Petroleum Hydrocarbon-contaminated Aquifer Research Articles

Metagenomic analysis revealed highly diverse carbon fixation microorganisms in a petroleum-hydrocarbon-contaminated aquifer

As one of the ultimate products of hydrocarbon biodegradation, inorganic carbon always be used to evaluate hydrocarbon biodegradation rates in petroleum-hydrocarbon-contaminated (PHC) aquifers. The evaluation method was challenged because of the existence of carbon fixation microorganisms, which may uptake inorganic carbons and consequently cause the biodegradation rates to be underestimated. We wonder if there are carbon fixation microorganisms in PHC aquifers. Although an extremely limited number of carbon fixation microorganisms in PHC sites have been studied in previous studies, the vast majority of microorganisms that participate in carbon fixation have not been systematically identified. To systematically reveal carbon fixation microorganisms and their survival environmental conditions, high-throughput metagenomic sequencing technologies, which are characterized by culture-independent, unbiased, and comprehensive methods for the detection and taxonomic characterization of microorganisms, were introduced to analyze the groundwater samples collected from a PHC aquifer. Results showed that 1041 genera were annotated as carbon fixation microorganisms, which accounted for 49% of the total number of genera in the PHC aquifer. Carbon fixation genes involved in Calvin-Benson-Bassham (CBB), 3-hydroxy propionate (3HP), reductive tricarboxylic acid (rTCA), and Wood-Ljungdahl (WL) cycles accounted for 2%, 41%, 34%, and 23% of the total carbon fixation genes, respectively, and 3HP, rTCA, and WL can be deemed as the dominant carbon fixation pathways. Most of the identified carbon fixation microorganisms are potential hydrocarbon biodegraders, and the most abundant carbon fixation microorganisms, such as Microbacterium, Novosphingobium, and Reyranella, were just the most abundant microorganisms in the aquifer system. It's deduced that most of the microorganisms in the aquifer were facultative autotrophic, and undertaking the dual responsibilities of degrading hydrocarbons to inorganic carbon and uptaking inorganic carbon to biomass.

14C Isotopes and microbial community structures as evidence for biodegradation in a petroleum hydrocarbon-contaminated aquifer

Petroleum hydrocarbon contamination of groundwater and soil constitutes a serious threat to human health and the natural environment. In situ biodegradation is a low-cost, low-risk and effective technique to remove petroleum hydrocarbon contaminants. The concentrations of total petroleum hydrocarbons (TPH), dissolved inorganic carbon (DIC) and \(^{14} {\text{C}}_{{{\text{HCO}}_{3} }}\) were analyzed, and polymerase chain reaction–denaturing gradient gel electrophoresis was used to investigate bacterial community structures. Downgradient from the contaminated source along the groundwater flow, the concentrations of TPH and DIC decreased and the 14C content increased from 44.3 to 64.3 pmc, which confirmed petroleum hydrocarbon biodegradation in the contaminated aquifers. The sequence analysis of the DGGE bands revealed that some species of known bacterial biodegrading strains, such as Dechloromonas aromatica RCB, Pseudomonas putida and Pseudomonas protegens, were the dominant bacterial groups in the field. Hydrogeochemical and stable isotope measurements, combined with analysis of bacterial community structures, could be useful tools to prove the occurrence of biodegradation processes in contaminated aquifers.

Microbial Communities Associated with Zones of Elevated Magnetic Susceptibility in Hydrocarbon-Contaminated Sediments

ABSTRACTRecent studies suggest that magnetic susceptibility (MS) measurements can play an important role in identifying zones where microbial-mediated iron mineral transformations are occurring. Here we investigated the microbial community variations within zones of elevated MS in a petroleum hydrocarbon-contaminated aquifer near Bemidji, Minnesota, USA. Our main objective was to 1) identify the key microbial populations that may play a role in hydrocarbon degradation, 2) analyze which microbial populations could be connected to the elevated MS and 3) explore the use of non-destructive geophysical techniques as a tool to guide microbial sampling. Clone libraries based on the 16S rRNA gene revealed the presence of iron-reducing β-Proteobacteria in the vadose zone, whereas the free petroleum phase on the water table was characterized by a methanogenic consortium, in which the syntrophic δ-proteobacterium Smithella and the hydrogenotrophic Methanoregula predominated. Nonmetric multidimensional scaling (NMDS) found a close relationship between elevated MS values and the methanogenic hydrocarbon-degrading consortium. Our results suggest that magnetic susceptibility measurements can guide microbiologists to zones of active microbial biodegradation in aged petroleum spills.

