Prevalence of anaerobic ammonium-oxidizing bacteria in contaminated groundwater

Anaerobic ammonium-oxidizing (anammox) bacteria perform an important step in the global nitrogen cycle: anaerobic oxidation of ammonium and reduction of nitrite to form dinitrogen gas (N(2)). Anammox organisms appear to be widely distributed in natural and artificial environments. However, their roles in groundwater ammonium attenuation remain unclear and only limited biomarker-based data confirmed their presence prior to this study. We used complementary molecular and isotope-based methods to assess anammox diversity and activity occurring at three ammonium-contaminated groundwater sites: quantitative PCR, denaturing gradient gel electrophoresis, sequencing of 16S rRNA genes, and (15)N-tracer incubations. Here we show that anammox performing organisms were abundant bacterial community members. Although all sites were dominated by Candidatus Brocadia-like sequences, the community at one site was particularly diverse, possessing four of five known genera of anammox bacteria. Isotope data showed that anammox produced up to 18 and 36% of N(2) at these sites. By combining molecular and isotopic results we have demonstrated the diversity, abundance, and activity of these autotrophic bacteria. Our results provide strong evidence for their important biogeochemical role in attenuating groundwater ammonium contamination.     

Field study of pulsed air sparging for remediation of petroleum hydrocarbon contaminated soil and groundwater

Recent laboratory-scale studies strongly suggested an advantage to operating air-sparging systems in a pulsed mode; however, little definitive field data existed to support the laboratory-scale observations. This study aimed to evaluate the performance of a field-scale pulsed air-sparging system during a short-term pilot test and during long-term system operation. The air-sparging system consisted of 32 sparging points and had been previously operated in a continuous mode for two years before the field study was performed. The field study used instruments with continuous data logging capabilities to monitor the dynamic responses of groundwater and soil vapor parameters to air injection. The optimum pulsing frequency was based on the evidence that the hydrocarbon volatilization and oxygen dissolution rates dramatically dropped after the air-sparging system reached steady state. The short-term pilot test results indicated a substantial increase in hydrocarbon volatilization and biodegradation in pulsed operation. On the basis of the results of the pilottest, the air-sparging system was set to operate in a pulsed mode at an optimum pulsing frequency. Operation parameters were collected 2, 8, and 12 months after the start of the pulsed operation. The long-term monitoring results showed thatthe pulsed operation increased the average hydrocarbon removal rate (kg/day) by a factor of up to 3 as compared to the previous continuous operation. The pulsed air sparging has resulted in higher reduction rates of dissolved benzene, toluene, ethylbenzene, and xylenes (BTEX) than were observed during the continuous operation. Among BTEX, benzene's reduction rate was the highest during the pulsed air-sparging operation.     

Spatial and temporal variability of arsenic solid-state speciation in historically lead arsenate contaminated soils

The arsenic (As) solid-state speciation (i.e., oxidation state, precipitates, and adsorption complexes) is one of the most important factors controlling dissolved As concentrations at As contaminated sites. In this case study, two representative subsurface samples (i.e., oxidized and semi-reduced sites) from former lead arsenate contaminated soils in the northeastern United States were chosen to investigate the effects of aging on As retention mechanisms using multiscale spectroscopic techniques. X-ray powder diffraction (XRD), synchrotron based microfocused (micro) XRD, in situ micro-synchrotron based X-ray fluorescence spectroscopy (SXRF), and micro-X-ray absorption near edge structure (XANES) spectroscopy were used to compliment the final bulk X-ray absorption spectroscopy (XAS) analyses. In the sample from an oxic area, As is predominantly (approximately 71%) present as As(V) adsorbed onto amorphous iron oxyhydroxides with a residue (approximately 29%) of an original contaminant, schultenite (PbHAsO4). Contrarily, there is no trace of schultenite in the sample from a semi-reduced area. Approximately 25% of the total As is present as adsorbed phases on amorphous iron oxyhydroxide and amorphous orpiment (As2S3). The rest of the fractions (approximately 46%) were identified as As(V)-Ca coprecipitates. This study shows that aging effects can significantly alter the original chemical constituent (schultenite) in soils, resulting in multi and site-specific As solid-state speciation. The variability in spatial and temporal scale may be important in assessing the environmental risk and in developing in situ remediation technologies.     

