Impact of Photooxidation and Biodegradation on the Fate of Oil Spilled During the Deepwater Horizon Incident: Advanced Stages of Weathering.

While the biogeochemical forces influencing the weathering of spilled oil have been investigated for decades, the environmental fate and effects of "oxyhydrocarbons" in sand patties deposited on beaches are not well-known. We collected sand patties deposited in the swash zone on Gulf of Mexico beaches following the Deepwater Horizon oil spill. When sand patties were exposed to simulated sunlight, a larger concentration of dissolved organic carbon was leached into seawater than the corresponding dark controls. This result was consistent with the general ease of movement of seawater through the sand patties as shown with a 35SO42- radiotracer. Ultrahigh-resolution mass spectrometry, as well as optical measurements revealed that the chemical composition of dissolved organic matter (DOM) leached from the sand patties under dark and irradiated conditions were substantially different, but neither had a significant inhibitory influence on the endogenous rate of aerobic or anaerobic microbial respiratory activity. Rather, the dissolved organic photooxidation products stimulated significantly more microbial O2 consumption (113 ± 4 µM) than either the dark (78 ± 2 µM) controls or the endogenous (38 µM ± 4) forms of DOM. The changes in the DOM quality and quantity were consistent with biodegradation as an explanation for the differences. These results confirm that sand patties undergo a gradual dissolution of DOM in both the dark and in the light, but photooxidation accelerates the production of water-soluble polar organic compounds that are relatively more amenable to aerobic biodegradation. As such, these processes represent previously unrecognized advanced weathering stages that are important in the ultimate transformation of spilled crude oil.

Design features of offshore oil production platforms influence their susceptibility to biocorrosion.

Offshore oil-producing platforms are designed for efficient and cost-effective separation of oil from water. However, design features and operating practices may create conditions that promote the proliferation and spread of biocorrosive microorganisms. The microbial communities and their potential for metal corrosion were characterized for three oil production platforms that varied in their oil-water separation processes, fluid recycling practices, and history of microbially influenced corrosion (MIC). Microbial diversity was evaluated by 16S rRNA gene sequencing, and numbers of total bacteria, archaea, and sulfate-reducing bacteria (SRB) were estimated by qPCR. The rates of 35S sulfate reduction assay (SRA) were measured as a proxy for metal biocorrosion potential. A variety of microorganisms common to oil production facilities were found, but distinct communities were associated with the design of the platform and varied with different locations in the processing stream. Stagnant, lower temperature (<37 °C) sites in all platforms had more SRB and higher SRA compared to samples from sites with higher temperatures and flow rates. However, high (5 mmol L-1) levels of hydrogen sulfide and high numbers (107 mL-1) of SRB were found in only one platform. This platform alone contained large separation tanks with long retention times and recycled fluids from stagnant sites to the beginning of the oil separation train, thus promoting distribution of biocorrosive microorganisms. These findings tell us that tracking microbial sulfate-reducing activity and community composition on off-shore oil production platforms can be used to identify operational practices that inadvertently promote the proliferation, distribution, and activity of biocorrosive microorganisms.

Involvement of thermophilic archaea in the biocorrosion of oil pipelines.

Two thermophilic archaea, strain PK and strain MG, were isolated from a culture enriched at 80°C from the inner surface material of a hot oil pipeline. Strain PK could ferment complex organic nitrogen sources (e.g. yeast extract, peptone, tryptone) and was able to reduce elemental sulfur (S°), Fe(3+) and Mn(4+) . Phylogenetic analysis revealed that the organism belonged to the order Thermococcales. Incubations of this strain with elemental iron (Fe°) resulted in the abiotic formation of ferrous iron and the accumulation of volatile fatty acids during yeast extract fermentation. The other isolate, strain MG, was a H(2) :CO(2) -utilizing methanogen, phylogenetically affiliated with the genus Methanothermobacter family. Co-cultures of the strains grew as aggregates that produced CH(4) without exogenous H(2) amendment. The co-culture produced the same suite but greater concentrations of fatty acids from yeast extract than did strain PK alone. Thus, the physiological characteristics of organisms both alone and in combination could conceivably contribute to pipeline corrosion. The Thermococcus strain PK could reduce elemental sulfur to sulfide, produce fatty acids and reduce ferric iron. The hydrogenotrophic methanogen strain MG enhanced fatty acid production by fermentative organisms but could not couple the dissolution Fe° with the consumption of water-derived H(2) like other methanogens.

Microbial communities in bulk fluids and biofilms of an oil facility have similar composition but different structure.

