Increasing electron donor concentration does not accelerate complete microbial reductive dechlorination in contaminated sediment with native organic carbon.

Experiments with Fe(III)-rich, chloroethene-contaminated sediment demonstrated that trichloroethylene (TCE) and vinyl chloride (VC) were completely reduced to ethene regardless of whether electron donor(s) were added at 1 × stoichiometry or 10 × stoichiometry relative to all-electron acceptors. Unamended controls uniformly reduced TCE to ethene with a mean time to complete dechlorination (operationally defined as the presence of stoichiometric ethene production) of 79 days. Adding 1 × and 10 × acetate hindered the rate and extent of TCE and VC reduction relative to unamended controls, with several only partially reduced when the experiments were terminated. Adding high molecular mass (soybean oil derivative) substrates did not increase microbial reductive dechlorination relative to unamended incubations, and in many cases, hindered microbial dechlorination in favor of methanogenesis. The mean time to complete dechlorination was comparable between low (× 1) and high (× 10) electron donor concentration for all lipid-based electron donors tested. Those tested included Newman Zone® Standard without sodium lactate (96 vs. 75 days, respectively), CAP 18 ME (85 vs. 94 days, respectively), EOS 598B42 (68 vs. 72 days, respectively), and acetate (134 vs. 125 days, respectively). These data suggest that the addition of an electron donor does not always increase the rate and extent of reductive dechlorination but will increase costs. In particular, increasing the concentration of electron donors higher than the stoichiometric demand only decreased complete microbial reductive dechlorination, which is the opposite of most standard "more time and more electrons" approaches. These data argue that site-specific electron donor demands must be evaluated, and in some cases, a monitored natural attenuation (MNA) approach is most favorable.

Combined Biotic-Abiotic 2,4-Dinitroanisole Degradation in the Presence of Hexahydro-1,3,5-trinitro-1,3,5-triazine.

The Department of Defense has developed new explosive formulations in which traditionally used cyclic nitramines such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) have been updated with the insensitive munition (IM) 2,4-dinitroanisole (DNAN). Understanding combined degradation of both compounds at explosive-contaminated sites will allow remediation approaches that simultaneously target both contaminants. DNAN reduction in the presence of RDX was evaluated in abiotic experiments using substoichiometric, stoichiometric, and superstoichiometric concentrations of ferrous iron and anthrahydroquinone disulfonate within a pH range from 7.0 to 9.0. Biological degradation was investigated in resting cell suspensions of Geobacter metallireducens strain GS-15, a model Fe(III)-reducing Bacteria. Cells were amended into anoxic tubes buffered at pH 7.0, with initial 100 µM DNAN and 40-50 µM RDX. In both abiotic and biological experiments, the DNAN was reduced through the intermediate 2-methoxy-5-nitroaniline or 4-methoxy-3-nitroaniline to 2,4-diaminoanisole. In biological experiments, the RDX was reduced to form methylenedinitramine, formaldehyde (HCHO), and ammonium (NH4+). Cells were able to reduce both DNAN and RDX most readily in the presence of extracellular electron shuttles and/or Fe(III). DNAN degradation (abiotic and biotic) was faster than degradation of RDX, suggesting that the reduction of IMs will not be inhibited by cyclic nitramines, but degradation dynamics did change in mixtures when compared to singular compounds.

Enhancing xylose and glucose utilization as well as solvent production using a simplified three-electrode potentiostat system during Clostridium fermentation.

A simple potentiostat was constructed as a strategy to enhance solvent production in a mediatorless and oxygen-exposed fermentation inoculated with the aerotolerant strain Clostridium sp. C10. Elevated n-butanol and acetone titers were recorded in all fermentations with either glucose or xylose in the presence of electrodes poised at + 500 mV (+ 814 mV vs SHE) relative to cells plus substrate only controls. Respective butanol titers and volumetric butanol productivities in studies performed with 30 g/L glucose or 30 g/L xylose were 1.67 and 2.27 times and 1.90 and 6.13 times greater in the presence of electrodes compared to controls. Glucose and xylose utilization in the presence of electrodes was 61 and 125% greater than no-electrode controls, respectively. Increasing substrate concentrations to 60 g/L decreased the butanol yields relative to the studies performed at 30 g/L. These data suggest that it may be more efficient to alter reactor reduction potential than increase substrate concentration for solvent output during industrial fermentations, which favors higher yield with few additional inputs.

Electron shuttling to ferrihydrite selects for fermentative rather than Fe3+ -reducing biomass in xylose-fed batch reactors derived from three different inoculum sources.

