Effects of graphene oxide and reduced graphene oxide on acetoclastic, hydrogenotrophic and methylotrophic methanogenesis.

This study describes the effects of graphene oxide (GO) and reduced graphene oxide (rGO) on the acetoclastic, hydrogenotrophic and methylotrophic pathways of methanogenesis by an anaerobic consortium. The results showed that GO negatively affected the hydrogenotrophic and acetoclastic pathways at a concentration of 300 mg/L, causing a decrease of ~ 38% on the maximum specific methanogenic activity (MMA) with respect to the controls lacking GO. However, the presence of rGO (300 mg/L) promoted an improvement of the MMA (> 45%) achieved with all substrates, except for the hydrogenotrophic pathway, which was relatively insensitive to rGO. The presence of either rGO or GO enhanced the methylotrophic pathway and resulted in an increase of the MMA of up to 55%. X-ray photoelectron spectroscopy (XPS) analysis revealed that GO underwent microbial reduction during the incubation period. Electrons derived from substrates oxidation were deviated from methanogenesis towards the reduction of GO, which may explain the MMA decreased observed in the presence of GO. Furthermore, XPS evidence indicated that the extent of GO reduction depended on the metabolic pathway triggered by a given substrate.

Diazole and triazole inhibition of nitrification process in return activated sludge.

Azoles are emerging contaminants that are resistant to biodegradation during wastewater treatment. Their presence has been widely reported in wastewater effluents and receiving waters. In this work, the potential inhibition of nitrification process by six different azole compounds in wastewater treatment plants was investigated in batch bioassays. The azoles studied included three diazoles: pyrazole (Pz); 1-methylpyrazole (MePz); 3,5-dimethylpyrazole (DMePz); and three triazoles: 1,2,4-triazole (Tz); benzotriazole (BTz); and 5-methyl benzotriazole (MeBTz). The concentration of azoles causing 50% inhibition (IC50) increased (azoles became less inhibitory) in the following order (mg L-1): BTz (1.99) < MeBTz (2.18) < Pz (2.69) < Tz (3.53) < DMePz (17.3) < MePz (49.6). No clear structure-inhibitory relationships were found using Log P and pKa as structural properties. The toxicity of any given azole may be related to the role of substituent groups on disabling/enabling binding to the active sites of metallo-enzymes in nitrifying microorganisms. This is exemplified by the low toxicity of MePz, which has a cyclic N blocked by a methyl group. The observed inhibition caused to nitrifying bacteria is more severe than their cytotoxicity to other target organisms (e.g., methanogens and heterotrophic bacteria), suggesting a specific inhibition to the copper-containing enzyme, ammonium monooxygenase, in ammonia oxidizing nitrifying microorganisms.

Adaptation of granular sludge microbial communities to nitrate, sulfide, and/or p-cresol removal.

Effluents from petroleum refineries contain a toxic mixture of sulfide, nitrogen, and phenolic compounds that require adequate treatment for their removal. Biological denitrification processes are a cost-effective option for the treatment of these effluents, but the knowledge on the microbial interactions in simultaneous sulfide and phenol oxidation in denitrifying reactors is still very limited. In this work, microbial community structure and macrostructure of granular biomass were studied in three denitrifying reactors treating a mixture of inorganic (sulfide) and organic (p-cresol) electron donors for their simultaneous removal. The differences in the available substrates resulted in different community assemblies that supported high removal efficiencies, indicating the community adaptation capacity to the fluctuating compositions of industrial effluents. The three reactors were dominated by nitrate reducing and denitrifying bacteria where Thiobacillus spp. were the prevalent denitrifying organisms. The toxicity and lack of adequate substrates caused the endogenous decay of the biomass, leading to release of organic matter that maintained a diverse although not very abundant group of heterotrophs. The endogenous digestion of the granules caused the degradation of its macrostructure, which should be considered to further develop the denitrification process in sulfur-based granular reactors for treatment of industrial wastewater with toxic compounds.

Gallium arsenide (GaAs) leaching behavior and surface chemistry changes in response to pH and O2.