Microbial CO2 fixation potential in a tar-oil-contaminated porous aquifer

CO(2) fixation is one of the most important processes on the Earth's surface, but our current understanding of the occurrence and importance of chemolithoautotrophy in the terrestrial subsurface is poor. Groundwater ecosystems, especially at organically polluted sites, have all the requirements for autotrophic growth processes, and CO(2) fixation is thus suggested to contribute significantly to carbon flux in these environments. We explored the potential for autotrophic CO(2) fixation in microbial communities of a petroleum hydrocarbon-contaminated aquifer by detection of functional marker genes (cbbL, cbbM), encoding different forms of the key enzyme RubisCO of the Calvin-Benson-Bassham cycle. Quantification of (red-like) cbbL genes revealed highest numbers at the upper fringe of the contaminant plume and the capillary fringe where reduced sulphur and iron species are regularly oxidized in the course of groundwater table changes. Functional gene sequences retrieved from this area were most closely related to sequences of different thiobacilli. Moreover, several cultures could be enriched from fresh aquifer material, all of which are able to grow under chemolithoautotrophic conditions. A novel, nitrate-reducing, thiosulfate-oxidizing bacterial strain, recently described as Thiobacillus thiophilus D24TN(T) sp. nov., was shown to carry and transcribe RubisCO large-subunit genes of form I and II. Enzyme tests proved the actual activity of RubisCO in this strain.

Recovery of in-situ methanotrophic activity following acetylene inhibition

Methane (CH4) is the second most important greenhouse gas after carbon dioxide (CO2). To understand CH4 cycling, quantitative information about microbial CH4 oxidation in soils is essential. Field methods such as the gas push-pull test (GPPT) to quantify CH4 oxidation are often used in combination with specific inhibitors, such as acetylene (C2H2). Acetylene irreversibly binds to the enzyme methane monooxygenase, but little is known about recovery of CH4 oxidation activity after C2H2 inhibition in situ, which is important when performing several experiments at the same location. To assess recovery of CH4 oxidation activity following C2H2 inhibition, we performed a series of GPPTs over 8 weeks at two different locations in the vadose zone above a petroleum hydrocarbon-contaminated aquifer in Studen, Switzerland. After 4 weeks a maximum recovery of 30% and 50% of the respective initial activity was reached, with a subsequent slight drop in activity at both locations. Likely, CH4 oxidation activity and CH4 concentrations were too low to allow for rapid recovery following C2H2 inhibition at the studied locations. Therefore, alternative competitive inhibitors have to be evaluated for application in conjunction with GPPTs, especially for sites with low activity.

Activity and Diversity of Methanogens in a Petroleum Hydrocarbon-Contaminated Aquifer

Methanogenic activity was investigated in a petroleum hydrocarbon-contaminated aquifer by using a series of four push-pull tests with acetate, formate, H(2) plus CO(2), or methanol to target different groups of methanogenic Archaea. Furthermore, the community composition of methanogens in water and aquifer material was explored by molecular analyses, i.e., fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes amplified with the Archaea-specific primer set ARCH915 and UNI-b-rev, and sequencing of DNA from dominant DGGE bands. Molecular analyses were subsequently compared with push-pull test data. Methane was produced in all tests except for a separate test where 2-bromoethanesulfonate, a specific inhibitor of methanogens, was added. Substrate consumption rates were 0.11 mM day(-1) for methanol, 0.38 mM day(-1) for acetate, 0.90 mM day(-1) for H(2), and 1.85 mM day(-1) for formate. Substrate consumption and CH(4) production during all tests suggested that at least three different physiologic types of methanogens were present: H(2) plus CO(2) or formate, acetate, and methanol utilizers. The presence of 15 to 20 bands in DGGE profiles indicated a diverse archaeal population. High H(2) and formate consumption rates agreed with a high diversity of methanogenic Archaea consuming these substrates (16S rRNA gene sequences related to several members of the Methanomicrobiaceae) and the detection of Methanomicrobiaceae by using FISH (1.4% of total DAPI [4',6-diamidino-2-phenylindole]-stained microorganisms in one water sample; probe MG1200). Considerable acetate consumption agreed with the presence of sequences related to the obligate acetate degrader Methanosaeata concilii and the detection of this species by FISH (5 to 22% of total microorganisms; probe Rotcl1). The results suggest that both aceticlastic and CO(2)-type substrate-consuming methanogens are likely involved in the terminal step of hydrocarbon degradation, while methanogenesis from methanol plays a minor role. DGGE profiles further indicate similar archaeal community compositions in water and aquifer material. The combination of hydrogeological and molecular methods employed in this study provide improved information on the community and the potential activity of methanogens in a petroleum hydrocarbon-contaminated aquifer.