Reports suggest that ferric iron and electron shuttling molecules will select for Fe3+ -reducer dominated microbial biomass. We investigated the influence of the redox mediators anthraquinone-2,6-disulfonate (AQDS) and riboflavin using xylose as the sole fermentation substrate, with or without ferric iron. Electron shuttling to insoluble ferrihydrite enhanced solventogenesis, acidogenesis, hydrogen production, and xylose consumption, relative to the cells plus xylose controls in fermentations inoculated with woodland marsh sediment, wetwood disease, or raw septic liquid, over multiple transfers in 15-day batch fermentations. 16S rRNA gene based community analyses indicated that ferrihydrite alone, and AQDS/riboflavin plus ferrihydrite, immediately shifted native heterogeneous communities to those predominantly belonging to the Clostdridiales, rather than stimulating Fe3+ respiring populations. Data were similar irrespective of the inoculum source, suggesting that Fe3+ and/or electron shuttling compounds select for rapid proliferation of fermentative genera when fermentable substrates are present, and increases the extent of xylose consumption and solvent production.

Solvent production from xylose.

Xylose is the second most abundant sugar derived from lignocellulose; it is considered less desirable than glucose for fermentation, and strategies that specifically increase xylose utilization in wild type or engineered cells are goals for biofuel production. Issues arise with xylose utilization because of carbohydrate catabolite repression, which is the preferential utilization of glucose relative to xylose in fermentations with both pure and mixed cultures. Taken together the low substrate utilization rates and solvent yields with xylose compared to glucose, many industrial fermentations ignore the xylolytic portion of the reaction in lieu of methods to maintain high glucose. This is shortsighted given the massive potential for xylose generation from a number of sustainable biomass feedstocks, based on utilization of the hemicellulose fraction(s) that enter pretreatment. A number of strategies have been developed in recent years to address xylose utilization and solvent production from xylose in systems with just xylose, or in systems with mixtures of glucose plus xylose, which are more typical of pretreated lignocellulose. The approaches vary in terms of complexity, stability, and ease of introduction to existing fermentation infrastructure (i.e., so-called drop-in fermentation strategies). Some approaches can be considered traditional engineering approaches (e.g., change the reaction conditions), while others are more subtle cellular approaches to eliminate the impacts of catabolite repression. Finally, genetic engineering has been used to increase xylose utilization, although this can be considered a relatively nascent approach compared to manipulations completed to date for glucose utilization.

Iron and Electron Shuttle Mediated (Bio)degradation of 2,4-Dinitroanisole (DNAN).

The Department of Defense has developed explosives with the insensitive munition 2,4-dinitroanisole (DNAN), to prevent accidental detonations during training and operations. Understanding the fate and transport of DNAN is necessary to assess the risk it may represent to groundwater once the new ordnance is routinely produced and used. Experiments with ferrous iron or anthrahydroquinone-2,6-disulfonate (AH2QDS) were conducted from pH 6.0 to 9.0 with initial DNAN concentrations of 100 µM. DNAN was degraded by 1.2 mM Fe(II) at pH 7, 8, and 9, and rates increased with increasing pH. Greater than 90% of the initial 100 µM DNAN was reduced within 10 min at pH 9, and all DNAN was reduced within 1 h. AH2QDS reduced DNAN at all pH values tested. Cells of Geobacter metallireducens were added in the presence and absence of Fe(III) and/or anthraquinone-2,6-disulfonate (AQDS), and DNAN was also reduced in all cell suspensions. Cells reduced the compound directly, but both AQDS and Fe(III) increased the reaction rate, via the production of AH2QDS and/or Fe(II). DNAN was degraded via two intermediates: 2-methoxy-5-nitroaniline and 4-methoxy-3-nitroaniline, to the amine product 2,4-diaminoanisole. These data suggest that an effective strategy can be developed for DNAN attenuation based on combined biological-abiotic reactions mediated by Fe(III)-reducing microorganisms.

Ferric iron and extracellular electron shuttling increase xylose utilization and butanol production during fermentation with multiple solventogenic bacteria.