Gallium arsenide (GaAs) is a material widely used in electronic devices. Disposal of electronic waste containing GaAs in municipal solid waste landfills raises concerns about the public health and ecological risks associated with the potential release of toxic arsenic (As) species. In this study, different tests were performed to investigate the leaching behavior of particulate GaAs in aqueous solutions. In the U.S. Toxicity Characteristic Leaching Procedure (TCLP) and California Waste Extraction Test (WET), the concentrations of As released from the GaAs particles were about 2.6-2.8-fold higher than the regulatory limit (5 mg/L). A much higher As concentration (72 mg/L), accounting for as much as 15.4% of the initial As in GaAs, was solubilized in a pH-7.6 synthetic landfill leachate under ambient atmosphere after 120 days. Additional tests performed to evaluate the dissolution of GaAs under a range of redox conditions, pH levels, ionic strength, and presence of organic constituents commonly found in landfills revealed that oxic environments and mildly alkaline conditions (pH 8.1-8.5) promote release of As (chiefly arsenite) and gallium species to the surrounding aqueous environment. The rate of As release in long-term exposure experiments was initially constant but later progressively diminished, likely due to the formation of a passivating layer on the surface of GaAs consisting of corrosion products rich in poorly soluble gallium oxides (Ga2O3 and Ga(OH)3). This hypothesis was confirmed by surface analysis of GaAs particles subjected to leaching using X-ray photoelectron spectroscopy (XPS). These findings suggest that further research is needed to assess the potential release of toxic As from electronic waste in municipal landfills.

Adsorption and oxidation of 3-nitro-1,2,4-triazole-5-one (NTO) and its transformation product (3-amino-1,2,4-triazole-5-one, ATO) at ferrihydrite and birnessite surfaces.

The emerging insensitive munitions compound (IMC) 3-nitro-1,2,4-triazole-5-one (NTO) is currently being used to replace conventional explosives such as 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX), but the environmental fate of this increasingly widespread IMC remains poorly understood. Upon release from unexploded solid phase ordinances, NTO exhibits high aqueous solubility and, hence, potential mobilization to groundwater. Adsorption and abiotic transformation at metal oxide surfaces are possible mechanisms for natural attenuation. Here, the reactions at ferrihydrite and birnessite surfaces of NTO and its biotransformation product, 3-amino-1, 2, 4-triazol-5-one (ATO), were studied in stirred batch reactor systems at controlled pH (7.0). The study was carried out at metal oxide solid to solution ratios (SSR) of 0.15, 1.5 and 15 g kg-1. The samples were collected at various time intervals up to 3 h after reaction initiation, and analyzed using HPLC with photodiode array and mass spectrometric detection. We found no detectable adsorption or transformation of NTO upon reaction with birnessite, whereas ATO was highly susceptible to oxidation by the same mineral, showing nearly complete transformation within 5 min at 15 g kg-1 SSR to urea, CO2(g) and N2(g). The mean surface-area-normalized pseudo-first order rate constant (k) for ATO oxidation by birnessite across all SSRs was 0.05 ±â€¯0.022 h-1 m-2, and oxidation kinetics were independent of dissolved O2 concentration. Both NTO and ATO were resistant to oxidation by ferrihydrite. However, NTO showed partial removal from solution upon reaction with ferrihydrite at 0.15 and 1.5 g kg-1 SSR and complete loss at 15 g kg-1 SSR due to strong adsorption. Conversely, ATO adsorption to ferrihydrite was much weaker than that measured for NTO.

Oxidation of reduced daughter products from 2,4-dinitroanisole (DNAN) by Mn(IV) and Fe(III) oxides.

Abiotic transformation of anthropogenic compounds by redox-active metal oxides affects contaminant fate in soil. The capacity of birnessite and ferrihydrite to oxidize the insensitive munitions compound, 2,4-dinitroanisol (DNAN), and its amine-containing daughter products, 2-methoxy-5-nitro aniline (MENA) and 2,4-diaminoanisole (DAAN), was studied in stirred reactors at controlled pH (7.0). Aqueous suspensions were reacted at metal oxide solid to solution mass ratios (SSR) of 0.15, 1.5 and 15 g kg-1 and solutions were analyzed after 0-3 h by high performance liquid chromatography coupled with photodiode array or mass spectrometry detection. Results indicate that DNAN was resistant to oxidation by birnessite and ferrihydrite. Ferrihydrite did not oxidize MENA, but MENA was susceptible to rapid oxidation by birnessite, with nitrogen largely mineralized to nitrite. This is the first report on mineralization of nonphenolic aromatics and the release of mineralized N from aromatic amines following reaction with birnessite. DAAN was oxidized by both solids, but ca. ten times higher rate was observed with birnessite as compared to ferrihydrite at an SSR of 1.5 g kg-1. At 15 g kg-1 SSR, DAAN was removed from solution within 5 min of reaction with birnessite. CO2(g) evolution experiments indicate mineralization of 15 and 12% of the carbon associated with MENA and DAAN, respectively, under oxic conditions with birnessite at SSR of 15 g kg-1. The results taken as a whole indicate that initial reductive (bio)transformation products of DNAN are readily oxidized by birnessite. The oxidizability of the reduced DNAN products was increased with progressive (bio)reduction as reflected by impacts on the oxidation rate.

