Response surface optimization of dissolved oxygen and nitrogen sources for the biodegradation of MTBE and BTEX

Response surface methodology (RSM) using central composite design was applied to obtain the optimal dissolved oxygen (DO) and nitrogen (N) concentrations for biodegrading MTBE (Methyl tert-butyl ether) and BTEX (benzene, ethylbenzene, toluene, p-xylene). Moreover, the effects of DO, N, and their interaction on the degradation process were evaluated. It was found that N, N², DO and DO² have significant effects on the efficiency of MTBE and BTEX removal. The removal efficiency when using biostimulation with bioaugmentation (BwB) is higher than with other processes, being greater than 82% at concentrations of 12 and 48 mg l⁻¹ for DO and N, respectively. However, it was also found that the interaction term of DO × N has no significant effect on the degradation processes.     

A Field Application of Hydrogen-Releasing Compound (HRCTM) for the Enhanced Bioremediation of Methyl Tertiary Butyl Ether (MTBE)

Due to a greater understanding of the behavior of the fuel oxygenate Methyl Tertiary Butyl Ether (MTBE) in groundwater, the United States Environmental Protection Agency (EPA) and the American Petroleum Institute (API) recently have acknowledged the need for the development and application of additional remedial strategies to address the more extensive, longer lived, and faster moving dissolved MTBE plumes often associated with oxygenated fuel releases (API, 2000 and USEPA, 2000a). The need for alternative methods for managing dissolved MTBE plumes is particularly evident in the case of the Upper Glacial aquifer of Long Island, New York. Hydrogeologic conditions in the this water table aquifer (i. e., high hydraulic conductivity, high average pore velocities, low organic carbon, and high rates of recharge) have been found to contribute to the formation of extensive, long, narrow, and three-dimensional dissolved MTBE plumes that plunge into the aquifer in response to recharge (Weaver et. al. 1999). The characteristics of MTBE plumes in the Upper Glacial aquifer in combination with abundant sensitive receptors (mainly drinking water supply wells), often renders monitored natural attenuation (MNA) plume management strategies inappropriate, resulting in the need for plume control, frequently via pumping and treating (NYSDEC, 2000). In such cases, remedial costs can rise well beyond those associated with similar fuel releases that did not contain MTBE (USEPA, 1998a). Consequently, the application of remedial technologies for MTBE other than MNA, or pumping and treating, are of great interest to those responsible for the management of dissolved MTBE plumes on Long Island or in similar hydrogeologic settings. An alternative strategy for the remediation of dissolved MTBE plumes was recently field tested at an oxygenated fuel spill site on Long Island. The strategy was enhanced biodegradation via the application of Hydrogen Release Compound (HRC^TM). HRC^TM is a form of polylactate ester that slowly releases biodegradation stimulating constituents into the aquifer and has been shown in other studies to foster methanogenic conditions that advance the reductive dechlorina-tion of perchloroethene (PCE) and trichloroethene (TCE) (Koenigsberg, 1998). Numerous reports have been written that discuss the biodegradation of MTBE under aerobic conditions, as well as microcosm studies in which MTBE biodegradation was observed under anaerobic conditions. However, there are limited reports that document the natural anaerobic biodegradation of dissolved MTBE (McLoughlin, 2000). Despite the lack of documented natural anaerobic biodegradation of MTBE, it has been observed that MTBE transport often occurs under anoxic conditions at oxygenated fuel releases as the result of the biodegradation of other fuel constituents, such as benzene, toluene, ethylbenzene and xylene (BTEX), which deplete the available dissolved oxygen as well as other electron acceptors (nitrate, ferric iron, manganese, etc.) (USEPA, 2000c and API, 1996). Therefore, an anaerobic biodegradation strategy is attractive due to its synergy with the existing geochemical conditions. Consequently, the study was conceived and designed to test the ability of HRC(tm) to foster the anaerobic bio-degradation of MTBE under methano-genic conditions (McLoughlin, 2000). The application of HRC(tm) did result in the formation of a large area of enhanced reducing conditions in the vicinity and down gradient of the application zone. However, under these site conditions, the HRC(tm) application did not induce measurable methanogenic conditions with the associated elevated dissolved hydrogen concentrations required for significant MTBE anaerobic biodegradation. The high hydraulic conductivity and high average pore velocity at the site were likely responsible. Despite this, the study can be viewed as a success since much was learned that can be used in future studies of anaerobic biodegradation of MTBE and the application of HRC(tm).     