Dual (C, H) isotope fractionation in anaerobic low molecular weight (poly)aromatic hydrocarbon (PAH) degradation: potential for field studies and mechanistic implications

Anaerobic polycyclic aromatic hydrocarbon (PAH) degradation is a key process for natural attenuation of oil spills and contaminated aquifers. Assessments by stable isotope fractionation, however, have largely been limited to monoaromatic hydrocarbons. Here, we report on measured hydrogen isotope fractionation during strictly anaerobic degradation of the PAH naphthalene. Remarkable large hydrogen isotopic enrichment factors contrasted with much smaller values for carbon: ε(H) = -100‰ ± 15‰, ε(C) = -5.0‰ ± 1.0‰ (enrichment culture N47); ε(H) = -73‰ ± 11‰, ε(C) = -0.7‰ ± 0.3‰ (pure culture NaphS2). This reveals a considerable potential of hydrogen isotope analysis to assess anaerobic degradation of PAHs. Furthermore, we investigated the conclusiveness of dual isotope fractionation to characterize anaerobic aromatics degradation. C and H isotope fractionation during benzene degradation (ε(C) = -2.5‰ ± 0.2‰; ε(H) = -55‰ ± 4‰ (sulfate-reducing strain BPL); ε(C) = -3.0‰ ± 0.5‰; ε(H) = -56‰ ± 8‰ (iron-reducing strain BF)) resulted in dual isotope slopes (Λ = 20 ± 2; 17 ± 1) similar to those reported for nitrate-reducers. This breaks apart the current picture that anaerobic benzene degradation by facultative anaerobes (denitrifiers) can be distinguished from that of strict anaerobes (sulfate-reducers, fermenters) based on the stable isotope enrichment factors.     

Sources of sulfate supporting anaerobic metabolism in a contaminated aquifer

Field and laboratory techniques were used to identify the biogeochemical factors affecting sulfate reduction in a shallow, unconsolidated alluvial aquifer contaminated with landfill leachate. Depth profiles of 35S-sulfate reduction rates in aquifer sediments were positively correlated with the concentration of dissolved sulfate. Manipulation of the sulfate concentration in samples revealed a Michaelis-Menten-like relationship with an apparent Km and Vmax of approximately 80 and 0.83 microM SO4(-2) x day(-1), respectively. The concentration of sulfate in the core of the leachate plume was well below 20 microM and coincided with very low reduction rates. Thus, the concentration and availability of this anion could limit in situ sulfate-reducing activity. Three sulfate sources were identified, including iron sulfide oxidation, barite dissolution, and advective flux of sulfate. The relative importance of these sources varied with depth in the alluvium. The relatively high concentration of dissolved sulfate at the water table is attributed to the microbial oxidation of iron sulfides in response to fluctuations of the water table. At intermediate depths, barite dissolves in undersaturated pore water containing relatively high concentrations of dissolved barium (approximately 100 microM) and low concentrations of sulfate. Dissolution is consistent with the surface texture of detrital barite grains in contact with leachate. Laboratory incubations of unamended and barite-amended aquifer slurries supported the field observation of increasing concentrations of barium in solution when sulfate reached low levels. At a deeper highly permeable interval just above the confining bottom layer of the aquifer, sulfate reduction rates were markedly higher than rates at intermediate depths. Sulfate is supplied to this deeper zone by advection of uncontaminated groundwater beneath the landfill. The measured rates of sulfate reduction in the aquifer also correlated with the abundance of accumulated iron sulfide in this zone. This suggests that the current and past distributions of sulfate-reducing activity are similar and that the supply of sulfate has been sustained at these sites.     

