Isolation and characterization of pure cultures for metabolizing 1,4-dioxane in oligotrophic environments.

1,4-Dioxane concentration in most contaminated water is much less than 1 mg/L, which cannot sustain the growth of most reported 1,4-dioxane-metabolizing pure cultures. These pure cultures were isolated following enrichment of mixed cultures at high concentrations (20 to 1,000 mg/L). This study is based on a different strategy: 1,4-dioxane-metabolizing mixed cultures were enriched by periodically spiking 1,4-dioxane at low concentrations (≤1 mg/L). Five 1,4-dioxane-metabolizing pure strains LCD6B, LCD6D, WC10G, WCD6H, and WD4H were isolated and characterized. The partial 16S rRNA gene sequencing showed that the five bacterial strains were related to Dokdonella sp. (98.3%), Acinetobacter sp. (99.0%), Afipia sp. (99.2%), Nitrobacter sp. (97.9%), and Pseudonocardia sp. (99.4%), respectively. Nitrobacter sp. WCD6H is the first reported 1,4-dioxane-metabolizing bacterium in the genus of Nitrobacter. The net specific growth rates of these five cultures are consistently higher than those reported in the literature at 1,4-dioxane concentrations <0.5 mg/L. Compared to the literature, our newly discovered strains have lower half-maximum-rate concentrations (1.8 to 8.2 mg-dioxane/L), lower maximum specific 1,4-dioxane utilization rates (0.24 to 0.47 mg-dioxane/(mg-protein ⋅ d)), higher biomass yields (0.29 to 0.38 mg-protein/mg-dioxane), and lower decay coefficients (0.01 to 0.02 d-1). These are characteristics of microorganisms living in oligotrophic environments.

Goethite Formed in the Periplasmic Space of Pseudomonas sp. JM-7 during Fe Cycling Enhances Its Denitrification in Water.

Denitrification-driven Fe(II) oxidation is an important microbial metabolism that connects iron and nitrogen cycling in the environment. The formation of Fe(III) minerals in the periplasmic space has a significant effect on microbial metabolism and electron transfer, but direct evidence of iron ions entering the periplasm and resulting in periplasmic mineral precipitation and electron conduction properties has yet to be conclusively determined. Here, we investigated the pathways and amounts of iron, with different valence states and morphologies, entering the periplasmic space of the denitrifier Pseudomonas sp. JM-7 (P. JM-7), and the possible effects on the electron transfer and the denitrifying ability. When consistently provided with Fe(II) ions (from siderite (FeCO3)), the dissolved Fe(II) ions entered the periplasmic space and were oxidized to Fe(III), leading to the formation of a 25 nm thick crystalline goethite crust, which functioned as a semiconductor, accelerating the transfer of electrons from the intracellular to the extracellular matrix. This consequently doubled the denitrification rate and increased the electron transport capacity by 4-30 times (0.015-0.04 µA). However, as the Fe(II) concentration further increased to above 4 mM, the Fe(II) ions tended to preferentially nucleate, oxidize, and crystallize on the outer surface of P. JM-7, leading to the formation of a densely crystallized goethite layer, which significantly slowed down the metabolism of P. JM-7. In contrast to the Fe(II) conditions, regardless of the initial concentration of Fe(III), it was challenging for Fe(III) ions to form goethite in the periplasmic space. This work has shed light on the likely effects of iron on environmental microorganisms, improved our understanding of globally significant iron and nitrogen geochemical cycles in water, and expanded our ability to study and control these important processes.

Simultaneous biodegradation kinetics of 1,4-dioxane and ethane.