The oil-water-gas environments of oil production facilities harbour abundant and diverse microbial communities that can participate in deleterious processes such as biocorrosion. Several molecular methods, including pyrosequencing of 16S rRNA libraries, were used to characterize the microbial communities from an oil production facility on the Alaskan North Slope. The communities in produced water and a sample from a 'pig envelope' were compared in order to identify specific populations or communities associated with biocorrosion. The 'pigs' are used for physical mitigation of pipeline corrosion and fouling and the samples are enriched in surface-associated solids (i.e. paraffins, minerals and biofilm) and coincidentally, microorganisms (over 10(5) -fold). Throughout the oil production facility, bacteria were more abundant (10- to 150-fold) than archaea, with thermophilic members of the phyla Firmicutes (Thermoanaerobacter and Thermacetogenium) and Synergistes (Thermovirga) dominating the community. However, the structure (relative abundances of taxa) of the microbial community in the pig envelope was distinct due to the increased relative abundances of the genera Thermacetogenium and Thermovirga. The data presented here suggest that bulk fluid is representative of the biofilm communities associated with biocorrosion but that certain populations are more abundant in biofilms, which should be the focus of monitoring and mitigation strategies.

Methanogenesis, sulfate reduction and crude oil biodegradation in hot Alaskan oilfields.

Petrochemical and geological evidence suggest that petroleum in most reservoirs is anaerobically biodegraded to some extent. However, the conditions for this metabolism and the cultivation of the requisite microorganisms are rarely established. Here, we report on microbial hydrocarbon metabolism in two distinct oilfields on the North Slope of Alaska (designated Fields A and B). Signature anaerobic hydrocarbon metabolites were detected in produced water from the two oilfields offering evidence of in situ biodegradation activity. Rate measurements revealed that sulfate reduction was an important electron accepting process in Field A (6-807 µmol S l(-1) day(-1)), but of lesser consequence in Field B (0.1-10 µmol S l(-1) day(-1)). Correspondingly, enrichments established at 55°C with a variety of hydrocarbon mixtures showed relatively high sulfate consumption but low methane production in Field A incubations, whereas the opposite was true of the Field B enrichments. Repeated transfer of a Field B enrichment showed ongoing methane production in the presence of crude oil that correlated with ≥ 50% depletion of several component hydrocarbons. Molecular-based microbial community analysis of the methanogenic oil-utilizing consortium revealed five bacterial taxa affiliating with the orders Thermotogales, Synergistales, Deferribacterales (two taxa) and Thermoanaerobacterales that have known fermentative or syntrophic capability and one methanogen that is most closely affiliated with uncultured clones in the H(2)-using family Methanobacteriaceae. The findings demonstrate that oilfield-associated microbial assemblages can metabolize crude oil under the thermophilic and anaerobic conditions prevalent in many petroleum reservoirs.

Field metabolomics and laboratory assessments of anaerobic intrinsic bioremediation of hydrocarbons at a petroleum-contaminated site.

Field metabolomics and laboratory assays were used to assess the in situ anaerobic attenuation of hydrocarbons in a contaminated aquifer underlying a former refinery. Benzene, ethylbenzene, 2-methylnaphthalene, 1,2,4- and 1,3,5-trimethylbenzene were targeted as contaminants of greatest regulatory concern (COC) whose intrinsic remediation has been previously reported. Metabolite profiles associated with anaerobic hydrocarbon decay revealed the microbial utilization of alkylbenzenes, including the trimethylbenzene COC, PAHs and several n-alkanes in the contaminated portions of the aquifer. Anaerobic biodegradation experiments designed to mimic in situ conditions showed no loss of exogenously amended COC; however, a substantive rate of endogenous electron acceptor reduction was measured (55 ± 8 µM SO(4) day(-1)). An assessment of hydrocarbon loss in laboratory experiments relative to a conserved internal marker revealed that non-COC hydrocarbons were being metabolized. Purge and trap analysis of laboratory assays showed a substantial loss of toluene, m- and o-xylene, as well as several alkanes (C(6)-C(12)). Multiple lines of evidence suggest that benzene is persistent under the prevailing site anaerobic conditions. We could find no in situ benzene intermediates (phenol or benzoate), the parent molecule proved recalcitrant in laboratory assays and low copy numbers of Desulfobacterium were found, a genus previously implicated in anaerobic benzene biodegradation. This study also showed that there was a reasonable correlation between field and laboratory findings, although with notable exception. Thus, while the intrinsic anaerobic bioremediation was clearly evident at the site, non-COC hydrocarbons were preferentially metabolized, even though there was ample literature precedence for the biodegradation of the target molecules.

Desulfoglaeba alkanexedens gen. nov., sp. nov., an n-alkane-degrading, sulfate-reducing bacterium.