In situ anaerobic transformation of trichlorofluoroethene in trichloroethene-contaminated groundwater

Methods are needed to obtain in situ information on the transformation rates of trichloroethene (TCE), the most commonly detected organic groundwater contaminant. The objective of this research was to investigate the potential for determining TCE transformation rates in groundwater by measuring the transformation rate of its fluorinated surrogate, trichlorofluoroethene (TCFE). To explore this hypothesis, the in situ transport behavior, transformation pathway, and transformation rate of injected TCFE were determined in TCE-contaminated groundwater using single-well, push-pull tests. Although transport behavior varied between wells, TCFE, dichlorofluoroethene (DCFE), and TCE were transported similarly to each other. In the shallow water-bearing zone, TCFE was reductively dechlorinated to cis-DCFE, trans-DCFE, and (E)-1-chloro-2-fluoroethene (CFE), while co-injected TCE was concurrently transformed to cis-dichloroethene (DCE), trans-DCE, 1,1-DCE, and a trace amount of chloroethene (CE). With added formate and the injected TCFE concentration being a factor of 20 higher than that of TCE, the TCFE transformation rate ranged from 0.053 to 0.30 mumol/L-day, while that of TCE ranged from 0.009 to 0.012 mumol/L-day. Without added formate, the TCFE transformation rate decreased to 0.036 mumol/L-day. In the deeper water-bearing zone, TCFE transformation occurred only after a lag time of 55 days with added formate. No TCFE transformation occurred in groundwater that had not previously been exposed to TCE. The potential applicability for TCFE as an in situ transport and transformation surrogate for TCE was demonstrated.     

Evidence of enantioselective degradation of alpha-hexachlorocyclohexane in groundwater

In the fall of 2000, 34 groundwater samples were collected from beneath an active pesticide reformulating and packaging facility in coastal northeastern Florida to measure the enantiomer fractions (EFs) of alpha-hexachlorocyclohexane (alpha-HCH) as an indicator of biodegradation of this chlorinated pesticide in groundwater. Concentrations of alpha-HCH as high as 500 microg/L were observed beneath the historical source area and decreased with distance downgradient. Seventy-eight percent of the EF values were greater than 0.504 and ranged up to 0.890, indicating that the (-)-alpha-HCH enantiomer is preferentially degraded relative to the (+)-alpha-HCH enantiomer at this site. Samples taken from the groundwater that flows north from the historical disposal facility to a local discharge point at a creek did not indicate enantioselective degradation (EF values ranged from 0.495 to 0.512). The acidity (pH 3.7-4.6) and short flow path to the creek for this lobe of the groundwater plume likely preclude biodegradation of alpha-HCH. In contrast, the neutral lobe of the groundwater plume, which flows eastward from the historical source area, demonstrated enantioselective degradation (EF values ranged from 0.500 to 0.890 and increased with distance from the source area). Groundwater conditions beneath this portion of the site are conducive to biodegradation of HCH owing to anaerobic reducing conditions and lengthy travel times, and the chiral signatures for alpha-HCH provide evidence that biological degradation is occurring beneath this portion of the site.     

Remediation of polycyclic aromatic hydrocarbon compounds in groundwater using poplar trees

A seven-year study was conducted to assess the effectiveness of hybrid poplar trees to remediate polycyclic aromatic hydrocarbon (PAH) compounds in soil and groundwater at a creosote-contaminated site. A reduction in the areal extent of the PAH plume was observed in the upper half of the 2-m-thick saturated zone, and PAH concentration levels in the groundwater declined throughout the plume. PAH concentrations began to decline during the period between the third and fourth growing seasons, which coincided with the propagation of the tree roots to the water table region. Remediation was limited to naphthalene and several three-ring PAHs (acenaphthylene and acenaphthene). PAH concentrations in soil and aquifer sediment samples also declined over time; however, levels of four-ring PAHs persisted at the lower depths during the study period. The naphthalene to total PAH concentration ratio in the most contaminated groundwater decreased from >0.90 at the beginning of the second growing season to approximately 0.70 at the end the study. Remediation in the lower region of the saturated zone was limited bythe presence of a 0.3-m-thick layer of creosote present as a dense nonaqueous phase liquid (DNAPL). The nearly steady-state condition of the PAH concentrations observed during the last three years of the study suggests that the effectiveness of the phytoremediation system is limited by the rate of PAH dissolution from the DNAPL source.     

