Martin H. Schroth

Activity and Diversity of Sulfate-Reducing Bacteria in a Petroleum Hydrocarbon-Contaminated Aquifer

ABSTRACT Microbial sulfate reduction is an important metabolic activity in petroleum hydrocarbon (PHC)-contaminated aquifers. We quantified carbon source-enhanced microbial SO42− 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.

Interaction between water flow and spatial distribution of microbial growth in a two-dimensional flow field in saturated porous media.

Bacterial growth and its interaction with water flow was investigated in a two-dimensional flow field in a saturated porous medium. A flow cell (56 x 44 x 1 cm) was filled with glass beads and operated under a continuous flow of a mineral medium containing nitrate as electron acceptor. A glucose solution was injected through an injection port, simulating a point source contamination. Visible light transmission was used to observe the distribution of the growing biomass and water flow during the experiment. At the end of the experiment (on day 31), porous medium samples were destructively collected and analyzed for abundance of total and active bacterial cells, bacterial cell volume and concentration of polysaccharides and proteins. Microbial growth was observed in two stripes along the length of the flow cell, starting at the glucose injection port, where highest biomass concentrations were obtained. The spatial distribution of biomass indicated that microbial activity was limited by transverse mixing between glucose and nitrate media, as only in the mixing zone between the media high biological activities were achieved. The ability of the biomass to change the flow pattern in the flow cell was observed, indicating that the biomass was locally reducing the hydraulic conductivity of the porous medium. This bioclogging effect became evident when the injection of the glucose solution was turned off and water flow still bypassed the area around the glucose injection port, preserving the flow pattern as it was during the injection of the glucose solution. As flow bypass was possible in this system, the average hydraulic properties of the flow cell were not affected by the produced biomass. Even in the vicinity of the injection port, the total volume of the bacterial cells remained below 0.01% of the pore space and was unlikely to be responsible for the bioclogging. However, the bacteria produced large amounts of extracellular polymeric substances (EPS), which likely caused the observed bioclogging effects.

Sulfur isotope fractionation during microbial sulfate reduction by toluene-degrading bacteria

Sulfate-reducing bacteria contribute considerably to the mineralization of petroleum hydrocarbons (PHC) in contaminated environments. Stable sulfur isotope fractionation during microbial sulfate reduction was investigated in microcosm experiments with different cultures of sulfate-reducing bacteria for various initial sulfate concentrations using toluene as the sole carbon source. Experiments were conducted with the marine strain Desulfobacula toluolica, the fresh water strain PRTOL1, and an enrichment culture from a PHC-contaminated aquifer. Sulfate reduction rates ranged from 7 ± 1 to 494 ± 9 nmol cm−3 d−1, whereas specific sulfate reduction rates (sSRR) ranged from 8.9 × 10−15 to 3.9 × 10−13 ± 9.2 × 10−14 mol cell−1 d−1. Calculated enrichment factors (ϵ) for the fractionation of stable sulfur isotopes during microbial sulfate reduction ranged from 19.8 ± 0.9 to 46.9 ± 2.1‰. In general, values of ϵ and sSRR obtained in our experiments were similar to those reported previously for sulfate-reducing bacteria incubated with readily available carbon sources under optimal growth conditions. Moreover, we found no obvious correlation between ϵ and sSRR values when data from all our microcosm experiments were combined or when we combined our data with several previously published data sets. In contrast, ϵ values determined in our enrichment culture experiments (average 23.5 ± 4.3‰) agreed well with ϵ values determined in a recent field study performed in situ in a PHC-contaminated aquifer. Thus, results from this laboratory study provide valuable information on stable sulfur isotope fractionation during microbial sulfate reduction under conditions that more closely resemble those in PHC-contaminated environments, i.e., for a variety of sulfate concentrations, including low sulfate concentrations, and for a an important PHC-constituent (toluene) used as sole carbon source.

In-situ oxidation of trichloroethene by permanganate: effects on porous medium hydraulic properties.