Field-scale isotopic labeling of phospholipid fatty acids from acetate-degrading sulfate-reducing bacteria

Isotopic labeling of biomarker molecules is a technique applied to link microbial community structure with activity. Previously, we successfully labeled phospholipid fatty acids (PLFA) of suspended nitrate-reducing bacteria in an aquifer. However, the application of the method to low energy-yielding processes such as sulfate reduction, and extension of the analysis to attached communities remained to be studied. To test the feasibility of the latter application, an anoxic test solution of 500 l of groundwater with addition of 0.5 mM Br − as a conservative tracer, 1.1 mM SO 4 2 - , and 2.0 mM [2- 13C]acetate was injected in the transition zone of a petroleum hydrocarbon-contaminated aquifer where sulfate-reducing and methanogenic conditions prevailed. Thousand liters of test solution/groundwater mixture were extracted in a stepwise fashion after 2–46 h incubation. Computed apparent first-order rate coefficients were 0.31 ± 0.04 day −1 for acetate and 0.34 ± 0.05 day −1 for SO 4 2 - consumption. The δ 13C increased from −71.03‰ to +3352.50‰ in CH 4 and from −16.15‰ to +32.13‰ in dissolved inorganic carbon (DIC). A mass balance suggested that 43% of the acetate-derived 13C appeared in DIC and 57% appeared in CH 4. Thus, acetate oxidation coupled to sulfate reduction and acetoclastic methanogenesis occurred simultaneously. The δ 13C of PLFA increased on average by 27‰ in groundwater samples and 4‰ in sediment samples. Hence, both suspended and attached communities actively degraded acetate. The PLFA labeling patterns and fluorescent in situ hybridization (FISH) analyses of sediment and groundwater samples suggested that the main sulfate-reducing bacteria degrading the acetate were Desulfotomaculum acetoxidans and Desulfobacter sp. in groundwater, and D. acetoxidans in sediment.

Field-scale 13C-labeling of phospholipid fatty acids (PLFA) and dissolved inorganic carbon: tracing acetate assimilation and mineralization in a petroleum hydrocarbon-contaminated aquifer

This study was conducted to determine the feasibility of labeling phospholipid-derived fatty acids (PLFA) of an active microbial population with a (13)C-labeled organic substrate in the denitrifying zone of a petroleum hydrocarbon-contaminated aquifer during a single-well push-pull test. Anoxic test solution was prepared from 500 l of groundwater with addition of 0.5 mM Br(-) as a conservative tracer, 0.5 mM NO(3) (-), and 0.25 mM [2-(13)C]acetate. At 4, 23 and 46 h after injection, 1000 l of test solution/groundwater mixture were sequentially extracted. During injection and extraction phases we measured Br(-), NO(3) (-) and acetate concentrations, characterized the microbial community structure by PLFA and fluorescent in situ hybridization (FISH) analyses, and determined (13)C/(12)C ratios in dissolved inorganic carbon (DIC) and PLFA. Computed first-order rate coefficients were 0.63+/-0.08 day(-1) for NO(3) (-) and 0.70+/-0.05 day(-1) for acetate consumption. Significant (13)C incorporation in DIC and PLFA was detected as early as 4 h after injection. At 46 h we measured delta(13)C values of up to 5614 per thousand in certain PLFA (especially monounsaturated fatty acids), and up to 59.8 per thousand in extracted DIC. Profiles of enriched PLFA and FISH analysis suggested the presence of active denitrifiers. Our results demonstrate the applicability of (13)C labeling of PLFA and DIC in combination with FISH to link microbial structure and activities at the field scale during a push-pull test.