Hydrous ferric oxide incorporated diatomite for remediation of arsenic contaminated groundwater

Two reactive media [zerovalent iron (ZVI, Fisher Fe0) and amorphous hydrous ferric oxide (HFO)-incorporated porous, naturally occurring aluminum silicate diatomite [designated as Fe (25%)-diatomite]], were tested for batch kinetic, pH-controlled differential column batch reactors (DCBRs), in small- and large-scale column tests (about 50 and 900 mL of bed volume) with groundwater from a hazardous waste site containing high concentrations of arsenic (both organic and inorganic species), as well as other toxic or carcinogenic volatile and semivolatile organic compounds (VOC/SVOCs). Granular activated carbon (GAC) was also included as a reactive media since a permeable reactive barrier (PRB) at the subject site would need to address the hazardous VOC/SVOC contamination as well as arsenic. The groundwater contained an extremely high arsenic concentration (341 mg L(-1)) and the results of ion chromatography and inductively coupled plasma mass spectrometry (IC-ICP-MS) analysis showed that the dominant arsenic species were arsenite (45.1%) and monomethyl arsenic acid (MMAA, 22.7%), while dimethyl arsenic acid (DMAA) and arsenate were only 2.4 and 1.3%, respectively. Based on these proportions of arsenic species and the initial As-to-Fe molar ratio (0.15 molAs mole(-1)), batch kinetic tests revealed that the sorption density (0.076 molAs molFe(-1)) for Fe (25%)-diatomite seems to be less than the expected value (0.086 molAs molFe(-1) calculated from the sorption density data reported by Lafferty and Loeppert (Environ. Sci. Technol. 2005, 39, 2120-2127), implying that natural organic matters (NOMs) might play a significant role in reducing arsenic removal efficiency. The results of pH-controlled DCBR tests using different synthetic species of arsenic solution showed that the humic acid inhibited the MMAA removal of Fe (25%)-diatomite more than arsenite. The mixed system of GAC and Fe (25%)-diatomite increased the arsenic sorption speed to more than that of either individual media alone. This increase might be deduced by the fact that the addition of GAC could enhance arsenic removal performance of Fe (25%)-diatomite through removing comparably high portions of NOMs. Small- and large-scale column studies demonstrated that the empty bed contact time (EBCT) significantly affected sorpton capacities at breakthrough (C = 0.5 C0) forthe Fe0/sand (50/50, w/w) mixture, but notfor GAC preloaded Fe (25%)-diatomite. In the large-scale column tests with actual groundwater conditions, the GAC preloaded Fe (25%)-diatomite effectively reduced arsenic to below 50 microg L(-1) for 44 days; additionally, most species of VOC/SVOCs were also simultaneously attenuated to levels below detection.     

Numerical model for biological fluidized-bed reactor treatment of perchlorate contaminated groundwater

Biological fluidized-bed reactor (BFBR) treatment with 1.3 mm granular activated carbon as support medium is being used for removal of 2.6 mg/L perchlorate from contaminated groundwater in California. The California drinking-water action level of 4 microg/L for perchlorate requires 99.9% perchlorate removal. Sufficient ethanol, the electron donor, is added to remove oxygen and nitrate as well as perchlorate, as all three serve as electron acceptors, but with biological preference for oxygen and nitrate. A numerical BFBR model based upon basic physical, chemical, and biological processes including reaction stoichiometry, biofilm kinetics, and sequential electron acceptor usage was developed and evaluated with the full-scale treatment results. A key fitting parameter was bacterial detachment rate, which impacts reaction stoichiometry. For best model fit this was found to vary between 0.062 and 0.31 d(-1), with an average of 0.22 d(-1). The model indicates that GAC particle size, reactor diameter, and perchlorate concentration affect BFBR performance. While empty-bed detention time might be decreased somewhat below 10 min by an increase in either GAC particle size or reactor diameter, the current design provides a good factor of safety in operation. With a 10 min detention time, the effluent goal of 4 microg/L should be achievable even with influent perchlorate concentration as high as 10 mg/L.     