Biodegradation of 1,4-Dioxane at environmentally relevant concentrations usually requires the addition of a primary electron-donor substrate to sustain biomass growth. Ethane is a promising substrate, since it is available as a degradation product of 1,4-Dioxane's common co-contaminants. This study reports kinetic parameters for ethane biodegradation and co-oxidations of ethane and 1,4-Dioxane. Based on experiments combined with mathematical modeling, we found that ethane promoted 1,4-Dioxane biodegradation when the initial mass ratio of ethane:1,4-Dioxane was < 9:1 mg COD/mg COD, while it inhibited 1,4-Dioxane degradation when the ratio was > 9:1. A model-independent estimator was used for kinetic-parameter estimation, and all parameter values for 1,4-Dioxane were consistent with literature-reported ranges. Estimated parameters support competitive inhibition between ethane as the primary substrate and 1,4-Dioxane as the secondary substrate. The results also support that bacteria that co-oxidize ethane and 1,4-Dioxane had a competitive advantage over bacteria that can use only one of the two substrates. The minimum concentration of ethane to sustain ethane-oxidizing bacteria and ethane and 1,4-Dioxane-co-oxidizing bacteria was 0.09 mg COD/L, which is approximately 20-fold lower than the minimum concentration reported for propane, another common substrate used to promote 1,4-Dioxane biodegradation. The minimum 1,4-Dioxane concentration required to sustain steady-state biomass with 1,4-Dioxane as the sole primary substrate was 1.3 mg COD/L. As 1,4-Dioxane concentrations at most groundwater sites are less than 0.18 mg COD/L, providing ethane as a primary substrate is vital to support biomass growth and consequently enable 1,4-Dioxane bioremediation.

Evidence and Mechanisms of Selenate Reduction to Extracellular Elemental Selenium Nanoparticles on the Biocathode.

Intracellular selenium nanoparticles (SeNPs) production is a roadblock to the recovery of selenium from biological water treatment processes because it is energy intensive to break microbial cells and then separate SeNPs. This study provided evidence of significantly more extracellular SeNP production on the biocathode (97-99%) compared to the conventional reactors (1-90%) using transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy. The cathodic microbial community analysis showed that relative abundance of Azospira oryzae, Desulfovibrio, Stenotrophomonas, and Rhodocyclaceae was <1% in the inoculum but enriched to 10-21% for each group when the bioelectrochemical reactor reached a steady state. These four groups of microorganisms simultaneously produce intracellular and extracellular SeNPs in conventional biofilm reactors per literature review but prefer to produce extracellular SeNPs on the cathode. This observation may be explained by the cellular energetics: by producing extracellular SeNPs on the biocathode, microbes do not need to transfer selenate and the electrons from the cathode into the cells, thereby saving energy. Extracellular SeNP production on the biocathode is feasible since we found high concentrations of C-type cytochrome, which is well known for its ability to transfer electrons from electrodes to microbial cells and reduce selenate to SeNPs on the cell membrane.

A Synergistic Platform for Continuous Co-removal of 1,1,1-Trichloroethane, Trichloroethene, and 1,4-Dioxane via Catalytic Dechlorination Followed by Biodegradation.

Groundwater co-contaminated with 1,4-dioxane, 1,1,1-trichloroethane (TCA), and trichloroethene (TCE) is among the most urgent environmental concerns of the U.S. Department of Defense (DoD), U.S. Environmental Protection Agency (EPA), and industries related to chlorinated solvents. Inspired by the pressing need to remove all three contaminants at many sites, we tested a synergistic platform: catalytic reduction of 1,1,1-TCA and TCE to ethane in a H2-based membrane palladium-film reactor (H2-MPfR), followed by aerobic biodegradation of ethane and 1,4-dioxane in an O2-based membrane biofilm reactor (O2-MBfR). During 130 days of continuous operation, 1,1,1-TCA and TCE were 95-98% reductively dechlorinated to ethane in the H2-MPfR, and ethane served as the endogenous primary electron donor for promoting 98.5% aerobic biodegradation of 1,4-dioxane in the O2-MBfR. In addition, the small concentrations of the chlorinated intermediate from the H2-MPfR, dichloroethane (DCA) and monochloroethane (MCA), were fully biodegraded through aerobic biodegradation in the O2-MBfR. The biofilms in the O2-MBfR were enriched in phylotypes closely related to the genera Pseudonocardia known to biodegrade 1,4-dioxane.

Long-Term Continuous Co-reduction of 1,1,1-Trichloroethane and Trichloroethene over Palladium Nanoparticles Spontaneously Deposited on H2-Transfer Membranes.