Leaching of cadmium and tellurium from cadmium telluride (CdTe) thin-film solar panels under simulated landfill conditions.

A crushed non-encapsulated CdTe thin-film solar cell was subjected to two standardized batch leaching tests (i.e., Toxicity Characteristic Leaching Procedure (TCLP) and California Waste Extraction Test (WET)) and to a continuous-flow column test to assess cadmium (Cd) and tellurium (Te) dissolution under conditions simulating the acidic- and the methanogenic phases of municipal solid waste landfills. Low levels of Cd and Te were solubilized in both batch leaching tests (<8.2% and <3.6% of added Cd and Te, respectively). On the other hand, over the course of 30days, 73% of the Cd and 21% of the Te were released to the synthetic leachate of a continuous-flow column simulating the acidic landfill phase. The dissolved Cd concentration was 3.24-fold higher than the TCLP limit (1mgL-1), and 650-fold higher than the maximum contaminant level established by the US-EPA for this metal in drinking water (0.005mgL-1). In contrast, the release of Cd and Te to the effluent of the continuous-flow column simulating the methanogenic phase of a landfill was negligible. The remarkable difference in the leaching behavior of CdTe in the columns is related to different aqueous pH and redox conditions promoted by the microbial communities in the columns, and is in agreement with thermodynamic predictions.

Transferable Training Modules: Building Environmental Education Opportunities With and for Mexican Community Health Workers (Promotores de Salud).

Community health workers (promotores de salud) have the ability to empower communities to mitigate negative health outcomes. Current training efforts in environmental topics are lacking. This project addressed this gap by developing 4 transferable training modules on environmental health. By applying a series of surveys, interviews, and trainings, we evaluated their relevance. Partners provided favorable feedback for 3 of the 4 modules. It was also learned that the development method could be improved by engaging technically trained promotores de salud in the role of co-creators. This project has implications for environmental justice communities as it can lessen information disparities.

Treatment of acid rock drainage using a sulfate-reducing bioreactor with zero-valent iron.

This study assessed the bioremediation of acid rock drainage (ARD) in flow-through columns testing zero-valent iron (ZVI) for the first time as the sole exogenous electron donor to drive sulfate-reducing bacteria in permeable reactive barriers. Columns containing ZVI, limestone or a mixture of both materials were inoculated with an anaerobic mixed culture and fed a synthetic ARD containing sulfuric acid and heavy metals (initially copper, and later also cadmium and lead). ZVI significantly enhanced sulfate reduction and the heavy metals were extensively removed (>99.7%). Solid-phase analyses showed that heavy metals were precipitated with biogenic sulfide in the columns packed with ZVI. Excess sulfide was sequestered by iron, preventing the discharge of dissolved sulfide. In the absence of ZVI, heavy metals were also significantly removed (>99.8%) due to precipitation with hydroxide and carbonate ions released from the limestone. Vertical-profiles of heavy metals in the columns packing, at the end of the experiment, demonstrated that the ZVI columns still had excess capacity to remove heavy metals, while the capacity of the limestone control column was approaching saturation. The ZVI provided conditions that enhanced sulfate reduction and generated alkalinity. Collectively, the results demonstrate an innovative passive ARD remediation process using ZVI as sole electron-donor.

Recovery of palladium(II) by methanogenic granular sludge.

This is the first report that demonstrates the ability of anaerobic methanogenic granular sludge to reduce Pd(II) to Pd(0). Different electron donors were evaluated for their effectiveness in promoting Pd reduction. Formate and H2 fostered both chemically and biologically mediated Pd reduction. Ethanol only promoted the reduction of Pd(II) under biotic conditions and the reduction was likely mediated by H2 released from ethanol fermentation. No reduction was observed in biotic or abiotic assays with all other substrates tested (acetate, lactate and pyruvate) although a large fraction of the total Pd was removed from the liquid medium likely due to biosorption. Pd(II) displayed severe inhibition towards acetoclastic and hydrogenotrophic methanogens, as indicated by 50% inhibiting concentrations as low as 0.96 and 2.7 mg/L, respectively. The results obtained indicate the potential of utilizing anaerobic granular sludge bioreactor technology as a practical and promising option for Pd(II) reduction and recovery offering advantages over pure cultures.