Field-scale 13C-labeling of phospholipid fatty acids (PLFA) and dissolved inorganic carbon: tracing acetate assimilation and mineralization in a petroleum hydrocarbon-contaminated aquifer

This study was conducted to determine the feasibility of labeling phospholipid-derived fatty acids (PLFA) of an active microbial population with a 13C-labeled organic substrate in the denitrifying zone of a petroleum hydrocarbon-contaminated aquifer during a single-well push-pull test. Anoxic test solution was prepared from 500 l of groundwater with addition of 0.5 mM Br − as a conservative tracer, 0.5 mM NO 3 −, and 0.25 mM [2- 13C]acetate. At 4, 23 and 46 h after injection, 1000 l of test solution/groundwater mixture were sequentially extracted. During injection and extraction phases we measured Br −, NO 3 − and acetate concentrations, characterized the microbial community structure by PLFA and fluorescent in situ hybridization (FISH) analyses, and determined 13C/ 12C ratios in dissolved inorganic carbon (DIC) and PLFA. Computed first-order rate coefficients were 0.63±0.08 day −1 for NO 3 − and 0.70±0.05 day −1 for acetate consumption. Significant 13C incorporation in DIC and PLFA was detected as early as 4 h after injection. At 46 h we measured δ 13C values of up to 5614‰ in certain PLFA (especially monounsaturated fatty acids), and up to 59.8‰ in extracted DIC. Profiles of enriched PLFA and FISH analysis suggested the presence of active denitrifiers. Our results demonstrate the applicability of 13C labeling of PLFA and DIC in combination with FISH to link microbial structure and activities at the field scale during a push-pull test.

Rapid evolution of redox processes in a petroleum hydrocarbon-contaminated aquifer.

Ground water chemistry data collected over a six-year period show that the distribution of contaminants and redox processes in a shallow petroleum hydrocarbon-contaminated aquifer has changed rapidly over time. Shortly after a gasoline release occurred in 1990, high concentrations of benzene were present near the contaminant source area. In this contaminated zone, dissolved oxygen in ground water was depleted, and by 1994 Fe(III) reduction and sulfate reduction were the predominant terminal electron accepting processes. Significantly, dissolved methane was below measurable levels in 1994, indicating the absence of significant methanogenesis. By 1996, however, depletion of solid-phase Fe(III)-oxyhydrox ides in aquifer sediments and depletion of dissolved sulfate in ground water resulted in the onset of methanogenesis. Between 1996 and 2000, water-chemistry data indicated that methanogenic metabolism became increasingly prevalent. Molecular analysis of 16S-rDNA extracted from sediments shows the presence of a more diverse methanogenic community inside as opposed to outside the plume core, and is consistent with water-chemistry data indicating a shift toward methanogenesis over time. This rapid evolution of redox processes reflects several factors including the large amounts of contaminants, relatively rapid ground water flow (approximately 0.3 m/day [approximately foot/day]), and low concentrations of microbially reducible Fe(III) oxyhydroxides ( approximately 1 micromol/g) initially present in aquifer sediments. These results illustrate that, under certain hydrologic conditions, redox conditions in petroleum hydrocarbon-contaminated aquifers can change rapidly in time and space, and that the availability of solid-phase Fe(III)-oxyhydroxides affects this rate of change.

Activity and diversity of sulfate-reducing bacteria in a petroleum hydrocarbon-contaminated aquifer.