Two novel sulfate-reducing bacteria, strains ALDC(T) and Lake, which were able to oxidize n-alkanes, were isolated from a naval oily wastewater-storage facility (VA, USA) and from oilfield production water (OK, USA), respectively. The type strain (ALDC(T)) had a narrow substrate specificity and could grow only with n-alkanes (from C(6) to C(12)), pyruvate, butyrate, hexanoic acid and 4-methyloctanoic acid. Cells of strain ALDC(T) stained Gram-negative and were slightly curved, short rods with oval ends (2.5-3.0x1.0-1.4 microm), often occurring in pairs. Cells tended to form aggregates or large clusters and were non-motile and did not form endospores. Optimum growth occurred between 31 and 37 degrees C and at pH 6.5-7.2. NaCl was not required for growth, but salt concentrations up to 55 g l(-1) could be tolerated. The DNA G+C content was 53.6 mol%. Phylogenetic analysis of the 16S rRNA genes revealed that strains ALDC(T) and Lake were closely related, but not identical (99.9 % similarity). The two strains were not closely related to other known alkane-degrading, sulfate-reducing bacteria or to other genera of the Deltaproteobacteria. Therefore, it is proposed that strain ALDC(T) (=JCM 13588(T)=ATCC BAA-1302(T)) represents the type strain of a novel species and genus, with the name Desulfoglaeba alkanexedens gen. nov., sp. nov.

Periplasmic cytochrome c3 of Desulfovibrio vulgaris is directly involved in H2-mediated metal but not sulfate reduction.

Kinetic parameters and the role of cytochrome c(3) in sulfate, Fe(III), and U(VI) reduction were investigated in Desulfovibrio vulgaris Hildenborough. While sulfate reduction followed Michaelis-Menten kinetics (K(m) = 220 micro M), loss of Fe(III) and U(VI) was first-order at all concentrations tested. Initial reduction rates of all electron acceptors were similar for cells grown with H(2) and sulfate, while cultures grown using lactate and sulfate had similar rates of metal loss but lower sulfate reduction activities. The similarities in metal, but not sulfate, reduction with H(2) and lactate suggest divergent pathways. Respiration assays and reduced minus oxidized spectra were carried out to determine c-type cytochrome involvement in electron acceptor reduction. c-type cytochrome oxidation was immediate with Fe(III) and U(VI) in the presence of H(2), lactate, or pyruvate. Sulfidogenesis occurred with all three electron donors and effectively oxidized the c-type cytochrome in lactate- or pyruvate-reduced, but not H(2)-reduced cells. Correspondingly, electron acceptor competition assays with lactate or pyruvate as electron donors showed that Fe(III) inhibited U(VI) reduction, and U(VI) inhibited sulfate loss. However, sulfate reduction was slowed but not halted when H(2) was the electron donor in the presence of Fe(III) or U(VI). U(VI) loss was still impeded by Fe(III) when H(2) was used. Hence, we propose a modified pathway for the reduction of sulfate, Fe(III), and U(VI) which helps explain why these bacteria cannot grow using these metals. We further propose that cytochrome c(3) is an electron carrier involved in lactate and pyruvate oxidation and is the reductase for alternate electron acceptors with higher redox potentials than sulfate.

Anaerobic oxidation of crude oil hydrocarbons by the resident microorganisms of a contaminated anoxic aquifer.

The biodegradation of two crude oils by microorganisms from an anoxic aquifer previously contaminated by natural gas condensate was examined under methanogenic and sulfate-reducing conditions. Artificially weathered Alaska North Slope crude oil greatly stimulated both methanogenesis and sulfate reduction. Gas chromatographic analysis revealed the entire n-alkane fraction of this oil (C13-C34) was consumed under both conditions. Naphthalene, 2-methylnaphthalene, and 2-ethylnaphthalene were also biodegraded but only in the presence of sulfate. Alba crude oil, which is naturally depleted in n-alkanes, resulted in a relatively modest stimulation of methanogenesis and sulfate reduction. Polycyclic aromatic hydrocarbon biodegradation was similar to that found for the Alaska North Slope crude oil, but a broader range of compounds was metabolized, including 2,6-dimethylnaphthalene and 2,7-dimethylnaphthalene in the presence of sulfate. These results indicate that n-alkanes are relatively labile, and their biodegradation in terrestrial environments is not necessarily limited by electron acceptor availability. Polycyclic aromatic hydrocarbons are relatively more recalcitrant, and the biodegradation of these substrates appeared to be sulfate-dependent and homologue-specific. This information should be useful for assessing the limits of in situ crude oil biodegradation in terrestrial environments and for making decisions regarding risk-based corrective actions.

Biodegradation of an alicyclic hydrocarbon by a sulfate-reducing enrichment from a gas condensate-contaminated aquifer.