SEQUENCE Visualization of Natural Attenuation Trends at Hill Air Force Base, Utah

For monitored natural attenuation to be considered as an acceptable remedial approach, the proponent must clearly document converging lines of evidence that illustrate the effectiveness of this measure. SEQUENCE, a visualization tool based on a modified radial diagram approach, is ideally suited for evaluating spatial and temporal trends that provide supporting evidence for the efficacy of monitored natural attenuation. SEQUENCE was applied to evaluate the natural attenuation of benzene, toluene, ethylbenzene, and total xylene (BTEX) concentrations observed in groundwater at Hill Air Force Base, Utah. SEQUENCE-BTEX maps provided an efficient means of documenting the declining BTEX concentrations downgradient from the source area. SE-QUENCE-Redox maps were used to facilitate a correlation between elevated BTEX concentrations; decreasing electron acceptor concentrations (oxygen, nitrate, and sulfate); and elevated metabolic byproduct concentrations (iron(II) and methane) providing a second line of evidence that suggests BTEX compounds are being destroyed through biodegradation processes downgradient from the source area. Site-specific guidelines for interpolating representative data sets for use with the SEQUENCE approach are discussed.     

Intrinsic bioremediation in a solvent-contaminated alluvial groundwater

An industrial site contaminated with a mixture of volatile organic compounds in its subsurface differed from previously reported locations in that the contamination consisted of a mixture of chlorinated, brominated, and non-halogenated aromatic and aliphatic solvents in an alluvial aquifer. The source area was adjacent to a river. Of the contaminants present in the aquifer, benzene, toluene, and chlorobenzene (BTC) were of primary concern. Studies of the physical, chemical, and microbiological characteristics of site groundwater were conducted. The studies concentrated on BTC, but also addressed the fate of the other aquifer VOCs. Gas chromatographic analyses performed on laboratory microcosms demonstrated that subsurface microorganisms were capable of BTC degradation. Mineralization of BTC was demonstrated by the release of ¹⁴CO₂ from radiolabelled BTC. In the field, distribution patterns of nutrients and electron acceptors were consistent with expression of in situ microbial metabolic activity: methane, conductivity, salinity and o-phosphate concentrations were all positively correlated with contaminant concentration; while oxidation-reduction potential, nitrate, dissolved oxygen and sulfate concentrations were negatively correlated. Total aerobes, aerotolerant anaerobes, BTC-specific degraders, and acridine orange direct microscopic microorganism counts were strongly and positively correlated with field contaminant concentrations. The relative concentrations of benzene and toluene were lower away from the core of the plume compared to the less readily metabolized compound, chlorobenzene. Hydrodynamic modeling of electron-acceptor depletion conservatively estimated that 450 kg of contaminant have been removed from the subsurface yearly. Models lacking a biodegradation term predicted that 360 kg of contaminant would reach the river annually, which would result in measurable contaminant concentrations. River surveillance, however, has only rarely detected these compounds in the sediment and then only at trace concentrations. Thus, the combination of field modeling, laboratory studies, and site surveillance data confirm that significant in situ biodegradation of the contaminants has occurred. These studies establish the presence of intrinsic bioremediation of groundwater contaminants in this unusual industrial site subsurface habitat. Received 01 December 1995/ Accepted in revised form 27 July 1996     

Implementation of natural attenuation at a JP-4 jet fuel release after active remediation