1,1,1-Trichloroethane (1,1,1-TCA) and trichloroethene (TCE) are common recalcitrant contaminants that coexist in groundwater. H2-induced reduction over precious-metal catalysts has proven advantageous, but its application to long-term continuous treatment has been limited due to poor H2-transfer efficiency and catalyst loss. Furthermore, catalytic reductions of aqueous 1,1,1-TCA alone or concomitant with TCE catalytic co-reductions are unstudied. Here, we investigated 1,1,1-TCA and TCE co-reduction using palladium nanoparticle (PdNP) catalysts spontaneously deposited on H2-transfer membranes that allow efficient H2 supply on demand in a bubble-free form. The catalytic activities for 1,1,1-TCA and TCE reductions reached 9.9 and 11 L/g-Pd/min, values significantly greater than that reported for other immobilized-PdNP systems. During 90 day continuous operation, removals were up to 95% for 1,1,1-TCA and 99% for TCE. The highest steady-state removal fluxes were 1.5 g/m2/day for 1,1,1-TCA and 1.7 g/m2/day for TCE. The major product was nontoxic ethane (94% selectivity). Only 4% of the originally deposited PdNPs were lost over 90 days of continuous operation. Documenting long-term continuous Pd-catalyzed dechlorination at high surface loading with minimal loss of the catalyst mass or activity, this work expands understanding of and provides a foundation for sustainable catalytic removal of co-existing chlorinated solvents.

Investigating promising substrates for promoting 1,4-dioxane biodegradation: effects of ethane and tetrahydrofuran on microbial consortia.

Cometabolic biodegradation of 1,4-dioxane (dioxane) in the presence of primary substrates is a promising strategy for treating dioxane at environmentally relevant concentrations. Seven aqueous amendments (i.e., tetrahydrofuran (THF), butanone, acetone, 1-butanol, 2-butanol, phenol and acetate) and five gaseous amendments (i.e., C1-C4 alkanes and ethylene) were evaluated as the primary substrates for dioxane degradation by mixed microbial consortia. The aqueous amendments were tested in microcosm bottles and the gaseous amendments were tested in a continuous-flow membrane biofilm reactor with hollow fibers pressurized by the gaseous amendments. Ethane was found to be the most effective gaseous substrate and THF was the only aqueous substrate that promoted dioxane degradation. A diverse microbial community consisting of several putative dioxane degraders-Mycobacterium, Flavobacterium and Bradyrhizobiaceae-were enriched in the presence of ethane. This is the first study showing that ethane was the most effective substrate among the short-chain alkanes and it promoted dioxane degradation by enriching dioxane-degraders that did not harbor the well-known dioxane/tetrahydrofuran monooxygenase.

Microbial Community Analysis Provides Insights into the Effects of Tetrahydrofuran on 1,4-Dioxane Biodegradation.

Tetrahydrofuran (THF) is known to induce the biodegradation of 1,4-dioxane (dioxane), an emerging contaminant, but the mechanisms by which THF affects dioxane biodegradation in microbial communities are not well understood. To fill this knowledge gap, changes in the microbial community structure in microcosm experiments with synthetic medium and landfill leachate were examined over time using 16S rRNA gene amplicon sequencing and functional gene quantitative PCR assays. The overarching hypothesis being tested was that THF promoted dioxane biodegradation by increasing the abundance of dioxane-degrading bacteria in the consortium. The data revealed that in experiments with synthetic medium, the addition of THF significantly increased the abundance of Pseudonocardia, a genus with several representatives that can grow on both dioxane and THF, and of Rhodococcus ruber, a species that can use THF as the primary growth substrate while cometabolizing dioxane. However, in similar experiments with landfill leachate, only R. ruber was significantly enriched. When the THF concentration was higher than the dioxane concentration, THF competitively inhibited dioxane degradation since dioxane degradation was negligible, while the dioxane-degrading bacteria and the corresponding THF/dioxane monooxygenase gene copies increased by a few orders of magnitude.IMPORTANCE Widespread in groundwater and carcinogenic to humans, 1,4-dioxane (dioxane) is attracting significant attention in recent years. Advanced oxidation processes can effectively remove dioxane but require high energy consumption and operation costs. Biological removal of dioxane is of particular interest due to the ability of some bacteria to mineralize dioxane at a low energy cost. Although dioxane is generally considered recalcitrant to biodegradation, more than 20 types of bacteria can degrade dioxane as the sole electron donor substrate or the secondary electron donor substrate. In the latter case, tetrahydrofuran (THF) is commonly studied as the primary electron donor substrate. Previous work has shown that THF promotes dioxane degradation at a low THF concentration but inhibits dioxane degradation at a high THF concentration. Our work expanded on the previous work by mechanically examining the effects of THF on dioxane degradation in a microbial community context.

Kinetics of anaerobic methane oxidation coupled to denitrification in the membrane biofilm reactor.