Arsenic remediation by formation of arsenic sulfide minerals in a continuous anaerobic bioreactor.

Arsenic (As) is a highly toxic metalloid that has been identified at high concentrations in groundwater in certain locations around the world. Concurrent microbial reduction of arsenate (As(V) ) and sulfate (SO4 (2-) ) can result in the formation of poorly soluble arsenic sulfide minerals (ASM). The objective of this research was to study As biomineralization in a minimal iron environment for the bioremediation of As-contaminated groundwater using simultaneous As(V) and SO4 (2-) reduction. A continuous-flow anaerobic bioreactor was maintained at slightly acidic pH (6.25-6.50) and fed with As(V) and SO4 (2-) , utilizing ethanol as an electron donor for over 250 d. A second bioreactor running under the same conditions but lacking SO4 (2-) was operated as a control to study the fate of As (without S). The reactor fed with SO4 (2-) removed an average 91.2% of the total soluble As at volumetric rates up to 2.9 mg As/(L · h), while less than 5% removal was observed in the control bioreactor. Soluble S removal occurred with an S to As molar ratio of 1.2, suggesting the formation of a mixture of orpiment- (As2 S3 ) and realgar-like (AsS) solid phases. Solid phase characterization using K-edge X-ray absorption spectroscopy confirmed the formation of a mixture of As2 S3 and AsS. These results indicate that a bioremediation process relying on the addition of a simple, low-cost electron donor offers potential to promote the removal of As from groundwater with naturally occurring or added SO4 (2-) by precipitation of ASM.

Biotransformation and Degradation of the Insensitive Munitions Compound, 3-Nitro-1,2,4-triazol-5-one, by Soil Bacterial Communities.

Insensitive munitions (IM) are a new class of explosives that are increasingly being adopted by the military. The ability of soil microbial communities to degrade IMs is relatively unknown. In this study, microbial communities from a wide range of soils were tested in microcosms for their ability to degrade the IM, 3-nitro-1,2,4-triazol-5-one (NTO). All seven soil inocula tested were able to readily reduce NTO to 3-amino-1,2,4-triazol-5-one (ATO) via 3-hydroxyamino-1,2,4-triazol-5-one (HTO), under anaerobic conditions with H2 as an electron donor. Numerous other electron donors were shown to be suitable for NTO-reducing bacteria. The addition of a small amount of yeast extract (10 mg/L) was critical to diminish lag times and increased the biotransformation rate of NTO in nearly all cases indicating yeast extract provided important nutrients for NTO-reducing bacteria. The main biotransformation product, ATO, was degradable only in aerobic conditions, as evidenced by a rise in the inorganic nitrogen species nitrite and nitrate, indicative of nitrogen-mineralization. NTO was nonbiodegradable in aerobic microcosms with all soil inocula.

Adaptation of a methanogenic consortium to arsenite inhibition.

Arsenic (As) is a ubiquitous metalloid known for its adverse effects to human health. Microorganisms are also impacted by As toxicity, including methanogenic archaea, which can affect the performance of process in which biological activity is required (i.e. stabilization of activated sludge in wastewater treatment plants). The novel ability of a mixed methanogenic granular sludge consortium to adapt to the inhibitory effect of arsenic (As) was investigated by exposing the culture to approximately 0.92 mM of AsIII for 160 d in an arsenate (AsV) reducing bioreactor using ethanol as the electron donor. The results of shaken batch bioassays indicated that the original, unexposed sludge was severely inhibited by arsenite (AsIII) as evidenced by the low 50% inhibition concentrations (IC50) determined, i.e., 19 and 90 µM for acetoclastic- and hydrogenotrophic methanogenesis, respectively. The tolerance of the acetoclastic and hydrogenotrophic methanogens in the sludge to AsIII increased 47-fold (IC50 = 910 µM) and 12-fold (IC50= 1100 µM), respectively, upon long-term exposure to As. In conclusion, the methanogenic community in the granular sludge demonstrated a considerable ability to adapt to the severe inhibitory effects of As after a prolonged exposure period.