Major issues regarding the efficiency of monitoring programs for nitrate contaminated groundwater

Major issues regarding the efficiency of moni toring programs for nitrate contaminated groundwater are analyzed in this paper: (i) representativeness of monitoring networks; (ii) correct interpretation of the monitoring data and resulting time series and trends; and (iii) differentiation among the different sources of nitrates in groundwater. Following an overview of the nitrate contamination problem and possible solutions, as well as some of the difficulties found, a relatively straightforward method for assessing monitoring network representativity is presented, namely interpolation standard error assessment. It is shown how nitrate-concentration time series resulting from periodic observations can be corrected with a conservative tracer, in order to avoid misinterpretation and confirm or correct apparent trends. Finally, coupled 1?N and 1?O isotope signatures of nitrate (NO??) in groundwater are used to differentiate among nitrogen (N) sources, to ensure correct targeting of restoration measures. The case study regards a Nitrate Vulnerable Zone in the south of Portugal, designated in compliance with the European Nitrates Directive, where coastal discharge of nutrient-rich groundwater threatens the good qualitative and ecological status of the Ria Formosa coastal lagoon. Results show that mineral fertilizer is the main source of N in groundwater, and that increases in N load can be masked by dilution phenomena.     

粪产碱菌对阿魏酸厌氧降解的影响

粪产碱菌在以阿魏酸为唯一碳源的厌氧发酵中培养7 d,阿魏酸的降解率约为70%;研究秸秆厌氧发酵产气（以产气量反应菌群的活性）发现,体系接种5%粪产碱菌后,菌群的活性最强,生物气增量最大,达130 mL,比接种3%和7%粪产碱菌的体系提高85.71%和116.67%;同时体系的产酸效率和阿魏酸的降解率均显著提升,分别比接种3%和7%粪产碱菌的体系提高136%和110.71%以及25%和33.33%。傅里叶红外光谱检测表明：厌氧发酵体系接种5%粪产碱菌后秸秆中木质素、阿魏酸的特征官能团结构被有效破坏。秸秆厌氧发酵体系接入粪产碱菌可以有效降解阿魏酸等木质素降解衍生物、解除木质素及其降解产物对厌氧菌群的毒性同时提高产气效率,具有应用价值。     

Sensitive detection of anaerobic monochlorobenzene degradation using stable isotope tracers

Microbial degradation of monochlorobenzene (MCB) under anaerobic conditions was investigated using a stable isotope tracer under in and ex situ conditions. In situ microcosms were incubated directly in an anoxic aquifer and carbon derived from [13C6]-MCB was found to be incorporated into the microbial biomass. In laboratory microcosms, amended with [13C6]-MCB, anaerobic mineralization of MCB was indicated by the production of 13CO2. Further, recovery of the 13C-label in the fatty acids confirmed the assimilation of MCB-derived carbon into microbial biomass. The described approach may be applied to various other organic groundwater contaminants of concern using carbon (13C) as well as other stable isotope tracers, such as nitrogen (15N), allowing direct and sensitive detection of biodegradation.     

Sustained removal of uranium from contaminated groundwater following stimulation of dissimilatory metal reduction

Previous field studies on in situ bioremediation of uranium-contaminated groundwater in an aquifer in Rifle, Colorado identified two distinct phases following the addition of acetate to stimulate microbial respiration. In phase I, Geobacter species are the predominant organisms, Fe(III) is reduced, and microbial reduction of soluble U(VI) to insoluble U(IV) removes uranium from the groundwater. In phase II, Fe(III) is depleted, sulfate is reduced, and sulfate-reducing bacteria predominate. Long-term monitoring revealed an unexpected third phase during which U(VI) removal continues even after acetate additions are stopped. All three of these phases were successfully reproduced in flow-through sediment columns. When sediments from the third phase were heat sterilized, the capacity for U(VI) removal was lost. In the live sediments U(VI) removed from the groundwater was recovered as U(VI) in the sediments. This contrasts to the recovery of U(IV) in sediments resulting from the reduction of U(VI) to U(IV) during the Fe(III) reduction phase in acetate-amended sediments. Analysis of 16S rRNA gene sequences in the sediments in which U(VI) was being adsorbed indicated that members of the Firmicutes were the predominant organisms whereas no Firmicutes sequences were detected in background sediments which did not have the capacity to sorb U(VI), suggesting that the U(VI) adsorption might be due to the presence of these living organisms or at least their intact cell components. This unexpected enhanced adsorption of U(VI) onto sediments following the stimulation of microbial growth in the subsurface may potentially enhance the cost effectiveness of in situ uranium bioremediation.     