Microbial sulfate reduction is an important metabolic activity in petroleum hydrocarbon (PHC)-contaminated aquifers. We quantified carbon source-enhanced microbial SO(4)(2-) reduction in a PHC-contaminated aquifer by using single-well push-pull tests and related the consumption of sulfate and added carbon sources to the presence of certain genera of sulfate-reducing bacteria (SRB). We also used molecular methods to assess suspended SRB diversity. In four consecutive tests, we injected anoxic test solutions (1,000 liters) containing bromide as a conservative tracer, sulfate, and either propionate, butyrate, lactate, or acetate as reactants into an existing monitoring well. After an initial incubation period, 1,000 liters of test solution-groundwater mixture was extracted from the same well. Average total test duration was 71 h. We measured concentrations of bromide, sulfate, and carbon sources in native groundwater as well as in injection and extraction phase samples and characterized the SRB population by using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Enhanced sulfate reduction concomitant with carbon source degradation was observed in all tests. Computed first-order rate coefficients ranged from 0.19 to 0.32 day(-1) for sulfate reduction and from 0.13 to 0.60 day(-1) for carbon source degradation. Sulfur isotope fractionation in unconsumed sulfate indicated that sulfate reduction was microbially mediated. Enhancement of sulfate reduction due to carbon source additions in all tests and variability of rate coefficients suggested the presence of specific SRB genera and a high diversity of SRB. We confirmed this by using FISH and DGGE. A large fraction of suspended bacteria hybridized with SRB-targeting probes SRB385 plus SRB385-Db (11 to 24% of total cells). FISH results showed that the activity of these bacteria was enhanced by addition of sulfate and carbon sources during push-pull tests. However, DGGE profiles indicated that the bacterial community structure of the dominant species did not change during the tests. Thus, the combination of push-pull tests with molecular methods provided valuable insights into microbial processes, activities, and diversity in the sulfate-reducing zone of a PHC-contaminated aquifer.

Characterizing Intrinsic Bioremediation in a Petroleum Hydrocarbon-Contaminated Aquifer by Combined Chemical, Isotopic, and Biological Analyses

Chemical, isotopic, and biological parameters were evaluated over a 1-year period to characterize microbial processes associated with intrinsic bioremediation in a petroleum hydrocarbon-contaminated aquifer located in Studen, Switzerland. Chemical parameters measured included oxidants such as O2, NO3 −, and SO4 2−, reduced species such as Fe2+ and CH4, and dissolved inorganic carbon (DIC). Stable carbon isotope analyses of DIC were used to differentiate between different processes that contribute to DIC production. Microbial populations were identified by sequence analysis of archaeal 16S rDNA and in situ hybridization using a general DNA binding dye (DAPI) and specific probes targeting the domain Archaea (Arch915) and Bacteria (Eub338), as well as the species Methanosaeta concilii (Rotcl1) and Methanospirillum sp. (Rotcl2). Groundwater exhibited reduced conditions and elevated concentrations of DIC within the contaminated zone. Spatially distinct values of δ13C ranging from −16.5l%c to −4.44%o were found, indicating the presence of different ongoing microbial processes. Detected microbial populations (% of DAPI-stained cells) within the contaminated zone belonged to Archaea (9±2% to 31±13%) and Bacteria (13±3% to 32±13%). In wells with methanogenic activity, Methanosaeta concilii accounted for up to 26% of all DAPI-detected microorganisms. These results demonstrate that this novel combination of chemical, isotopic, and biological analysis provides valuable insights that can be used for the characterization of microbial processes in contaminated aquifers.

Engineered in situ bioremediation of a petroleum hydrocarbon-contaminated aquifer: assessment of mineralization based on alkalinity, inorganic carbon and stable carbon isotope balances

A concept is proposed to assess in situ petroleum hydrocarbon mineralization by combining data on oxidant consumption, production of reduced species, CH 4, alkalinity and dissolved inorganic carbon (DIC) with measurements of stable isotope ratios. The concept was applied to a diesel fuel contaminated aquifer in Menziken, Switzerland, which was treated by engineered in situ bioremediation. In the contaminated aquifer, added oxidants (O 2 and NO 3 −) were consumed, elevated concentrations of Fe(II), Mn(II), CH 4, alkalinity and DIC were detected and the DIC was generally depleted in 13 C compared to the background. The DIC production was larger than expected based on the consumption of dissolved oxidants and the production of reduced species. Stable carbon isotope balances revealed that the DIC production in the aquifer originated mainly from microbial petroleum hydrocarbon mineralization, and that geochemical reactions such as carbonate dissolution produced little DIC. This suggests that petroleum hydrocarbon mineralization can be underestimated if it is determined based on concentrations of dissolved oxidants and reduced species.