Biomineralization of arsenate to arsenic sulfides is greatly enhanced at mildly acidic conditions.

Arsenic (As) is an important water contaminant due to its high toxicity and widespread occurrence. Arsenic-sulfide minerals (ASM) are formed during microbial reduction of arsenate (As(V)) and sulfate (SO4(2-)). The objective of this research is to study the effect of the pH on the removal of As due to the formation of ASM in an iron-poor system. A series of batch experiments was used to study the reduction of SO4(2-) and As(V) by an anaerobic biofilm mixed culture in a range of pH conditions (6.1-7.2), using ethanol as the electron donor. Total soluble concentrations and speciation of S and As were monitored. Solid phase speciation of arsenic was characterized by x-ray adsorption spectroscopy (XAS). A marked decrease of the total aqueous concentrations of As and S was observed in the inoculated treatments amended with ethanol, but not in the non-inoculated controls, indicating that the As-removal was biologically mediated. The pH dramatically affected the extent and rate of As removal, as well as the stoichiometric composition of the precipitate. The amount of As removed was 2-fold higher and the rate of the As removal was up to 17-fold greater at pH 6.1 than at pH 7.2. Stoichiometric analysis and XAS results confirmed the precipitate was composed of a mixture of orpiment and realgar, and the proportion of orpiment in the sample increased with increasing pH. The results taken as a whole suggest that ASM formation is greatly enhanced at mildly acidic pH conditions.

Socially responsible mining: the relationship between mining and poverty, human health and the environment.

Increasing global demand for metals is putting strain on the ability of the mining industry to physically keep up with demand (physical scarcity). Conversely, social issues including the environmental and human health consequences of mining as well as the disparity in income distribution from mining revenues are disproportionately felt at the local community level. This has created social rifts, particularly in the developing world, between affected communities and both industry and governments. Such rifts can result in a disruption of the steady supply of metals (situational scarcity). Here we discuss the importance of mining in relationship to poverty, identify steps that have been taken to create a framework for socially responsible mining, and then discuss the need for academia to work in partnership with communities, government, and industry to develop transdisciplinary research-based step change solutions to the intertwined problems of physical and situational scarcity.

Toxicity of TiO2, ZrO2, Fe°, Fe2O3, and Mn2O3 nanoparticles to the yeast, Saccharomyces cerevisiae.

The growing application of engineered nanomaterials is leading to an increased occurrence of nanoparticles (NPs) in the environment. Thus, there is a need to better understand their potential impact on the environment. This study evaluated the toxicity of nanosized TiO2, ZrO2, Fe(0), Fe2O3, and Mn2O3 towards the yeast Saccharomyces cerevisiae based on O2 consumption and cell membrane integrity. In addition, the state of dispersion of the nanoparticles in the bioassay medium was characterized. All the nanomaterials showed high tendency to aggregate in the bioassay medium. A non-toxic polyacrylate dispersant was used to improve the NP dispersion stability and test the influence of the aggregation state in their toxicity. Mn2O3 NPs showed the highest inhibition of O2 consumption (50% at 170 mg L(-1)) and cell membrane damage (approximately 30% of cells with compromised membrane at 1000 mg L(-1)), while the other NPs caused low (Fe(0)) or no toxicity (TiO2, ZrO2, and Fe2O3) to the yeast. Dispersant supplementation decreased the inhibition caused by Mn2O3 NPs at low concentrations, which could indicate that dispersant association with the particles may have an impact on the interaction between the NPs and the cells.

Toxicity assessment of inorganic nanoparticles to acetoclastic and hydrogenotrophic methanogenic activity in anaerobic granular sludge.

Release of engineered nanoparticles (NPs) to municipal wastewater from industrial and residential sources could impact biological systems in wastewater treatment plants. Methanogenic inhibition can cause failure of anaerobic waste(water) treatment. This study investigated the inhibitory effect of a wide array of inorganic NPs (Ag(0), Al2O3, CeO2, Cu(0), CuO, Fe(0), Fe2O3, Mn2O3, SiO2, TiO2, and ZnO supplied up to 1500 mgL(-1)) to acetoclastic and hydrogenotrophic methanogenic activity of anaerobic granular sludge. Of all the NPs tested, only Cu(0) and ZnO caused severe methanogenic inhibition. The 50% inhibiting concentrations determined towards acetoclastic and hydrogenotrophic methanogens were 62 and 68 mgL(-1) for Cu(0) NP; and 87 and 250 mgL(-1) for ZnO NP, respectively. CuO NPs also caused inhibition of acetoclastic methanogens. Cu(2+) and Zn(2+) salts caused similar levels of inhibition as Cu(0) and ZnO NPs based on equilibrium soluble metal concentrations measured during the assays, suggesting that the toxicity was due to the release of metal ions by NP-corrosion. A commercial dispersant, Dispex, intended to increase NP stability did not affect the inhibitory impact of the NPs. The results taken as a whole suggest that Zn- and Cu-containing NPs can release metal ions that are inhibitory for methanogenesis.