Performance of a sequential reactive barrier for bioremediation of coal tar contaminated groundwater

Following a thorough site investigation, a biological Sequential Reactive Barrier (SEREBAR), designed to remove Polycyclic Aromatic Hydrocarbons (PAHs) and BTEX compounds, was installed at a Former Manufactured Gas Plant (FMGP) site. The novel design of the barrier comprises, in series, an interceptor and six reactive chambers. The first four chambers (2 nonaerated-2 aerated) were filled with sand to encourage microbial colonization. Sorbant Granular Activated Carbon (GAC) was present in the final two chambers in order to remove any recalcitrant compounds. The SEREBAR has been in continuous operation for 2 years at different operational flow rates (ranging from 320 L/d to 4000 L/d, with corresponding residence times in each chamber of 19 days and 1.5 days, respectively). Under low flow rate conditions (320-520 L/d) the majority of contaminant removal (>93%) occurred biotically within the interceptor and the aerated chambers. Under high flow rates (1000-4000 L/d) and following the installation of a new interceptor to prevent passive aeration, the majority of contaminant removal (>80%) again occurred biotically within the aerated chambers. The sorption zone (GAC) proved to be an effective polishing step, removing any remaining contaminants to acceptable concentrations before discharge down-gradient of the SEREBAR (overall removals >95%).     

Bioremediation of groundwater contaminated with gasoline hydrocarbons and oxygenates using a membrane-based reactor

The objective of this study was to operate a novel, field-scale, aerobic bioreactor and assess its performance in the ex situ treatment of groundwater contaminated with gasoline from a leaking underground storage tank in Pascoag, RI. The groundwater contained elevated concentrations of MTBE (methyl tert-butyl ether), TBA (tert-butyl alcohol), TBF (tert-butyl formate), BTEX (benzene, toluene, ethyl benzene, and xylene isomers), and other gasoline additives (tert-amyl methyl ether, di-isopropyl ether, tert-amyl alcohol, methanol, and acetone). The bioreactor was a gravity-flow membrane-based system called a Biomass Concentrator Reactor (BCR) designed to retain all biomass within the reactor. It was operated for six months at an influent flow rate that ultimately reached 5 gpm. The goal was to achieve a removal of all contaminants to <5 microg/L, which is the California Drinking Water advisory for MTBE. The concentration of TBA, an MTBE biodegradation byproduct, was consistently lower than that of MTBE. The other daughter compound detected in the influent, TBF, was degraded to concentrations below the detection limit of 0.02 microg/L. BTEX were consistently degraded to significantly lower levels in the effluent throughout the duration of the study (<1 microg/L). A similar high removal efficiency of the other gasoline oxygenates present in the groundwater (TAME, DIPE, and TAA) was also achieved. Dissolved organic carbon analysis demonstrated the ability of the bioreactor to produce high quality effluents with nonpurgeable organic carbon (NPOC) averaging approximately 50% lowerthan the NPOC concentrations in the influent contaminated groundwater.     

Microscale analysis of in vitro anaerobic degradation of lignocellulosic wastes by rumen microorganisms

Anaerobic degradation of lignin in waste straw by ruminal microbes was directly observed using atomic force microscope (AFM). A series of high-resolution AFM images of the straw surface in the biodegradation show that the wax flakelets and lignin granules covering the straw surface were removed by the rumen microorganisms. Such degradation resulted in an exposure of cellulose fibers located inside the straw. The appearance of holes and microfibers in fermentation reveals that tunneling might be one of the ways for rumen microorganisms to attack the straw. Increases in the atomic ratio of oxygen to carbon (O/C) and the ratio C2/C3 in C1s spectra of X-ray photoelectron spectroscopy confirm that more cellulose was exposed on the surface after the anaerobic fermentation of straw. Gas chromatography/mass spectrometry analytical results demonstrate the decomposition of lignin by rumen microorganisms. Fourier transform infrared spectroscopy spectra and the measurement of degradation efficiency of the main straw components further verify these microscaled observations.