Evaluation of methanotrophic bacterial communities capable of biodegrading trichloroethene (TCE) in acidic aquifers.

While bioremediation technologies for trichloroethene (TCE), a suspected carcinogen, have been successfully demonstrated in neutral pH aquifers, these technologies are often ineffective for remediating TCE contamination in acidic aquifers (i.e., pH < 5.5). Acidophilic methanotrophs have been detected in several low pH environments, but their presence and potential role in TCE degradation in acidic aquifers is unknown. This study applied a stable isotope probing-based technique to identify active methanotrophs that are capable of degrading TCE in microcosms prepared from two low pH aquifers. A total of thirty-five clones of methanotrophs were derived from low pH microcosms in which methane and TCE degradation had been observed, with 29 clustered in γ-Proteobacteria and 6 clustered in α-Proteobacteria. None of the clones has a high similarity to known acidophilic methanotrophs from other environments. The presence and diversity of particulate MMO and soluble MMO were also investigated. The pmoA gene was detected predominantly at one site, and the presence of a specific form of mmoX in numerous samples suggested that Methylocella spp. may be common in acidic aquifers. Finally, a methane-grown culture at pH 4 was enriched from an acidic aquifer and its ability to biodegrade various chlorinated ethenes was tested. Interestingly, the mixed culture rapidly degraded TCE and vinyl chloride, but not cis-dichloroethene after growth on methane. The data suggest that aerobic biodegradation of TCE and other chlorinated solvents in low pH groundwater may be facilitated by methanotrophic bacteria, and that there are potentially a wide variety of different strains that inhabit acidic aquifers.

Carbon Isotope Fractionation of 1,2-Dibromoethane by Biological and Abiotic Processes.

1,2-Dibromethane (EDB) is a toxic fuel additive that likely occurs at many sites where leaded fuels have impacted groundwater. This study quantified carbon (C) isotope fractionation of EDB associated with anaerobic and aerobic biodegradation, abiotic degradation by iron sulfides, and abiotic hydrolysis. These processes likely contribute to EDB degradation in source zones (biodegradation) and in more dilute plumes (hydrolysis). Mixed anaerobic cultures containing dehalogenating organisms (e.g., Dehaloccoides spp.) were examined, as were aerobic cultures that degrade EDB cometabolically. Bulk C isotope enrichment factors (εbulk) associated with biological degradation covered a large range, with mixed anaerobic cultures fractionating more (εbulk from -8 to -20‰) than aerobic cultures (εbulk from -3 to -6‰). εbulk magnitudes associated with the abiotic processes (dihaloelimination by FeS/FeS2 and hydrolysis) were large but fairly well constrained (εbulk from -19 to -29‰). As expected, oxidative mechanisms fractionated EDB less than dihaloelimination and substitution mechanisms, and biological systems exhibited a larger range of fractionation, potentially due to isotope masking effects. In addition to quantifying and discussing εbulk values, which are highly relevant for quantifying in situ EDB degradation, an innovative approach for constraining the age of EDB in the aqueous phase, based on fractionation during hydrolysis, is described.

Potential for cometabolic biodegradation of 1,4-dioxane in aquifers with methane or ethane as primary substrates.

The objective of this research was to evaluate the potential for two gases, methane and ethane, to stimulate the biological degradation of 1,4-dioxane (1,4-D) in groundwater aquifers via aerobic cometabolism. Experiments with aquifer microcosms, enrichment cultures from aquifers, mesophilic pure cultures, and purified enzyme (soluble methane monooxygenase; sMMO) were conducted. During an aquifer microcosm study, ethane was observed to stimulate the aerobic biodegradation of 1,4-D. An ethane-oxidizing enrichment culture from these samples, and a pure culture capable of growing on ethane (Mycobacterium sphagni ENV482) that was isolated from a different aquifer also biodegraded 1,4-D. Unlike ethane, methane was not observed to appreciably stimulate the biodegradation of 1,4-D in aquifer microcosms or in methane-oxidizing mixed cultures enriched from two different aquifers. Three different pure cultures of mesophilic methanotrophs also did not degrade 1,4-D, although each rapidly oxidized 1,1,2-trichloroethene (TCE). Subsequent studies showed that 1,4-D is not a substrate for purified sMMO enzyme from Methylosinus trichosporium OB3b, at least not at the concentrations evaluated, which significantly exceeded those typically observed at contaminated sites. Thus, our data indicate that ethane, which is a common daughter product of the biotic or abiotic reductive dechlorination of chlorinated ethanes and ethenes, may serve as a substrate to enhance 1,4-D degradation in aquifers, particularly in zones where these products mix with aerobic groundwater. It may also be possible to stimulate 1,4-D biodegradation in an aerobic aquifer through addition of ethane gas. Conversely, our results suggest that methane may have limited importance in natural attenuation or for enhancing biodegradation of 1,4-D in groundwater environments.