Anaerobic oxidation of methane coupled to denitrification (AOM-D) in a membrane biofilm reactor (MBfR), a platform used for efficiently coupling gas delivery and biofilm development, has attracted attention in recent years due to the low cost and high availability of methane. However, experimental studies have shown that the nitrate-removal flux in the CH4 -based MBfR (<1.0 g N/m2 -day) is about one order of magnitude smaller than that in the H2 -based MBfR (1.1-6.7 g N/m2 -day). A one-dimensional multispecies biofilm model predicts that the nitrate-removal flux in the CH4 -based MBfR is limited to <1.7 g N/m2 -day, consistent with the experimental studies reported in the literature. The model also determines the two major limiting factors for the nitrate-removal flux: The methane half-maximum-rate concentration (K2 ) and the specific maximum methane utilization rate of the AOM-D syntrophic consortium (kmax2 ), with kmax2 being more important. Model simulations show that increasing kmax2 to >3 g chemical oxygen demand (COD)/g cell-day (from its current 1.8 g COD/g cell-day) and developing a new membrane with doubled methane-delivery capacity (Dm ) could bring the nitrate-removal flux to ≥4.0 g N/m2 -day, which is close to the nitrate-removal flux for the H2 -based MBfR. Further increase of the maximum nitrate-removal flux can be achieved when Dm and kmax2 increase together.

Direct solid-state evidence of H2 -induced partial U(VI) reduction concomitant with adsorption by extracellular polymeric substances (EPS).

Adsorption of hexavalent uranium (U(VI)) by extracellular polymeric substances (EPS) has been studied, but the possibility of simultaneous U(VI) reduction mediated by EPS has not had experimental confirmation, as the reduction products have not yet been directly proven. Here, we reported the first direct evidence of lower-valent products of U(VI) immobilization by loosely associated EPS (laEPS) isolated from a fermenter strain of Klebsiella sp. J1 when the laEPS was exposed to H2 . During the 120-min tests for similarly 86% adsorption under O2 , N2 , and H2 , 8% more U was immobilized through a non-adsorptive pathway by the EPS for H2 than for N2 and O2 . A set of solid-state characterization tools (FT-IR, XPS, EELS, and TEM-EDX) confirmed partial reduction of U(VI) to lower-valence U, with the main reduced form being uraninite (UIV O2 ) nanoparticles, and the results reinforced the role of the reduction in accelerating U immobilization and shaping the characteristics of immobilized U in terms of valency, size, and crystallization. The laEPS, mostly comprised of carbohydrate and protein, contained non-cytochrome enzymes and electron carriers that could be responsible for electron transfer to U(VI). Taken together, our results directly confirm that EPS was able to mediate partial U(VI) reduction in the presence of H2 through non-cytochrome catalysis and that reduction enhanced overall U immobilization. Our study fills in some gaps of the microbe-mediated U cycle and will be useful to understand and control U removal in engineered reactors and in-situ bioremediation.

Chemical-Free Recovery of Elemental Selenium from Selenate-Contaminated Water by a System Combining a Biological Reactor, a Bacterium-Nanoparticle Separator, and a Tangential Flow Filter.

Biological selenate (SeO42-) reduction to elemental selenium nanoparticles (SeNPs) has been intensively studied but little practiced because of the additional cost associated with separation of SeNPs from water. Recovery of the SeNPs as a valuable resource has been researched to make the approach more competitive. Separation of the intracellular SeNPs from the biomass usually requires the addition of chemicals. In this research, a novel approach that combined a biological reactor, a bacterium-SeNP separator, and a tangential flow ultrafiltration module (TFU) was investigated to biologically reduce selenate and separate the SeNPs, biomass, and water from each other. This approach efficiently removed and recovered selenium while eliminating the use of chemicals for separation. The three units in the approach worked in synergism to achieve the separation and recovery. The TFU module retained the biomass in the system, which increased the biomass retention time and allowed for more biomass decay through which intracellular SeNPs could be released and recovered. SeNP aggregates were separated from bacterial aggregates due to their different interactions with a tilted polyethylene sheet in the bacterium-SeNP separator. SeNP aggregates stayed on the polyethylene sheet while bacterial aggregates settled down to the bottom of the separator.

Modeling multidimensional and multispecies biofilms in porous media.