After eighteen months of active remediation at a JP-4 jet-fuel spill, aresidual of unremediated hydrocarbon remained. Further site characterizationwas conducted to evaluate the contribution of natural attenuation to controlexposure to hazards associated with the residual contamination in thesubsurface. Activities included the detailed characterization ofground-water flow through the spill; the distribution of fuel contaminantsin groundwater; and the analysis of soluble electron acceptors moving intothe spill from upgradient. These activities allowed a rigorous evaluation ofthe transport of contaminants from the spill to the receptor of groundwater,the Pasquotank River. The transport of dissolved contaminants of concern,that is benzene, toluene, ethyl benzene, xylene isomers (BTEX) andmethyl-tertiary-butyl ether (MTBE), into the river from the source area wascontrolled by equilibrium dissolution from the fuel spill to the adjacentgroundwater, diffusion in groundwater from the spill to permeable layers inthe aquifer, and advective transport in the permeable layers. The estimatedyearly loading of BTEX compounds and MTBE into the receptor was trivial evenwithout considering biological degradation. The biodegradation ofhydrocarbon dissolved in groundwater through aerobic respiration,denitrification, sulfate reduction, and iron reduction was estimated fromchanges in ground-water chemistry along the flow path. The concentrations oftarget components in permanent monitoring wells continue to decline overtime. Long term monitoring will ensure that the plume is under control, andno further active remediation is required.     

Detection of intermediate metabolites of benzene biodegradation under microaerophilic conditions

The intermediate metabolites of benzene transformation by a microaerophilic bacterial consortium, adapted to degrade gasoline and benzene at low concentrations of dissolved oxygen (<1 mg l^-1), were identified. The examined range of initial DO concentration, 0.05 to 1 mg l^-1, was considerably lower than the previously reported values believed to be necessary to initiate benzene biodegradation. An extensive transformation of benzene, higher than the theoretical predictions for its aerobic oxidation, was observed. Phenol was identified as the most stable and the major intermediate metabolite which was subsequently transformed into catechol and benzoate. The use of ¹³C-labeled compounds identified benzene as the source of phenol, and phenol as the source of catechol and benzoate, suggesting the involvement of a monooxygenase enzymatic system in biodegradation of benzene at low DO concentrations. A metabolic sequence was proposed to describe the simultaneous detection of catechol and benzoate during the microaerophilic transformation of benzene. The results of this work demonstrate that it is possible to transform benzene, a highly carcinogenic hydrocarbon and a major contaminant of groundwater, to more easily biodegradable compounds in the presence of very small amounts of oxygen.     

Aerobic Biotransformation of Gasoline Aromatics in MultiComponent Mixtures

The primary objective of this study was to evaluate the impact of substrate interactions on the biotransformation rates and mineralization potentials of gasoline monoaromatics and methyl tert-butyl ether (MTBE), compounds that commonly co-exist in groundwater contaminant plumes. A mixed culture was derived from gasoline-contaminated aquifer material using toluene as the enrichment substrate. Two pure cultures, Rhodococcus sp. RR1 and RR2, were isolated from the mixed culture. The three toluene-grown cultures were shown to biotransform all of the six BTEX compounds (benzene, toluene, ethylbenzene, o-xylene, m-xylene, and p-xylene), both individually and in mixtures, over a broad range of concentrations. The mixed culture was shown to degrade all of the BTEX compounds to ¹⁴CO₂, while the two isolates mineralized BTE(m-/p-)X, but biotransformed o-xylene without production of carbon dioxide. Studies to evaluate substrate interactions caused by the concurrent presence of multiple BTEX compounds during their biodegradation revealed a number of patterns,including competitive inhibition and cometabolism. Ethylbenzene was shown to significantly inhibit BTX degradation in mixtures. MTBE was not biodegraded by any of the three toluene-grown cultures over a range of MTBE concentrations. Furthermore, the presence of MTBE at concentrations of 2 to 100?mg/L had no effect on BTEX biotransformation rates.     