Enhancing aerobic biodegradation of 1,2-dibromoethane in groundwater using ethane or propane and inorganic nutrients.

1,2-Dibromoethane (ethylene dibromide; EDB) is a probable human carcinogen that was previously used as both a soil fumigant and a scavenger in leaded gasoline. EDB has been observed to persist in soils and groundwater, particularly under oxic conditions. The objective of this study was to evaluate options to enhance the aerobic degradation of EDB in groundwater, with a particular focus on possible in situ remediation strategies. Propane gas and ethane gas were observed to significantly stimulate the biodegradation of EDB in microcosms constructed with aquifer solids and groundwater from the FS-12 EDB plume at Joint Base Cape Cod (Cape Cod, MA), but only after inorganic nutrients were added. Ethene gas was also effective, but rates were appreciably slower than for ethane and propane. EDB was reduced to <0.02 µg/L, the Massachusetts state Maximum Contaminant Level (MCL), in microcosms that received ethane gas and inorganic nutrients. An enrichment culture (BE-3R) that grew on ethane or propane gas but not EDB was obtained from the site materials. The degradation of EDB by this culture was inhibited by acetylene gas, suggesting that degradation is catalyzed by a monooxygenase enzyme. The BE-3R culture was also observed to biodegrade 1,2-dichloroethane (DCA), a compound commonly used in conjunction with EDB as a lead scavenger in gasoline. The data suggest that addition of ethane or propane gas with inorganic nutrients may be a viable option to enhance degradation of EDB in groundwater aquifers to below current state or federal MCL values.

Aerobic biodegradation of iso-butanol and ethanol and their relative effects on BTEX biodegradation in aquifer materials.

The aerobic biodegradability of iso-butanol, a new biofuel, and its impact on benzene, toluene, ethylbenzene and xylenes (BTEX) degradation was investigated in aerobic microcosms consisting of groundwater and sediment from a California site with a history of gasoline contamination. To the best of our knowledge this is the first study directly examining the effects of iso-butanol on BTEX degradation. Microcosms that received either low (68 µM) or high (3400 µM) concentrations of iso-butanol showed complete biodegradation of iso-butanol within 7 and 23 d, respectively, of incubation at 15°C under aerobic conditions. A maximum utilization rate coefficient of 2.3±0.1×10⁻7 µmol cell⁻¹ h⁻¹ and a half saturation constant of 610±54 µM were regressed from the iso-butanol data. Iso-butanol biodegradation resulted in transient formation of the degradation intermediate products iso-butylaldehyde and iso-butyric acid, and both compounds were subsequently degraded within the timeframe of the experiments. Ethanol was biodegraded more slowly than iso-butanol. Ethanol also exhibited greater adverse impacts on BTEX biodegradation than iso-butanol. Results of the study suggest that iso-butanol added to fuels will be readily biodegraded in the environment under aerobic conditions without the accumulation of major intermediate products (iso-butylaldehyde and iso-butyric acid), and that it will pose less impacts on BTEX biodegradation than ethanol.

Anaerobic biodegradation of iso-butanol and ethanol and their relative effects on BTEX biodegradation in aquifer materials.

