed intermediates formed during anaerobic coal biodegradation. Instead of the expected rapid mineralization of these substrates, methanogenesis was inordinately slow (∼1 μmol day-1), following long lag periods (>100 days), and methane yields typically did not reach stoichiometrically expected levels. However, a gene microarray confirmed the potential for a wide variety of microbiological functions, including methanogenesis, at all sites. The Cook Inlet incubations produced methane at a relatively rapid rate when amended with butyrate (r = 0.98; p = 0.001) or valerate (r = 0.84; p = 0.04), a result that significantly correlated with the number of positive mcr gene sequence probes from the functional gene microarray and was consistent with the in situ detection of C4-C5 alkanoic acids. This finding highlighted the role of syntrophy for the biodegradation of the softer lignite and subbituminous coal in this formation, but methanogenesis from the harder subbituminous and bituminous coals in the other fields was less apparent. We conclude that coal methanogenesis is probably not limited by the inherent lack of metabolic potential, the presence of alternate electron acceptors or the lack of available nutrients, but more likely restricted by the inherent recalcitrance of the coal itself

Municipal Solid Waste Landfills Harbor Distinct Microbiomes

Landfills are the final repository for most of the discarded material from human society and its built environments. Microorganisms subsequently degrade this discarded material in the landfill, releasing gases (largely CH4 and CO2) and a complex mixture of soluble chemical compounds in leachate. Characterization of landfill microbiomes and their comparison across several landfills should allow the identification of environmental or operational properties that influence the composition of these microbiomes and potentially their biodegradation capabilities. To this end, the composition of landfill microbiomes was characterized as part of an ongoing USGS national survey studying the chemical composition of leachates from 19 non-hazardous landfills across 16 states in the continental U.S. The landfills varied in parameters such as size, waste composition, management strategy, geography, and climate zone. The diversity and composition of bacterial and archaeal populations in leachate samples were characterized by 16S rRNA gene sequence analysis, and compared against a variety of physical and chemical parameters in an attempt to identify their impact on selection. Members of the Epsilonproteobacteria, Gammaproteobacteria, Clostridia, and candidate division OP3 were the most abundant. The distribution of the observed phylogenetic diversity could best be explained by a combination of variables and was correlated most strongly with the concentrations of chloride and barium, rate of evapotranspiration, age of waste, and the number of detected household chemicals. This study illustrates how leachate microbiomes are distinct from those of other natural or built environments, and sheds light on the major selective forces responsible for this microbial diversity

Impact of Organosulfur Content on Diesel Fuel Stability and Implications for Carbon Steel Corrosion

Ultralow sulfur diesel (ULSD) fuel has been integrated into the worldwide fuel infrastructure to help meet a variety of environmental regulations. However, desulfurization alters the properties of diesel fuel in ways that could potentially impact its biological stability. Fuel desulfurization might predispose ULSD to biodeterioration relative to sulfur-rich fuels and in marine systems accelerate rates of sulfate reduction, sulfide production, and carbon steel biocorrosion. To test such prospects, an inoculum from a seawater-compensated ballast tank was amended with fuel from the same ship or with refinery fractions of ULSD, low- (LSD), and high sulfur diesel (HSD) and monitored for sulfate depletion. The rates of sulfate removal in incubations amended with the refinery fuels were elevated relative to the fuel-unamended controls but statistically indistinguishable (∼50 μM SO₄/day), but they were found to be roughly twice as fast (∼100 μM SO₄/day) when the ship’s own diesel was used as a source of carbon and energy. Thus, anaerobic hydrocarbon metabolism likely occurred in these incubations regardless of fuel sulfur content. Microbial community structure from each incubation was also largely independent of the fuel amendment type, based on molecular analysis of 16S rRNA sequences. Two other inocula known to catalyze anaerobic hydrocarbon metabolism showed no differences in fuel-associated sulfate reduction or methanogenesis rates between ULSD, LSD, and HSD. These findings suggest that the stability of diesel is independent of the fuel organosulfur compound status and reasons for the accelerated biocorrosion associated with the use of ULSD should be sought elsewhere