We used ethylcyclopentane (ECP) as a model alicyclic hydrocarbon and investigated its metabolism by a sulfate-reducing bacterial enrichment obtained from a gas condensate-contaminated aquifer. The enrichment coupled the consumption of ECP with the stoichiometrically expected amount of sulfate reduced. During ECP biodegradation, we observed the transient accumulation of metabolite peaks by gas chromatography-mass spectrometry, three of which had identical mass spectrometry profiles. Mass-spectral similarities to analogous authentic standards allowed us to identify these metabolites as ethylcyclopentylsuccinic acids, ethylcyclopentylpropionic acid, ethylcyclopentylcarboxylic acid, and ethylsuccinic acid. Based on these findings, we propose a pathway for the degradation of this alicyclic hydrocarbon. Furthermore, a putative metabolite similar to ethylcyclopentylsuccinic acid was also found in samples of contaminated groundwater from the aquifer. However, no such finding was evident for samples collected from wells located upgradient of the gas condensate spill. Microbial community analysis of the ECP-degrading enrichment by denaturing gradient gel electrophoresis revealed the presence of at least three different organisms using universal eubacterial primers targeting 550 bp of the 16S rRNA gene. Based on sequence analysis, these organisms are phylogenetically related to the genera Syntrophobacter and Desulfotomaculum as well as a member of the Cytophaga-Flexibacter-Bacteroides group. The evidence suggests that alicyclic hydrocarbons such as ECP can be anaerobically activated by the addition to the double bond of fumarate to form alkylsuccinate derivatives under sulfate-reducing conditions and that the reaction occurs in the laboratory and in hydrocarbon-impacted environments.

Detection of anaerobic metabolites of saturated and aromatic hydrocarbons in petroleum-contaminated aquifers.

Recent investigations have demonstrated that several classes of petroleum hydrocarbons are susceptible to anaerobic decay, including alkanes and mono- and polycyclic aromatic compounds. In previous work, benzylsuccinates were shown to be useful indicators of in situ anaerobic alkylbenzene metabolism. In the present study, we sought to determine whether metabolites of alkanes and naphthalenes could similarly be used as indicators of the intrinsic decomposition of these compounds in petroleum-contaminated aquifers. Such metabolites include succinate derivatives of n-alkanes, cyclic alkanes, and alkylaromatic hydrocarbons as well as naphthoic acids. Using gas chromatography-mass spectrometry (GC-MS), we analyzed trimethylsilyl-derivatized organic extracts from six hydrocarbon-contaminated groundwaters for MS fragment ions indicative of such anaerobic metabolites. Geochemical indicators in these aquifers suggested the prevalence of anaerobic processes. In the groundwaters of the contaminated sites, we found compounds whose MS profiles suggested that they were indeed alkylsuccinic acids, ranging from C3 to C11 succinates. Propyl-, hexyl-, octyl-, and decylsuccinic acids were positively identified in the groundwaters by GC-MS matches with chemical or biologically produced standards. In two of the aquifers, we also detected components whose MS profiles matched with authentic standards of naphthoic acids and tetrahydronaphthoic acids. Metabolites were detected in nanomolar concentrations. The finding of these putative anaerobic metabolites of alkanes and naphthalenes signifies the in situ biodegradation of these hydrocarbons and attests to their value as indicators of intrinsic bioremediation.

In-situ evidence for uranium immobilization and remobilization.

The in-situ microbial reduction and immobilization of uranium was assessed as a means of preventing the migration of this element in the terrestrial subsurface. Uranium immobilization (putatively identified as reduction) and microbial respiratory activities were evaluated in the presence of exogenous electron donors and acceptors with field push-pull tests using wells installed in an anoxic aquifer contaminated with landfill leachate. Uranium(VI) amended at 1.5 microM was reduced to less than 1 nM in groundwater in less than 8 d during all field experiments. Amendments of 0.5 mM sulfate or 5 mM nitrate slowed U(VI) immobilization and allowed for the recovery of 10% and 54% of the injected element, respectively, as compared to 4% in the unamended treatment. Laboratory incubations confirmed the field tests and showed that the majority of the U(VI) immobilized was due to microbial reduction. In these tests, nitrate treatment (7.5 mM) inhibited U(VI) reduction, and nitrite was transiently produced. Further push-pull tests were performed in which either 1 or 5 mM nitrate was added with 1.0 uM U(VI) to sediments that already contained immobilized uranium. After an initial loss of the amendments, the concentration of soluble U(VI) increased and eventually exceeded the injected concentration, indicating that previously immobilized uranium was remobilized as nitrate was reduced. Laboratory experiments using heat-inactivated sediment slurries suggested that the intermediates of dissimilatory nitrate reduction (denitrification or dissimilatory nitrate reduction to ammonia), nitrite, nitrous oxide, and nitric oxide were all capable of oxidizing and mobilizing U(IV). These findings indicate that in-situ subsurface U(VI) immobilization can be expected to take place under anaerobic conditions, but the permanence of the approach can be impaired by disimilatory nitrate reduction intermediates that can mobilize previously reduced uranium.