Biologically produced iso-butanol is currently being considered as an additive in gasoline blends. To evaluate its potential environmental fate in groundwater aquifers, a laboratory microcosm study was performed to evaluate iso-butanol biodegradation under various anaerobic conditions (nitrate-reducing, sulfate-reducing and methanogenic). The impacts of iso-butanol on benzene, toluene, ethylbenzene, and total xylenes (BTEX) biodegradation were also assessed, and microcosms prepared using ethanol instead of iso-butanol were evaluated to provide a basis for comparison. Iso-butanol was biodegraded under all conditions studied, with an observed apparent first-order rate constant ranging from approximately 0.2 d⁻¹ (nitrate-reducing) to approximately 0.02 d⁻¹ (sulfate-reducing). Iso-butanol typically was degraded in a time frame that was shorter than or similar to BTEX compounds. Iso-butyric acid and trace levels of iso-butylaldehyde were identified as transient intermediates, and both of these compounds were subsequently degraded within the time frame of the experiments. Iso-butanol and ethanol were biodegraded in similar time frames under methanogenic conditions. Under sulfate-reducing conditions, iso-butanol biodegradation initially proceeded more slowly than ethanol, and then increased to a rate greater than that observed for ethanol; this observation likely was due to the growth of iso-butanol degrading bacteria. Iso-butanol generally exhibited less adverse impacts on BTEX biodegradations than ethanol under the anaerobic conditions studied. In some cases, addition of iso-butanol enhanced the rate of TEX biodegradation.

Aerobic biodegradation of N-nitrosodimethylamine by the propanotroph Rhodococcus ruber ENV425.

The propanotroph Rhodococcus ruber ENV425 was observed to rapidly biodegrade N-nitrosodimethylamine (NDMA) after growth on propane, tryptic soy broth, or glucose. The key degradation intermediates were methylamine, nitric oxide, nitrite, nitrate, and formate. Small quantities of formaldehyde and dimethylamine were also detected. A denitrosation reaction, initiated by hydrogen atom abstraction from one of the two methyl groups, is hypothesized to result in the formation of n-methylformaldimine and nitric oxide, the former of which decomposes in water to methylamine and formaldehyde and the latter of which is then oxidized further to nitrite and then nitrate. Although the strain mineralized more than 60% of the carbon in [(14)C]NDMA to (14)CO(2), growth of strain ENV425 on NDMA as a sole carbon and energy source could not be confirmed. The bacterium was capable of utilizing NDMA, as well as the degradation intermediates methylamine and nitrate, as sources of nitrogen during growth on propane. In addition, ENV425 reduced environmentally relevant microgram/liter concentrations of NDMA to <2 ng/liter in batch cultures, suggesting that the bacterium may have applications for groundwater remediation.

Oxygen and chlorine isotopic fractionation during perchlorate biodegradation: laboratory results and implications for forensics and natural attenuation studies.

Perchlorate is a widespread environmental contaminant having both anthropogenic and natural sources. Stable isotope ratios of O and Cl in a given sample of perchlorate may be used to distinguish its source(s). Isotopic ratios may also be useful for identifying the extent of biodegradation of perchlorate, which is critical for assessing natural attenuation of this contaminant in groundwater. For this approach to be useful, however, the kinetic isotopic fractionations of O and Cl during perchlorate biodegradation must first be determined as a function of environmental variables such as temperature and bacterial species. A laboratory study was performed in which the O and Cl isotope ratios of perchlorate were monitored as a function of degradation by two separate bacterial strains (Azospira suillum JPLRND and Dechlorospirillum sp. FBR2) at both 10 degrees C and 22 degrees C with acetate as the electron donor. Perchlorate was completely reduced by both strains within 280 h at 22 degrees C and 615 h at 10 degrees C. Measured values of isotopic fractionation factors were epsilon(18)O = -36.6 to -29.0% per hundred and epsilon(37)Cl = -14.5 to -11.5% per hundred, and these showed no apparent systematic variation with either temperature or bacterial strain. An experiment using (18)O-enriched water (delta(18)O = +198% per hundred) gave results indistinguishable from those observed in the isotopically normal water (delta(18)O = -8.1% per hundred) used in the other experiments, indicating negligible isotope exchange between perchlorate and water during biodegradation. The fractionation factor ratio epsilon(18)O/epsilon(37)Cl was nearly invariant in all experiments at 2.50 +/- 0.04. These data indicate that isotope ratio analysis will be useful for documenting perchlorate biodegradation in soils and groundwater. The establishment of a microbial fractionation factor ratio (epsilon(18)O/ epsilon(37)Cl) also has significant implications for forensic studies.

Biotransformation of N-nitrosodimethylamine by Pseudomonas mendocina KR1.

