In-situ sonication for enhanced recovery of aquifer microbial communities.

Sampling methods for characterization of microbial communities in aquifers should target both suspended and attached microorganisms (biofilms). We investigated the effectiveness and reproducibility of low-frequency (200 Hz) sonication pulses on improving extraction efficiency and quality of microorganisms from a petroleum-contaminated aquifer in Studen (Switzerland). Sonication pulses at different power levels (0.65, 0.9, and 1.1 kW) were applied to three different groundwater monitoring wells. Groundwater samples extracted after each pulse were compared with background groundwater samples for cell and adenosine tri-phosphate concentration. Turbidity values were obtained to assess the release of sediment fines and associated microorganisms. The bacterial community in extracted groundwater samples was analyzed by terminal-restriction-fragment-length polymorphism and compared with communities obtained from background groundwater samples and from sediment cores. Sonication enhanced the extraction efficiency up to 13-fold, with most of the biomass being associated with the sediment fines extracted with groundwater. Consecutive pulses at constant power were decreasingly effective, while pulses with higher power yielded the best results both in terms of extraction efficiency and quality. Our results indicate that low-frequency sonication may be a viable and cost-effective tool to improve the extraction of microorganisms from aquifers, taking advantage of existing groundwater monitoring wells.

Influence of arsenate adsorption to ferrihydrite, goethite, and boehmite on the kinetics of arsenate reduction by Shewanella putrefaciens strain CN-32.

The kinetics of As(V) reduction by Shewanella putrefaciens strain CN-32 was investigated in suspensions of 0.2, 2, or 20 g L(-1) ferrihydrite, goethite, or boehmite at low As (10 µM) and lactate (25 µM) concentrations. Experimental data were compared with model predictions based on independently determined sorption isotherms and rates of As(V) desorption, As(III) adsorption, and microbial reduction of dissolved As(V), respectively. The low lactate concentration was chosen to prevent significant Fe(III) reduction, but still allowing complete As(V) reduction. Reduction of dissolved As(V) followed first-order kinetics with a 3 h half-life of As(V). Addition of mineral sorbents resulted in pronounced decreases in reduction rates (32-1540 h As(V) half-life). The magnitude of this effect increased with increasing sorbent concentration and sorption capacity (goethite < boehmite < ferrihydrite). The model consistently underestimated the concentrations of dissolved As(V) and the rates of microbial As(V) reduction after addition of S. putrefaciens (∼5 × 10(9) cells mL(-1)), suggesting that attachment of S. putrefaciens cells to oxide mineral surfaces promoted As(V) desorption and thereby facilitated As(V) reduction. The interplay between As(V) sorption to mineral surfaces and bacterially induced desorption may thus be critical in controlling the kinetics of As reduction and release in reducing soils and sediments.

New field method: gas push-pull test for the in-situ quantification of microbial activities in the vadose zone.

Quantitative information on microbial processes in the field is important. Here we propose a new field method, the "gas push-pull test" (GPPT) for the in-situ quantification of microbial activities in the vadose zone. To evaluate the new method, we studied microbial methane oxidation above an anaerobic, petroleum-contaminated aquifer. A GPPT consists of the injection of a gas mixture of reactants (e.g., methane, oxygen) and nonreactive tracer gases (e.g., neon, argon) into the vadose zone and the subsequent extraction of the injection gas mixture together with soil air from the same location. Rate constants of gas conversion are calculated from breakthrough curves of extracted reactants and tracers. In agreement with expectations from previously measured gas profiles, we determined first-order rate constants of 0.68 h(-1) at 1.1 m below soil surface and 2.19 h(-1) at 2.7 m, close to the groundwater table. Co-injection of a specific inhibitor (acetylene) for methanotrophs showed that the observed methane consumption was microbially mediated. This was confirmed by increases of stable carbon isotope ratios in methane by up to 42.6 %. In the future, GPPTs should provide useful quantitative information on a range of microbial processes in the vadose zone.

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.

Methods to assess the amenability of petroleum hydrocarbons to bioremediation.

Bioremediation has achieved acceptance as a cost-effective technique for the remediation of soils and groundwater contaminated with petroleum hydrocarbons (PHC). A range of laboratory techniques to assess the biodegradability and bioavailability of PHCs are presented. Biodegradability and bioavailability are important determinants of the bioremediation performance of PHCs. Novel methods for the assessment of the bioavailability of PHC components are described. The techniques are demonstrated for a hydraulic fluid and a spindle oil from a contaminated site. Biodegradation is measured by oxygen consumption and carbon dioxide production. Bioavailability of the PHCs is estimated based on the PHC-water partitioning of tracer compounds and a novel analysis of gas chromatograms based on Raoult's law. The PHCs tested were only partially biodegradable (< 25% in 78 d) due to the low solubility and likely recalcitrance of some of their components. The combination of techniques outlined is expected to be of use in assessing the likely bioremediation performance of PHCs for which published data are scarce or inadequate.

Engineered and subsequent intrinsic in situ bioremediation of a diesel fuel contaminated aquifer.

A diesel fuel contaminated aquifer in Menziken, Switzerland was treated for 4.5 years by injecting aerated groundwater, supplemented with KNO3 and NH4H2PO4 to stimulate indigenous populations of petroleum hydrocarbon (PHC) degrading microorganisms. After dissolved PHC concentrations had stabilized at a low level, engineered in situ bioremediation was terminated. The main objective of this study was to evaluate the efficacy of intrinsic in situ bioremediation as a follow-up measure to remove PHC remaining in the aquifer after terminating engineered in situ bioremediation. In the first 7 months of intrinsic in situ bioremediation, redox conditions in the source area became more reducing as indicated by lower concentrations of SO4(2-) and higher concentrations of Fe(II) and CH4. In the core of the source area, strongly reducing conditions prevailed during the remaining study period (3 years) and dissolved PHC concentrations were higher than during engineered in situ bioremediation. This suggests that biodegradation in the core zone was limited by the availability of oxidants. In lateral zones of the source area, however, gradually more oxidized conditions were reestablished again, suggesting that PHC availability increasingly limited biodegradation. The total DIC production rate in the aquifer decreased within 2 years to about 25% of that during engineered in situ bioremediation and remained at that level. Stable carbon isotope analysis confirmed that the produced DIC mainly originated from PHC mineralization. The total rate of DIC and CH4 production in the source area was more than 300 times larger than the rate of PHC elution. This indicates that biodegradation coupled to consumption of naturally occurring oxidants was an important process for removal of PHC which remained in the aquifer after terminating engineered measures.

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.