Effects of environmental settings on MTBE removal for a mixed culture and its monoculture isolation

A mixed culture was utilized to evaluate methyl tert-butyl ether (MTBE) removal under various conditions and to isolate a MTBE-degrading pure culture. The results showed that high MTBE removal efficiencies can be reached even in the presence of other substrates. The biodegradation sequence of the target compounds by the mixed culture, in order of removal rate, was toluene, ethyl benzene, p-xylene, benzene, MTBE, ethyl ether, tert-amyl methyl ether, and ethyl tert-butyl ether. In addition, preincubation of the mixed cultures with benzene and toluene showed no negative effect on MTBE removal; on the contrary, it could even increase the degradation rate of MTBE. The kinetic behavior showed that the maximum specific growth rate and the saturation constant of the mixed culture degrading MTBE are 0.000778 h⁻¹ and 0.029 mg l⁻¹, respectively. However, a high MTBE concentration (60 mg l⁻¹) was slightly inhibiting to the growth of the mixed culture. The pure culture isolated from the enrichments in the bubble-air bioreactor showed better efficiency in MTBE removal than the mixed culture; whereas, tert-butyl alcohol was formed as a metabolic intermediate during the breakdown of MTBE.     

Assessment of Nitrification Potential in Ground Water Using Short Term, Single-Well Injection Experiments

Nitrification was measured within a sand and gravel aquifer on Cape Cod, MA, using a series of single-well injection tests. The aquifer contained a wastewater-derived contaminant plume, the core of which was anoxic and contained ammonium. The study was conducted near the downgradient end of the ammonium zone, which was characterized by inversely trending vertical gradients of oxygen (270 to 0 μM) and ammonium (19 to 625 μM) and appeared to be a potentially active zone for nitrification. The tests were conducted by injecting a tracer solution (ambient ground water + added constituents) into selected locations within the gradients using multilevel samplers. After injection, the tracers moved by natural ground water flow and were sampled with time from the injection port. Rates of nitrification were determined from changes in nitrate and nitrite concentration relative to bromide. Initial tests were conducted with ¹⁵N-enriched ammonium; subsequent tests examined the effect of adding ammonium, nitrite, or oxygen above background concentrations and of adding difluoromethane, a nitrification inhibitor. In situ net nitrate production exceeded net nitrite production by 3- to 6- fold and production rates of both decreased in the presence of difluoromethane. Nitrification rates were 0.02–0.28 μmol (L aquifer)⁻¹ h⁻¹ with in situ oxygen concentrations and up to 0.81 μmol (L aquifer)⁻¹ h⁻¹ with non-limiting substrate concentrations. Geochemical considerations indicate that the rates derived from single-well injection tests yielded overestimates of in situ rates, possibly because the injections promoted small-scale mixing within a transport-limited reaction zone. Nonetheless, these tests were useful for characterizing ground water nitrification in situ and for comparing potential rates of activity when the tracer cloud included non-limiting ammonium and oxygen concentrations.     

Vertical change in dissolved organic carbon and oxygen at the water table region of an aquifer recharged with stormwater: biological uptake or mixing?

Decreases in dissolved organic carbon (DOC) and dissolved oxygen (DO) with increasing depth below the groundwater table are often considered as evidence for aerobic respiration; however, they may reflect mixing of infiltrating water and groundwater. We found that groundwater DOC concentration was on average 0.3 mg C l^?1 higher and DO concentration 1.5 mg O₂ l^?1 lower at recharge sites replenished with stormwater than at reference sites fed by direct infiltration of rain water from the land surface. Groundwater DOC increased and DO decreased with increasing vadose zone thickness (VZT) at both recharge and reference sites. There was no significant interaction between the effects of stormwater infiltration and VZT. Vertical changes in DOC and DO below the groundwater table at recharge sites could account for by simple mixing of infiltrating stormwater and groundwater. Moreover, aquifer sediment respiration (SR) was not significantly higher at recharge sites than at reference sites. However, slow filtration column experiments showed that SR increased significantly with an increasing supply of easily biodegradable DOC. We conclude that the observed reduction in DOC below the groundwater table at recharge sites was essentially due to water mixing rather than biological uptake because of the low biodegradability of the DOC and the short transit time of stormwater in the upper layers of groundwater. Our results highlight the need to distinguish between the effect of hydrological and biological processes on DOC and DO patterns below the groundwater before conclusions are made on the efficiency of groundwater in degrading surface-derived DOC.     