Modeling multidimensional and multispecies biofilm in porous media at the pore scale is challenging due to the need to simultaneously track the microbial community in the biofilms and the interfaces between the biofilms and the fluid. Therefore, researchers usually assume that the model has only one dimension in space or has only one microbial species. This work uses bioremediation of U(VI)-contaminated groundwater as the context to develop a two-dimensional and multispecies biofilm model. The model simulates the transverse mixing zone in which U(VI) is mixed with propionate, a nutrient externally supplied to stimulate the growth of microorganisms. The model considers multiple interactions among fluid flow, transport and reaction of chemical species, and growth of biofilm. The biofilm consists of two types of active biomass (syntrophs and dissimilatory metal reducing bacteria [DMBR]) and inert biomass. The two types of active biomass collaboratively remove U(VI). The model outputs biomass distribution, chemical species concentrations, and fluid flow at the pore scale to fundamentally study the multiple interactions. The model also outputs the contaminant removal rate that can be potentially used for up-scaling studies. The simulated results are generally consistent with experimental observations from other studies in trend. The trend can be explained by the multiple interactions based on thermodynamics and microbial kinetics. Biotechnol. Bioeng. 2017;114: 1679-1687. © 2017 Wiley Periodicals, Inc.

Bioreduction of Chromate in a Methane-Based Membrane Biofilm Reactor.

For the first time, we demonstrate chromate (Cr(VI)) bioreduction using methane (CH4) as the sole electron donor in a membrane biofilm reactor (MBfR). The experiments were divided into five stages lasting a total of 90 days, and each stage achieved a steady state for at least 15 days. Due to continued acclimation of the microbial community, the Cr(VI)-reducing capacity of the biofilm kept increasing. Cr(VI) removal at the end of the 90-day test reached 95% at an influent Cr(VI) concentration of 3 mg Cr/L and a surface loading of 0.37g of Cr m(-2) day(-1). Meiothermus (Deinococci), a potential Cr(VI)-reducing bacterium, was negligible in the inoculum but dominated the MBfR biofilm after Cr(VI) was added to the reactor, while Methylosinus, a type II methanotrophs, represented 11%-21% of the total bacterial DNA in the biofilm. Synergy within a microbial consortia likely was responsible for Cr(VI) reduction based on CH4 oxidation. In the synergy, methanotrophs fermented CH4 to produce metabolic intermediates that were used by the Cr(VI)-reducing bacteria as electron donors. Solid Cr(III) was the main product, accounting for more than 88% of the reduced Cr in most cases. Transmission electron microscope (TEM) and energy dispersive X-ray (EDS) analysis showed that Cr(III) accumulated inside and outside of some bacterial cells, implying that different Cr(VI)-reducing mechanisms were involved.

Selenate and Nitrate Bioreductions Using Methane as the Electron Donor in a Membrane Biofilm Reactor.

Selenate (SeO4(2-)) bioreduction is possible with oxidation of a range of organic or inorganic electron donors, but it never has been reported with methane gas (CH4) as the electron donor. In this study, we achieved complete SeO4(2-) bioreduction in a membrane biofilm reactor (MBfR) using CH4 as the sole added electron donor. The introduction of nitrate (NO3(-)) slightly inhibited SeO4(2-) reduction, but the two oxyanions were simultaneously reduced, even when the supply rate of CH4 was limited. The main SeO4(2-)-reduction product was nanospherical Se(0), which was identified by scanning electron microscopy coupled to energy dispersive X-ray analysis (SEM-EDS). Community analysis provided evidence for two mechanisms for SeO4(2-) bioreduction in the CH4-based MBfR: a single methanotrophic genus, such as Methylomonas, performed CH4 oxidation directly coupled to SeO4(2-) reduction, and a methanotroph oxidized CH4 to form organic metabolites that were electron donors for a synergistic SeO4(2-)-reducing bacterium.

Immobilization of selenite via two parallel pathways during in situ bioremediation.