In-situ oxidation of dense nonaqueous-phase liquids (DNAPLs) by strong oxidants such as potassium permanganate (KMnO4) has been proposed as a possible DNAPL remediation strategy. In this study, we investigated the effects of in-situ trichloroethene (TCE) oxidation by KMnO4 on porous medium hydraulic properties. In particular, we wanted to determine the overall effects of concurrent solid phase (MnO2) precipitation, gas (CO2) evolution and TCE dissolution resulting from the oxidation reaction on the porous mediums aqueous-phase relative permeability, krw. Three TCE removal experiments were conducted in a 95-cm long, 5.1-cm i.d. glass column, which was homogeneously packed with well-characterized 30/40-mesh silica sand. TCE was emplaced in the sand-pack in residual, entrapped form through a sequence of water/TCE imbibition and drainage steps. The column was then flushed under constant aqueous flux conditions for up to 104 h with either deionized water (reference experiment), deionized water containing 5 mM KMnO4 or deionized water containing 5 mM KMnO4 and 300 mM Na2HPO4. Aqueous-phase relative permeabilities were computed from measured flow rates and measurements of aqueous-phase pressure head, h obtained using pressure transducers connected to tensiometers distributed along the column length. A dual-energy gamma radiation system was used to monitor changes in fluid saturation that occurred during each experiment. In addition, column effluent samples were collected for chemical analyses. Dissolution of TCE during deionized water flushing led to an increase in krw by approximately 22% and a local reduction in h. On the other hand, vigorous CO2 gas production and precipitation of MnO2 was visually observed during flushing with deionized water that contained 5 mM KMnO4. As a consequence, krw declined by approximately 96% and h increased locally by more than 1000 cm H2O during the first 24 h of the experiment, causing sand-pack ruptures and pump failure. Conversely, less CO2 gas production and MnO2 precipitation was visually observed during flushing with deionized water that contained 5 mM KMnO4 and 300 mM Na2HPO4. Consequently, only small increases in h (< 15 cm H2O) were observed in this experiment due to a reduction in krw of approximately 53%. While we must attribute changes in h due to variations in krw to our specific experimental design (constant aqueous flux, one-dimensional flow experiments), these experiments nevertheless confirm that successful application of in situ chemical oxidation of TCE requires consideration of detrimental processes such as MnO2 precipitation and CO2 gas formation. In addition, our results indicate that utilization of a buffered oxidant solution may improve the effectiveness of in-situ oxidation of TCE by KMnO4 in otherwise weakly buffered porous media.

In situ assessment of microbial sulfate reduction in a petroleum-contaminated aquifer using push–pull tests and stable sulfur isotope analyses

Anaerobic microbial activities such as sulfate reduction are important for the degradation of petroleum hydrocarbons (PHC) in contaminated aquifers. The objective of this study was to evaluate the feasibility of single-well push-pull tests in combination with stable sulfur isotope analyses for the in situ quantification of microbial sulfate reduction. A series of push-pull tests was performed in an existing monitoring well of a PHC-contaminated aquifer in Studen (Switzerland). Sulfate transport behavior was evaluated in a first test. In three subsequent tests, we injected anoxic test solutions (up to 1000 l), which contained 0.5 mM bromide (Br-) as conservative tracer and 1 mM sulfate (SO4(2-)) as reactant. After an initial incubation period of 42.5 to 67.9 h, up to 1100 l of test solution/groundwater mixture was extracted in each test from the same location. During the extraction phases, we measured concentrations of relevant species including Br-, SO4(2-) and sulfide (S(-II)), as well as stable sulfur isotope ratios (delta 34S) of extracted, unconsumed SO4(2-) and extracted S(-II). Results indicated sulfate reduction activity in the vicinity of the test well. Computed first-order rate coefficients for sulfate reduction ranged from 0.043 +/- 0.013 to 0.130 +/- 0.015 day-1. Isotope enrichment factors (epsilon) computed from sulfur isotope fractionation of extracted, unconsumed SO4(2-) ranged from 20.2 +/- 5.5@1000 to 22.8 +/- 3.4@1000. Together with observed fractionation in extracted S(-II), isotope enrichment factors provided strong evidence for microbially mediated sulfate reduction. Thus, push-pull tests combined with stable sulfur isotope analyses proved useful for the in situ quantification of microbial sulfate reduction in a PHC-contaminated aquifer.

In situ evaluation of solute retardation using single-well push–pull tests

Abstract More efficient methods are needed for the in situ evaluation of solute sorption to aquifer sediments. The objective of this study was to develop a simplified method for estimating retardation factors for injected solutes from “push–pull” test extraction phase breakthrough curves (BTCs). Sensitivity analyses based on numerical simulations were used to evaluate the method performance for a variety of test conditions. Simulations were conducted for varying retardation factors, aquifer parameters and injection phase durations, for tests performed under nonideal transport conditions such as nonlinear equilibrium and linear nonequilibrium sorption, and for a test performed in a physically heterogeneous aquifer. Predicted retardation factors showed errors ⩽14% in tests performed under ideal transport conditions (physically homogeneous aquifer with spatially uniform dispersivity that does not vary from solute to solute, spatially uniform linear equilibrium sorption). The method performed more poorly for solutes with large retardation factors ( R >20) and for tests conducted under nonideal transport conditions, and is expected to perform poorly in aquifers with highly heterogeneous sorption. In an example application, we used the method to estimate the distribution coefficient for 85 Sr using data from a field test performed by Pickens JF, Jackson RE, Inch KJ, Merritt WF. (Water Resour Res 1981;17:529–44). Reasonable agreement was found between distribution coefficients obtained using the simplified method of estimation and those obtained by Pickens et al. (1981).