Kinetics of benzene biotransformation under microaerophilic and oxygen-limited conditions

A special microbial consortium adapted to degrade petroleum hydrocarbons at limited availability of oxygen, transformed benzene, a highly toxic and carcinogenic contaminant of groundwater and soil, at low initial dissolved oxygen (DO) concentrations of 0.05-2 mg/L. The employed initial concentrations of dissolved oxygen were considerably lower than the previously reported values. Under these conditions, the overall transformation of benzene ranged from 34% +/- 1.7% to 100%, considerably higher than the theoretical predictions for complete mineralization of benzene based on the requirement of 3.08 mg oxygen/mg benzene. Unlike biotransformation that proceeded at the lowest examined DO concentration of 0.05 mg/L, the mineralization of benzene, defined by its conversion to CO(2) and water, required a minimum DO concentration of 0.2 mg/L. The mineralization of benzene under microaerophilic conditions (DO < 2 mg/L), ranged from 0.83% +/- 0.06% to 89% +/- 1.3%, which was less than the theoretical predictions at any given initial DO concentration. The regulatory effects of dissolved oxygen concentration or its partial pressure on the activities of enzymes catalyzing the biotransformation of aromatic hydrocarbons was postulated to account for the reduced mineralization of benzene. The ratio between the transformed benzene and the consumed oxygen increased with the decrease of initial DO concentration, reaching a value of 2.8, considerably higher than the theoretical value of 0.33 obtained for a complete aerobic oxidation of benzene. Phenol was the major and the most stable intermediate metabolite during the biotransformation of benzene at low concentrations of DO. While benzene transformation stopped after the depletion of oxygen in the experimental system, phenol continued to accumulate under strictly anaerobic conditions, indicating its formation from an alternative carbon source, possibly biomass.     

Methyl tert-butyl ether (MTBE) degradation by a microbial consortium

The widespread use of methyl tert-butyl ether (MTBE) as a gasoline additive has resulted in a large number of cases of groundwater contamination. Bioremediation is often proposed as the most promising alternative after treatment. However, MTBE biodegradation appears to be quite different from the biodegradation of usual gasoline contaminants such as benzene, toluene, ethyl benzene and xylene (BTEX). In the present paper, the characteristics of a consortium degrading MTBE in liquid cultures are presented and discussed. MTBE degradation rate was fast and followed zero order kinetics when added at 100 mg l(-1). The residual MTBE concentration in batch degradation experiments ranged from below the detection limit (1 microg l(-1)) to 50 microg l(-1). The specific activity of the consortium ranged from 7 to 52 mgMTBE g(dw)(-1) h(-1) (i.e. 19-141 mgCOD g(dw) (-1) h(-1)). Radioisotope experiments showed that 79% of the carbon-MTBE was converted to carbon-carbon dioxide. The consortium was also capable of degrading a variety of hydrocarbons, including tert-butyl alcohol (TBA), tert-amyl methyl ether (TAME) and gasoline constituents such as benzene, toluene, ethylbenzene and xylene (BTEX). The consortium was also characterized by a very slow growth rate (0.1 d(-1)), a low overall biomass yield (0.11 gdw g(-1)MTBE; i.e. 0.040 gdw gCOD(-1)), a high affinity for MTBE and a low affinity for oxygen, which may be a reason for the slow or absence of MTBE biodegradation in situ. Still, the results presented here show promising perspectives for engineering the in situ bioremediation of MTBE.     

Effects of Oxygenation on Hydrocarbon Biodegradation in a Hypoxic Environment

In 1984, an underground storage tank leaked approximately 41,000 L of gasoline into the ground water at the Naval Construction Battalion Command in Port Hueneme, CA (USA). Benzene, toluene, ethylbenzene, and xylenes (BTEX) contamination stimulated remedial action. In 1995, a ground water circulation well (GCW) and network of surrounding monitoring wells were installed. After year of operation, dissolved oxygen and nitrate concentrations remained low in all monitoring wells. Benzene utilization (the sum of respiration, uptake, and conversion to polar compounds) ranged from 0.03 to 4.6 µg L^-1 h^-1, and toluene utilization ranged from 0.01 to 5.2 µg L^-1 h^-1. Heterotrophic bacterial productivity (total carbon assimilation) increased dramatically in the GCW, although benzene and toluene utilization decreased markedly relative to surrounding wells. Benzene and toluene uptake accounted for a significant proportion (mean=22%) of the heterotrophic bacterial productivity except within the GCW, indicating other fuel contaminant or indigenous organic carbon and not BTEX compounds served as primary carbon source. The GCW effectively air-stripped BTEX compounds, but failed to stimulate benzene and toluene biodegradation and thus would not be effective for stimulating BTEX bioremediation under current deployment parameters. Air stripping was three orders of magnitude more effective than biodegradation for removing benzene and toluene in the GCW.     