It is widely understood that selenite can be biologically reduced to elemental selenium. Limited studies have shown that selenite can also be immobilized through abiotic precipitation with sulfide, a product of biological sulfate reduction. We demonstrate that both pathways significantly contribute to selenite immobilization in a microfluidic flow cell having a transverse mixing zone between propionate and selenite that mimics the reaction zone along the margins of a selenite plume undergoing bioremediation in the presence of background sulfate. The experiment showed that red particles of amorphous elemental selenium precipitate on the selenite-rich side of the mixing zone, while long crystals of selenium sulfides precipitate on the propionate-rich side of the mixing zone. We developed a continuum-scale reactive transport model that includes both pathways. The simulated results are consistent with the experimental results, and indicate that spatial segregation of the two selenium precipitates is due to the segregation of the more thermodynamic favorable selenite reduction and the less thermodynamically favorable sulfate reduction. The improved understanding of selenite immobilization and the improved model can help to better design in situ bioremediation processes for groundwater contaminated by selenite or other contaminants (e.g., uranium(IV)) that can be immobilized via similar pathways.

Nitrate shaped the selenate-reducing microbial community in a hydrogen-based biofilm reactor.

To study the effect of nitrate (NO3(-)) on selenate (SeO4(2-)) reduction, we tested a H2-based biofilm with a range of influent NO3(-) loadings. When SeO4(2-) was the only electron acceptor (stage 1), 40% of the influent SeO4(2-) was reduced to insoluble elemental selenium (Se(0)). SeO4(2-) reduction was dramatically inhibited when NO3(-) was added at a surface loading larger than 1.14 g of N m(-2) day(-1), when H2 delivery became limiting and only 80% of the input NO3(-) was reduced (stage 2). In stage 3, when NO3(-) was again removed from the influent, SeO4(2-) reduction was re-established and increased to 60% conversion to Se(0). SeO4(2-) reduction remained stable at 60% in stages 4 and 5, when the NO3(-) surface loading was re-introduced at ≤ 0.53 g of N m(-2) day(-1), allowing for complete NO3(-) reduction. The selenate-reducing microbial community was significantly reshaped by the high NO3(-) surface loading in stage 2, and it remained stable through stages 3-5. In particular, the abundance of α-Proteobacteria decreased from 30% in stage 1 to less than 10% of total bacteria in stage 2. ß-Proteobacteria, which represented about 55% of total bacteria in the biofilm in stage 1, increased to more than 90% of phylotypes in stage 2. Hydrogenophaga, an autotrophic denitrifier, was positively correlated with NO3(-) flux. Thus, introducing a NO3(-) loading high enough to cause H2 limitation and suppress SeO4(2-) reduction had a long-lasting effect on the microbial community structure, which was confirmed by principal coordinate analysis, although SeO4(2-) reduction remained intact.

Pyrosequencing analysis yields comprehensive assessment of microbial communities in pilot-scale two-stage membrane biofilm reactors.

We studied the microbial community structure of pilot two-stage membrane biofilm reactors (MBfRs) designed to reduce nitrate (NO3(-)) and perchlorate (ClO4(-)) in contaminated groundwater. The groundwater also contained oxygen (O2) and sulfate (SO4(2-)), which became important electron sinks that affected the NO3(-) and ClO4(-) removal rates. Using pyrosequencing, we elucidated how important phylotypes of each "primary" microbial group, i.e., denitrifying bacteria (DB), perchlorate-reducing bacteria (PRB), and sulfate-reducing bacteria (SRB), responded to changes in electron-acceptor loading. UniFrac, principal coordinate analysis (PCoA), and diversity analyses documented that the microbial community of biofilms sampled when the MBfRs had a high acceptor loading were phylogenetically distant from and less diverse than the microbial community of biofilm samples with lower acceptor loadings. Diminished acceptor loading led to SO4(2-) reduction in the lag MBfR, which allowed Desulfovibrionales (an SRB) and Thiothrichales (sulfur-oxidizers) to thrive through S cycling. As a result of this cooperative relationship, they competed effectively with DB/PRB phylotypes such as Xanthomonadales and Rhodobacterales. Thus, pyrosequencing illustrated that while DB, PRB, and SRB responded predictably to changes in acceptor loading, a decrease in total acceptor loading led to important shifts within the "primary" groups, the onset of other members (e.g., Thiothrichales), and overall greater diversity.

Perchlorate reduction from a highly contaminated groundwater in the presence of sulfate-reducing bacteria in a hydrogen-fed biofilm.