Fate of cerium dioxide (CeO2) nanoparticles in municipal wastewater during activated sludge treatment.

This study investigated the fate of nano-CeO(2) during municipal wastewater treatment using a laboratory-scale activated sludge (A/S) system fed with primarily-treated municipal wastewater and nano-CeO(2) (55.0 mg Ce/L). Nano-CeO(2) was highly removed during A/S treatment (96.6% total Ce). Extensive removal of CeO(2) <200 nm was also attained and the concentration escaping treatment was only 0.11 mg Ce/L. Elimination occurred mainly by aggregation and settling of CeO(2) particles, promoted by circumneutral pH values and by nanoparticle interactions with organic and/or inorganic wastewater constituents. Biosorption also contributed to CeO(2) removal as shown by sludge analysis and batch adsorption studies. Batch bioassays demonstrated that nano-CeO(2) only exerted inhibition of O(2) uptake by A/S at concentrations exceeding those in the bioreactor feed (50% inhibition at 950 mg CeO(2)/L). These findings indicate that A/S treatment is expected to provide extensive removal of nano-CeO(2) in municipal wastewaters.

Low toxicity of HfO2, SiO2, Al2O3 and CeO2 nanoparticles to the yeast, Saccharomyces cerevisiae.

Increasing use of nanomaterials necessitates an improved understanding of their potential impact on environment health. This study evaluated the cytotoxicity of nanosized HfO(2), SiO(2), Al(2)O(3) and CeO(2) towards the eukaryotic model organism Saccharomyces cerevisiae, and characterized their state of dispersion in bioassay medium. Nanotoxicity was assessed by monitoring oxygen consumption in batch cultures and by analysis of cell membrane integrity. CeO(2), Al(2)O(3), and HfO(2) nanoparticles were highly unstable in yeast medium and formed micron-sized, settleable agglomerates. A non-toxic polyacrylate dispersant (Dispex A40) was used to improve nanoparticle stability and determine the impact of enhanced dispersion on toxicity. None of the NPs tested without dispersant inhibited O(2) uptake by yeast at concentrations as high as 1000 mg/L. Dispersant supplementation only enhanced the toxicity of CeO(2) (47% at 1000 mg/L). Dispersed SiO(2) and Al(2)O(3) (1000 mg/L) caused cell membrane damage, whereas dispersed HfO(2) and CeO(2) did not cause significant disruption of membrane integrity at the same concentration. These results suggest that the O(2) uptake inhibition observed with dispersed CeO(2) NPs was not due to reduced cell viability. This is the first study evaluating toxicity of nanoscale HfO(2), SiO(2), Al(2)O(3) and CeO(2) to S. cerevisiae. Overall the results obtained demonstrate that these nanomaterials display low or no toxicity to yeast.

Uranium bioremediation in continuously fed upflow sand columns inoculated with anaerobic granules.

Reductive precipitation of soluble hexavalent uranium (U(VI)) to insoluble tetravalent uranium (U(IV)) containing minerals is one of the more promising approaches to uranium remediation. The objective of this study was to evaluate the long-term performance of methanogenic granules for the continuous treatment of U(VI). For this purpose, three sand-packed columns inoculated with anaerobic biofilm were operated with or without ethanol and one column was exposed to nitrate co-contamination. The columns were operated for 373 days and efficiently removed U (24 mg L(-1)) in excess of 99.8%. No long-term benefit of ethanol addition was observed, suggesting that endogenous substrates in the biofilm were sufficient to drive the reduction reactions. Nitrate addition was found to inhibit U(VI) reduction and cause re-oxidation of some U(IV) deposited in the column. Taken as a whole, the results indicate that methanogenic biofilms can be reliably applied in bioreactor technology for sustained U removal from groundwater.