Denitrification in a Sand and Gravel Aquifer

Denitrification was assayed by the acetylene blockage technique in slurried core material obtained from a freshwater sand and gravel aquifer. The aquifer, which has been contaminated with treated sewage for more than 50 years, had a contaminant plume greater than 3.5-km long. Near the contaminant source, groundwater nitrate concentrations were greater than 1 mM, whereas 0.25 km downgradient the central portion of the contaminant plume was anoxic and contained no detectable nitrate. Samples were obtained along the longitudinal axis of the plume (0 to 0.25 km) at several depths from four sites. Denitrification was evident at in situ nitrate concentrations at all sites tested; rates ranged from 2.3 to 260 pmol of N₂O produced (g of wet sediment)⁻¹ h⁻¹. Rates were highest nearest the contaminant source and decreased with increasing distance downgradient. Denitrification was the predominant nitrate-reducing activity; no evidence was found for nitrate reduction to ammonium at any site. Denitrifying activity was carbon limited and not nitrate limited, except when the ambient nitrate level was less than the detection limit, in which case, even when amended with high concentrations of glucose and nitrate, the capacity to denitrify on a short-term basis was lacking. These results demonstrate that denitrification can occur in groundwater systems and, thereby, serve as a mechanism for nitrate removal from groundwater.     

Aerobic Biodegradation of Methyl tert-Butyl Ether by Aquifer Bacteria from Leaking Underground Storage Tank Sites

The potential for aerobic methyl tert-butyl ether (MTBE) degradation was investigated with microcosms containing aquifer sediment and groundwater from four MTBE-contaminated sites characterized by oxygen-limited in situ conditions. MTBE depletion was observed for sediments from two sites (e.g., 4.5 mg/liter degraded in 15 days after a 4-day lag period), whereas no consumption of MTBE was observed for sediments from the other sites after 75 days. For sediments in which MTBE was consumed, 43 to 54% of added [U-¹⁴C]MTBE was mineralized to ¹⁴CO₂. Molecular phylogenetic analyses of these sediments indicated the enrichment of species closely related to a known MTBE-degrading bacterium, strain PM1. At only one site, the presence of water-soluble gasoline components significantly inhibited MTBE degradation and led to a more pronounced accumulation of the metabolite tert-butyl alcohol. Overall, these results suggest that the effects of oxygen and water-soluble gasoline components on in situ MTBE degradation will vary from site to site and that phylogenetic analysis may be a promising predictor of MTBE biodegradation potential.     

Modeling the Impact of Oxygen Reaeration on Natural Attenuation

Based on studies of leaking petroleum storage tank (LPST) sites in Texas and California, the average plume of benzene, toluene, ethylene, and xylenes (BTEX) is between 61 and 132 m (200 and 400 ft) long. Standard modeling of BTEX plumes produces plumes well in excess of observed plume lengths. The amount of oxygen carried into the plume zone with clean upgradient water often is insufficient to account for the levels of biodegradation observed in these studies. Traditional recharge of oxygen-containing water into an aquifer adds insufficient oxygen to the system and cannot account for the observed plume lengths. Research has shown that anaerobic processes can contribute to biodegradation in certain cases; however, anaerobic pathways are not included in this work. Reaeration of oxygen-depleted aquifers by diffusive transport of oxygen through the vadose zone has generally been neglected as a way to introduce oxygen into surficial aquifers. The observed plume lengths and preliminary laboratory results indicate that this source of oxygen should be accounted for in any natural attenuation model of BTEX contamination. This approach to modeling reaeration has been incorporated into the finite-element groundwater flow and contaminant transport code, FLOTRAN. Adding diffusion-driven reaeration to the modeling process produces BTEX plumes consistent with observed plume lengths.     