Intrinsic bioremediation of a petroleum hydrocarbon-contaminated aquifer and assessment of mineralization based on stable carbon isotopes.

This study presents a stepwise concept to assess the in situ microbial mineralization of petroleum hydrocarbons (PHC) in aquifers. A new graphical method based on stable carbon isotope ratios (delta 13C) was developed to verify the origin of dissolved inorganic carbon (DIC). The concept and the isotope method were applied to an aquifer in Student, Switzerland, in which more than 34,000 liters of heating oil were accidentally released. Chemical analyses of ground water revealed that in this aquifer locally, anaerobic conditions prevailed, and that PHC mineralization was linked to the consumption of oxidants such as O2, NO3-, and SO4(2-) and the production of reduced species such as Fe2+, Mn2+, H2S and CH4. However, alkalinity and DIC balances showed a quantitative disagreement in the link between oxidant consumption and DIC production, indicating that chemical data alone may not be a reliable assessment tool. delta 13C ratios in DIC have been used before for bioremediation assessment, but results were reported to be negatively influenced by methanogenesis. Using the new graphical method to display delta 13C data, it was possible to identify anomalies found in methanogenic monitoring wells. It could be shown that 88% of the DIC produced in the contaminated aquifer originated from microbial PHC mineralization. Thus, the new graphical method to display delta 13C ratios appears to be a useful tool for the assessment of microbial hydrocarbon mineralization in a complex environment.

Lack of Correlation between Organic Acid Concentrations and Predominant Electron-Accepting Processes in a Contaminated Aquifer

Long-term (1992−1995) monitoring data from a petroleum hydrocarbon-contaminated aquifer were used to examine the hypothesis that concentrations of low molecular weight (LMW) aliphatic organic acids reflect terminal electron-accepting processes. During the period of study, concentrations of dissolved hydrogen (H2) indicated that methanogenic, sulfate-reducing, and iron(III)-reducing condi tions predominated at the site. However, there was no correlation between LMW organic acid concentrations and concentrations of dissolved H2. These results indicate that organic acid concentrations are not a reliable indicator of local redox conditions at this site.

Influence of Electron Donor on the Minimum Sulfate Concentration Required for Sulfate Reduction in a Petroleum Hydrocarbon-Contaminated Aquifer

Fluctuations in the availability of electron donor (petroleum hydrocarbons) affected the competition between sulfate-reducing bacteria (SRB) and methanogenic bacteria (MB) for control of electron flow in a petroleum hydrocarbon-contaminated aquifer. The data suggest that abundant electron donor availability allowed MB to sequester a portion of the electron flow even when sulfate was present in sufficient concentrations to support sulfate reduction. For example, in an area of abundant electron-donor availability, SRB appeared to be unable to sequester the electron flow from MB in the presence of 1.4 mg/L sulfate. The data also suggest that when electron-donor availability was limited, SRB outcompeted MB for available substrate at a lower concentration of sulfate than when electron donor was plentiful. For example, in an area of limited electron-donor availability, SRB appeared to maintain dominance of electron flow at sulfate concentrations less than 1 mg/L. The presence of abundant electron donor and a limited amount of sulfate reduced competition for available substrate, allowing both SRB and MB to metabolize available substrates concurrently. 34 refs., 5 figs.

Rapid Toluene Mineralization by Aquifer Microorganisms at Adak, Alaska: Implications for Intrinsic Bioremediation in Cold Environments

Sediments from a relatively cold (5 °C), petroleum hydrocarbon-contaminated aquifer in Adak, AK, mineralized [ 14 C]toluene at an aerobic rate (16.3% day -1 at 5 °C) comparable to that (5.1% day -1 at 20 °C) of sediments from a more temperate aquifer at Hanahan, SC. In addition, rates of overall microbial metabolism in sediments from the two aquifers, as estimated by [1- 14 C]acetate mineralization, were similar (∼10.6% h -1 ) at their respective in situ temperatures. These results are not consistent with the common assumption that biodegradation rates in cold groundwater systems are depressed relative to more temperate systems. Furthermore, these results suggest that intrinsic bioremediation of petroleum hydrocarbon contaminants in cold groundwater systems may be technically feasible, in some cases.