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

ABSTRACT Methanogenic activity was investigated in a petroleum hydrocarbon-contaminated aquifer by using a series of four push-pull tests with acetate, formate, H2 plus CO2, 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 H2, and 1.85 mM day−1 for formate. Substrate consumption and CH4 production during all tests suggested that at least three different physiologic types of methanogens were present: H2 plus CO2 or formate, acetate, and methanol utilizers. The presence of 15 to 20 bands in DGGE profiles indicated a diverse archaeal population. High H2 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 CO2-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.

Sulfate-reducing bacterial community response to carbon source amendments in contaminated aquifer microcosms

Abstract Microbial sulfate reduction is an important metabolic activity in many reduced habitats. However, little is known about the sulfate-reducing communities inhabiting petroleum hydrocarbon (PHC)-contaminated freshwater aquifer sediments. The purpose of this study was to identify the groups of sulfate-reducing bacteria (SRB) selectively stimulated when sediment from a PHC-contaminated freshwater aquifer was incubated in sulfate-reducing aquifer microcosms that were amended with specific carbon sources (acetate, butyrate, propionate, lactate, and citrate). After 2 months of incubation, the SRB community was characterized using phospholipid fatty acid (PLFA) analysis combined with multivariate statistics as well as fluorescence in situ hybridization (FISH). Molybdate was used to specifically inhibit SRB in separate microcosms to investigate the contribution of non-SRB to carbon source degradation. Results indicated that sulfate reduction in the original sediment was an important process but was limited by the availability of sulfate. Substantially lower amounts of acetate and butyrate were degraded in molybdate treatments as compared to treatments without molybdate, suggesting that SRB were the major bacterial group responsible for carbon source turnover in microcosms. All of the added carbon sources induced changes in the SRB community structure. Members of the genus Desulfobulbus were present but not active in the original sediment but an increase of the fatty acids 15:1omega6c and 17:1omega6c and FISH results showed an enrichment of these bacteria in microcosms amended with propionate or lactate. The appearance of cy17:0 revealed that bacteria affiliated with the Desulfobacteriaceae were responsible for acetate degradation. Desulfovibrio and Desulfotomaculum spp. were not important populations within the SRB community in microcosms because they did not proliferate on carbon sources usually favored by these organisms. Metabolic, PLFA, and FISH results provided information on the SRB community in a PHC-contaminated freshwater environment, which exhibited stimulation patterns similar to other (e.g. marine) environments.

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.

Geometry and position of light nonaqueous-phase liquid lenses in water-wetted porous media

Predicting the movement of LNAPLs (light nonaqueous-phase liquids) in the subsurface environment is critical for the design of effective remediatory action. The objective of this study was to develop a method for predicting the shape and extent of LNAPL lenses in the capillary fringe of the vadose zone. Two-dimensional experiments were performed in a glass chamber (50 cm × 60 cm × 0.95 cm) using four Miller-similar silica sands (1220, 2030, 3040 and 4050 sieve sizes) and two LNAPLs (Soltrol® 220 and Duoprime® 55 mineral oil). LNAPLs were released in water-wetted sands to simulate a point-source discharge above a water table. Observation of light transmission was used to delineate the changing LNAPL lens boundary during infiltration until equilibrium was established. At equilibrium, no zone above the capillary fringe remained at a NAPL saturation higher than the residual saturation. A previously published model for predicting vertical lens dimensions was tested and good agreement was found between measured and predicted lens thicknesses when the time-dependent nature of LNAPL—water interfacial tensions was considered. Less agreement between measured and predicted lens thicknesses was found when model equations were modified to a fully explicit, predictive form. Observed spatial variability in the emplacement of LNAPLs in the capillary fringe, compounded by the strong time dependence of a key variable, limits the use of the model as a predictive tool, but provides important insight into the low precision in prediction which is attainable even if a more complete model was developed. The results provide a means to better understand LNAPL behavior in the subsurface environment.