Incorporation of Oxygen from Water into Toluene and Benzene during Anaerobic Fermentative Transformation

Toluene and benzene were anaerobically transformed and eventually mineralized in mixed methanogenic cultures. However, the source of oxygen for the initial oxidation step had been unknown, owing to the presence of both methanol and water. No exogenous electron acceptors other than carbon dioxide, toluene, and benzene were present in the defined mineral medium. Through the use of ¹⁸O-labeled water, the oxygen incorporated into the monoaromatic compounds was shown to come from water. The cresol from the toluene and the phenol from the benzene contained up to 8% ¹⁸O label after incubation in 9% ¹⁸O-labeled medium. Gas chromatography-mass spectrometry was used to detect the ¹⁸O-labeled aromatic metabolites.     

Oxygen supply and the adaptations of animals in groundwater

1. The first part of this review focuses on the oxygen status of natural groundwater systems (mainly porous aquifers) and hyporheic zones of streams. The second part examines the sensitivity of groundwater organisms, especially crustaceans, to low oxygen concentrations (< 3.0 mg L^?1 O₂). 2. Dissolved oxygen (DO) in groundwater is spatially heterogeneous at macro- (km), meso- (m) and micro- (cm) scales. This heterogeneity, an essential feature of the groundwater environment, reflects changes in sediment composition and structure, groundwater flow velocity, organic matter content, and the abundance and activity of micro-organisms. Dissolved oxygen also exhibits strong temporal changes in the hyporheic zone of streams as well as in the recharge area of aquifers, but these fluctuations should be strongly attenuated with increasing distance from the stream and the recharge zone. 3. Dissolved oxygen gradients along flow paths in groundwater systems and hyporheic zones vary over several orders of magnitude (e.g. declines of 9 × 10^?5 to 1.5 ×10^?2 mg L^?1 O₂ m^?1 in confined aquifers and 2 × 10^?2 to 1 mg L^?1 O₂ m^?1 in parafluvial water). Several factors explain this strong variation. Where the water table is close to the surface, oxygen is likely to be consumed rapidly in the first few metres below the water table because of incomplete degradation of soil-generated labile dissolved organic carbon (DOC) in the vadose zone. Where the water table is far from the surface, strong oxygen depletion in the vicinity of the water table does not occur, DO being then gradually consumed as groundwater flows down the hydraulic gradient. In unconfined groundwater systems, oxygen consumption along flow paths may be compensated by down-gradient replenishment of DO, resulting either from the ingress of atmospheric oxygen or water recharge through the vadose zone. In confined groundwater systems, where replenishment of oxygen is impossible, the removal time of DO varies from a few years to more than 10 000 years, depending mainly on the organic carbon content of the sediment. Comparison of the hyporheic zones between systems also revealed strong differences in the removal time and length of underground pathways for DO. This strong variability among systems seems related to differences in contact time of water with sediment. 4. Although groundwater macro-crustaceans are much more resistant to hypoxia than epigean species, they cannot survive severe hypoxia (DO < 0.01 mg L^?1 O₂) for very long (lethal time for 50% of the population ranged from 46.7 to 61.7 h). In severe hypoxia, none of the hypogean crustaceans examined utilized a high-ATP yielding metabolic pathway. High survival times are mainly a result of the combination of three mechanisms: a high storage of fermentable fuels (glycogen and phosphagen), a low metabolic rate in normoxia, and a further reduction in metabolic rate by reducing locomotion and ventilation. It is suggested here that the low metabolic rate of many hypogean species may be an adaptation to low oxygen and not necessarily result from an impoverished food supply. 5. An interesting physiological feature of hypogean crustaceans is their ability to recover from anaerobic stress and, more specifically, rapidly to resynthesize glycogen stores during post-hypoxic recovery. A high storage and rapid restoration of fermentable fuels (without feeding) allows groundwater crustaceans to exploit a moving mosaic of suboxic (< 0.3 mg L?1 O₂), dysoxic (0.3–3.0 mg L^?1 O₂) and oxic (> 3 mg L^?1 O₂) patches. 6. It is concluded that although hypogean animals are probably unsuited for life in extensively or permanently suboxic groundwater, they can be found in small or temporarily suboxic patches. Indeed, their adaptations to hypoxia are clearly suited for life in groundwater characterized by spatially heterogeneous or highly dynamic DO concentrations. Their capacity to survive severe hypoxia for a few days and to recover rapidly would explain partly why ecological field studies often reveal the occurrence of interstitial taxa in groundwater with a wide range of DO.     