N-Nitrosodimethylamine (NDMA) is a potent carcinogen and an emerging contaminant in groundwater and drinking water. The metabolism of NDMA in mammalian cells has been widely studied, but little information is available concerning the microbial transformation of this compound. The objective of this study was to elucidate the pathway(s) of NDMA biotransformation by Pseudomonas mendocina KR1, a strain that possesses toluene-4-monooxygenase (T4MO). P. mendocina KR1 was observed to initially oxidize NDMA to N-nitrodimethylamine (NTDMA), a novel metabolite. The use of 18O2 and H(2)18O revealed that the oxygen added to NDMA to produce NTDMA was derived from atmospheric O2. Experiments performed with a pseudomonad expressing cloned T4MO confirmed that T4MO catalyzes this initial reaction. The NTDMA produced by P. mendocina KR1 did not accumulate, but rather it was metabolized further to produce N-nitromethylamine (88 to 94% recovery) and a trace amount of formaldehyde (HCHO). Small quantities of methanol (CH3OH) were also detected when the strain was incubated with NDMA but not during incubation with either NTDMA or HCHO. The formation of methanol is hypothesized to occur via a second, minor pathway mediated by an initial alpha-hydroxylation of the nitrosamine. Strain KR1 did not grow on NDMA or mineralize significant quantities of the compound to carbon dioxide, suggesting that the degradation process is cometabolic.

Application of a vital fluorescent staining method for simultaneous, near-real-time concentration monitoring of two bacterial strains in an Atlantic coastal plain aquifer in Oyster, Virginia.

Two differentially labeled bacterial strains were monitored in near-real time during two field-scale bacterial transport experiments in a shallow aquifer in July 2000 and July 2001. Comamonas sp. strain DA001 and Acidovorax sp. strain OY-107 were grown and labeled with the vital fluorescent stain TAMRA/SE (5 [and -6]-carboxytetramethylrhodamine, succinimidyl ester) or CFDA/SE (5 [and -6]-carboxyfluorescein diacetate, succinimidyl ester). Fluorescently labeled cells and a conservative bromide tracer were introduced into a suboxic superficial aquifer, followed by groundwater collection from down-gradient multilevel samplers. Cells were enumerated in the field by microplate spectrofluorometry, with confirmatory analyses for selected samples done in the laboratory by epifluorescence microscopy, flow cytometry, and ferrographic capture. There was general agreement in the results from all of the vital-stain-based enumeration methods, with differences ranging from <10% up to 40% for the analysis of identical samples between different tracking methods. Field analysis by microplate spectrofluorometry was robust and efficient, allowing thousands of samples to be analyzed in quadruplicate for both of the injected strains. The near-real-time data acquisition allowed adjustments to the predetermined sampling schedule to be made. The microplate spectrofluorometry data sets for the July 2000 and July 2001 experiments allowed the transport of the injected cells to be related to the site hydrogeology and injection conditions and enabled the assessment of differences in the transport of the two strains. This near-real-time method should prove effective for a number of microbial ecology applications.

Enhancing transport of hydrogenophaga flava ENV735 for bioaugmentation of aquifers contaminated with methyl tert-butyl ether.

The gasoline oxygenate methyl tert-butyl ether (MTBE) has become a widespread contaminant in groundwater throughout the United States. Bioaugmentation of aquifers with MTBE-degrading cultures may be necessary to enhance degradation of the oxygenate in some locations. However, poor cell transport has sometimes limited bioaugmentation efforts in the past. The objective of this study was to evaluate the transport characteristics of Hydrogenophaga flava ENV735, a pure culture capable of growth on MTBE, and to improve movement of the strain through aquifer solids. The wild-type culture moved only a few centimeters in columns of aquifer sediment. An adhesion-deficient variant (H. flava ENV735:24) of the wild-type strain that moved more readily through sediments was obtained by sequential passage of cells through columns of sterile sediment. Hydrophobic and electrostatic interaction chromatography revealed that the wild-type strain is much more hydrophobic than the adhesion-deficient variant. Electrophoretic mobility assays and transmission electron microscopy showed that the wild-type bacterium contains two distinct subpopulations, whereas the adhesion-deficient strain has only a single, homogeneous population. Both the wild-type strain and adhesion-deficient variant degraded MTBE, and both were identified by 16S rRNA analysis as pure cultures of H. flava. The effectiveness of surfactants for enhancing transport of the wild-type strain was also evaluated. Many of the surfactants tested were toxic to ENV735; however, one nonionic surfactant, Tween 20, enhanced cell transport in sand columns. Improving microbial transport may lead to a more effective bioaugmentation strategy for MTBE-contaminated sites where indigenous oxygenate degraders are absent.