We used a hydrogen (H2 )-based biofilm to treat a groundwater contaminated with perchlorate (ClO(4)(-) ) at ~10 mg/L, an unusually high concentration. To enhance ClO(4)(-) removal, we either increased the H2 pressure or decreased the electron-acceptor surface loading. The ClO(4)(-) removal increased from 94% to 98% when the H2 pressure was increased from 1.3 to 1.7 atm when the total acceptor surface loading was 0.49 g H2 /m(2) day. We then decreased the acceptor surface loading stepwise from 0.49 to 0.07 g H2 /m(2) day, and the ClO(4)(-) removal improved to 99.6%, giving an effluent ClO(4)(-) concentration of 41 µg/L. However, the tradeoff was that sulfate (SO(4)(2-) ) reduction occurred, reaching 85% conversion at the lowest acceptor surface loading (0.07 g H(2) /m(2) day). In two steady states with the highest ClO(4)(-) reduction, we assayed for the presence of perchlorate-reducing bacteria (PRB), denitrifying bacteria (DB), and sulfate-reducing bacteria (SRB) by quantitative polymerase chain reaction (qPCR) targeting characteristic reductases. The qPCR results documented competition between PRB and SRB for space within the biofilm. A simple model analysis for a steady-state biofilm suggests that competition from SRB pushed the PRB to locations having a higher detachment rate, which prevented them from driving the ClO(4)(-) concentration below 41 µg/L.

A biofilm model to understand the onset of sulfate reduction in denitrifying membrane biofilm reactors.

This work presents a multispecies biofilm model that describes the co-existence of nitrate- and sulfate-reducing bacteria in the H(2)-based membrane biofilm reactor (MBfR). The new model adapts the framework of a biofilm model for simultaneous nitrate and perchlorate removal by considering the unique metabolic and physiological characteristics of autotrophic sulfate-reducing bacteria that use H(2) as their electron donor. To evaluate the model, the simulated effluent H(2), UAP (substrate-utilization-associated products), and BAP (biomass-associated products) concentrations are compared to experimental results, and the simulated biomass distributions are compared to real-time quantitative polymerase chain reaction (qPCR) data in the experiments for parameter optimization. Model outputs and experimental results match for all major trends and explain when sulfate reduction does or does not occur in parallel with denitrification. The onset of sulfate reduction occurs only when the nitrate concentration at the fiber's outer surface is low enough so that the growth rate of the denitrifying bacteria is equal to that of the sulfate-reducing bacteria. An example shows how to use the model to design an MBfR that achieves satisfactory nitrate reduction, but suppresses sulfate reduction.

Using a two-stage hydrogen-based membrane biofilm reactor (MBfR) to achieve complete perchlorate reduction in the presence of nitrate and sulfate.

We evaluated a strategy for achieving complete reduction of perchlorate (ClO(4)(-)) in the presence of much higher concentrations of sulfate (SO(4)(2-)) and nitrate (NO(3)(-)) in a hydrogen-based membrane biofilm reactor (MBfR). Full ClO(4)(-) reduction was achieved by using a two-stage MBfR with controlled NO(3)(-) surface loadings to each stage. With an equivalent NO(3)(-) surface loading larger than 0.65 ± 0.04 g N/m(2)-day, the lead MBfR removed about 87 ± 4% of NO(3)(-) and 30 ± 8% of ClO(4)(-). This decreased the equivalent surface loading of NO(3)(-) to 0.34 ± 0.04-0.53 ± 0.03 g N/m(2)-day for the lag MBfR, in which ClO(4)(-) was reduced to nondetectable. SO(4)(2-) reduction was eliminated without compromising full ClO(4)(-) reduction using a higher flow rate that gave an equivalent NO(3)(-) surface loading of 0.94 ± 0.05 g N/m(2)-day in the lead MBfR and 0.53 ± 0.03 g N/m(2)-day in the lag MBfR. Results from qPCR and pyrosequencing showed that the lead and lag MBfRs had distinctly different microbial communities when SO(4)(2-) reduction took place. Denitrifying bacteria (DB), quantified using the nirS and nirK genes, dominated the biofilm in the lead MBfR, but perchlorate-reducing bacteria (PRB), quantified using the pcrA gene, became more important in the lag MBfR. The facultative anaerobic bacteria Dechloromonas, Rubrivivax, and Enterobacter were dominant genera in the lead MBfR, where their main function was to reduce NO(3)(-). With a small NO(3)(-) surface loading and full ClO(4)(-) reduction, the dominant genera shifted to ClO(4)(-)-reducing bacteria Sphaerotilus, Rhodocyclaceae, and Rhodobacter in the lag MBfR.