Biotreatment of groundwater contaminated with MTBE: interaction of common environmental co-contaminants

Contamination of groundwater with the gasoline additive methyl tert-butyl ether (MTBE) is often accompanied by many aromatic components such as benzene, toluene, ethylbenzene, o-xylene, m-xylene and p-xylene (BTEX). In this study, a laboratory-scale biotrickling filter for groundwater treatment inoculated with a microbial consortium degrading MTBE was studied. Individual or mixtures of BTEX compounds were transiently loaded in combination with MTBE. The results indicated that single BTEX compound or BTEX mixtures inhibited MTBE degradation to varying degrees, but none of them completely repressed the metabolic degradation in the biotrickling filter. Tert-butyl alcohol (TBA), a frequent co-contaminant of MTBE had no inhibitory effect on MTBE degradation. The bacterial consortium was stable and showed promising capabilities to remove TBA, ethylbenzene and toluene, and partially degraded benzene and xylenes without significant lag time. The study suggests that it is feasible to deploy a mixed bacterial consortia to degrade MTBE, BTEX and TBA at the same time.     

Dissolved oxygen and dissolved inorganic carbon stable isotope composition and concentration fluxes across several shallow floodplain aquifers and in a diffusion experiment

Recent studies have documented the occurrence of dissolved molecular oxygen (DO) in shallow groundwater that is isotopically lighter than can be explained by atmospheric gas exchange or by biogeochemical reactions that consume ¹⁶O¹⁶O faster than ¹⁶O¹⁸O. In the present study, spatial gradients in the isotopic composition of DO (δ¹⁸O-DO) and dissolved inorganic carbon (δ¹³C-DIC) were measured in three shallow floodplain aquifers: (1) the Nyack aquifer, of the Middle Fork of the Flathead River in northwest Montana; (2) the Silver Bow Creek floodplain in southwest Montana; and (3) the Umatilla River floodplain in northeast Oregon. The field data show general trends of increasing DIC concentration, decreasing δ¹³C-DIC, and decreasing DO concentration with increase in groundwater path length. These trends are consistent with consumption of DO and production of DIC by microbial respiration. Although the expected trend of an increase in δ¹⁸O-DO with increase in path length was found at an area adjacent to hyporheic recharge at the Nyack floodplain, the majority of groundwater samples collected at Nyack and from the other sites distal to recharge zones had anomalously low δ¹⁸O-DO values well below 24.2 ‰, the value corresponding to atmospheric isotopic equilibrium. At the Nyack site, ³H-³He dates were used to estimate groundwater travel time: all groundwater samples with apparent age >1 year had δ¹⁸O-DO<24.2 ‰. Previously it has been suggested that diffusion of O₂ could be a viable mechanism to explain the existence of isotopically light DO in shallow groundwater. To test this hypothesis, laboratory experiments were conducted to measure the isotopic fractionation of O₂ as it diffuses from air across a simulated capillary fringe (made from a floating layer of foam beads) into a stirred, initially anoxic, water column. As expected, ¹⁶O¹⁶O diffused faster than ¹⁶O¹⁸O, and the magnitude of isotope fractionation associated with diffusion increased with a decrease in temperature. Fractionation factors (α) calculated from these diffusion experiments were 1.0030 at 15–19 °C and 1.0048 at 8 °C. The combined field and laboratory data suggest that diffusion is an important mechanism to maintain aerobic conditions in shallow groundwater systems, allowing microbial respiration to continue at long distances (km scale) from the source of groundwater recharge.