Simultaneous transport of two bacterial strains in intact cores from Oyster, Virginia: biological effects and numerical modeling.

The transport characteristics of two adhesion-deficient, indigenous groundwater strains, Comamonas sp. strain DA001 and Erwinia herbicola OYS2-A, were studied by using intact sediment cores (7 by 50 cm) from Oyster, Va. Both strains are gram-negative rods (1.10 by 0.56 and 1.56 by 0.46 microm, respectively) with strongly hydrophilic membranes and a slightly negative surface charge. The two strains exhibited markedly different behaviors when they were transported through granular porous sediment. To eliminate any effects of physical and chemical heterogeneity on bacterial transport and thus isolate the biological effect, the two strains were simultaneously injected into the same core. DA001 cells were metabolically labeled with (35)S and tagged with a vital fluorescent stain, while OYS2-A cells were metabolically labeled with (14)C. The fast decay of (35)S allowed deconvolution of the two isotopes (and therefore the two strains). Dramatic differences in the transport behaviors were observed. The breakthrough of DA001 and the breakthrough of OYS2-A both occurred before the breakthrough of a conservative tracer (termed differential advection), with effluent recoveries of 55 and 30%, respectively. The retained bacterial concentration of OYS2-A in the sediment was twofold higher than that of DA001. Among the cell properties analyzed, the statistically significant differences between the two strains were cell length and diameter. The shorter, larger-diameter DA001 cells displayed a higher effluent recovery than the longer, smaller-diameter OYS2-A cells. CXTFIT modeling results indicated that compared to the DA001 cells, the OYS2-A cells experienced lower pore velocity, higher porosity, a higher attachment rate, and a lower detachment rate. All these factors may contribute to the observed differences in transport.

Relative dominance of physical versus chemical effects on the transport of adhesion-deficient bacteria in intact cores from South Oyster, Virginia.

Bacterial transport experiments were conducted using intact sediment cores collected from sites on the Delmarva Peninsula near South Oyster, VA, to delineate the relative importance of physical and chemical heterogeneity in controlling transport of an adhesion-deficient bacterial strain. Electron microscopy revealed that the sediments consisted of quartz and feldspar with a variable amount of clay and iron and aluminum hydroxide coatings on the grains. A nonmotile, gram-negative indigenous groundwater strain, designated as Comamonas sp. DA001, was injected into the cores along with a conservative tracer bromide (Br). DA001 cells were 1.2 x 0.6 microm in size with a hydrophilic surface and a slightly negative surface charge. Bacterial breakthrough preceded that of Br. This differential advection phenomenon can be accounted for by reduction of the effective porosity for the bacteria relative to Br. The distribution of cells remaining in the core as determined by scintillation counting and phosphor imaging techniques was variable, ranging from nearly uniform concentrations throughout the core to exponentially decreasing concentrations with distance from the point of injection. The fraction of bacterial retention in the core was positively correlated with the abundance of the metal hydroxides and negatively correlated with grain size. Because grain size was inversely correlated with the abundance of the metal hydroxide coatings, it was necessary to separate the effects of grain size and mineralogy. The fraction of the bacterial retention accounting for the effect of grain size, the collision efficiency, exhibited no correlation with the abundance of the metal hydroxides, indicating that the bacterial retention was primarily controlled by grain size. Reasons for the lack of influence of mineralogy on bacterial transport include (i) the slightly negatively charged bacterial surfaces; (ii) an insufficient heterogeneity of sediment surface properties; and (iii) the masking of the positive charge of the metal hydroxide surfaces by adsorbed organic carbon (up to 1180 ppm). This study demonstrates that the laboratory-based bacterial transport experiments are effective in delineating physical versus chemical controlling factors and provide an important link to field-based bacterial transport studies.