Xylose is the second most abundant sugar derived from lignocellulose; it is considered less desirable than glucose for fermentation, and strategies that specifically increase xylose utilization in wild-type cells are goals for biofuel production. Xylose consumption, butanol production, and hydrogen production increased in both Clostridium beijerinckii and a novel solventogenic bacterium (strain DC-1) when anthraquinone-2,6,-disulfonate (AQDS) or riboflavin were used as redox mediators to transfer electrons to poorly crystalline Fe(OH)3 as an extracellular electron sink. Strain DC-1 was most closely related to Rhizobiales bacterium Mfc52 based on 95% 16S rRNA gene sequence similarity, which demonstrates that this response is not limited to a single genus of xylose-fermenting bacteria. Xylose utilization and butanol production were negligible in control incubations containing cells plus 3% (w/v) xylose alone during a 10-day batch fermentation, for both strains tested (n-butanol titers of 0.05 g L-1). Micromolar concentrations of AQDS and riboflavin were added as electron shuttling compounds with poorly crystalline Fe(OH)3 as an insoluble electron acceptor, and respective n-butanol titers increased to 6.35 and 7.46 g L-1. Increases in xylose consumption for the iron treatments were relatively high, from less than 0.49 g L-1 (xylose alone, no iron or electron shuttling molecules) to 25.98 and 29.15 g L-1 for the AQDS and riboflavin treatments, respectively. Hydrogen production was also 3.68 times greater for the AQDS treatment and 5.27 greater for the riboflavin treatment relative to controls. Strain DC-1 data were similar, again indicating that the effects are not specific to the genus Clostridium.

Hydrogenophaga carboriunda sp. nov., a tertiary butyl alcohol-oxidizing, psychrotolerant aerobe derived from granular-activated carbon (GAC).

A Gram-negative, rod-shaped bacterium was isolated from a mixed culture that degraded tert-butyl alcohol (TBA) in a granular-activated carbon (GAC) sample from a Biological-GAC reactor. Strain YZ2(T) was assigned to the Betaproteobacteria within the family Comamonadaceae based on 16S rRNA gene similarities. The nearest phylogenetic relative (95.0 % similarity) with a valid name was Hydrogenophaga taeniospiralis. The DNA G+C content was 66.4 mol%. DNA:DNA hybridization indicated that the level of relatedness to members of the genus Hydrogenophaga ranged from 1.1 to 10.8 %. The dominant cellular fatty acids were: 18:1 w7c (75 %), 16:0 (4.9 %), 17:0 (3.85 %), 18:0 (2.93 %), 11 methyl 18:1 w7c (2.69 %), Summed Feature 2 (2.27 %), and 18:0 3OH (1.35 %). The primary substrate used was TBA, which is a fuel oxygenate and groundwater contaminant. YZ2(T) was non-motile, without apparent flagella. It is a psychrotolerant, facultative aerobe that grew between pH 6.5 and 9.5, and 4 and 30 °C. The culture grew on and mineralized TBA at 4 °C, which is the first report of psychrotolerant TBA degradation. Hydrogen was used as an alternative electron donor. The culture also grew well in defined freshwater medium with ethanol, butanol, hydroxy isobutyric acid, acetate, pyruvate, citrate, lactate, isopropanol, and benzoic acid as electron donors. Nitrate was reduced with hydrogen as the sole electron donor. On the basis of morphological, physiological, and chemotaxonomic data, a new species, Hydrogenophaga carboriunda is proposed, with YZ2(T) as the type strain.

Interactions between Clostridium beijerinckii and Geobacter metallireducens in co-culture fermentation with anthrahydroquinone-2, 6-disulfonate (AH2QDS) for enhanced biohydrogen production from xylose.

To enhance biohydrogen production, Clostridium beijerinckii was co-cultured with Geobacter metallireducens in the presence of the reduced extracellular electron shuttle anthrahydroquinone-2, 6-disulfonate (AH(2)QDS). In the co-culture system, increases of up to 52.3% for maximum cumulative hydrogen production, 38.4% for specific hydrogen production rate, 15.4% for substrate utilization rate, 39.0% for substrate utilization extent, and 34.8% for hydrogen molar yield in co-culture fermentation were observed compared to a pure culture of C. beijerinckii without AH(2)QDS. G. metallireducens grew in the co-culture system, resulting in a decrease in acetate concentration under co-culture conditions and a presumed regeneration of AH(2)QDS from AQDS. These co-culture results demonstrate metabolic crosstalk between the fermentative bacterium C. beijerinckii and the respiratory bacterium G. metallireducens and suggest a strategy for industrial biohydrogen production.

Electron shuttle-mediated biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine adsorbed to granular activated carbon.

Granular activated carbon (GAC) effectively removes hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) from groundwater but generates RDX-laden GAC that must be disposed of or regenerated. Batch reactors containing GAC to which RDX was preadsorbed were used in experiments to test the potential for adsorbed RDX reduction and daughter product formation using (i) chemically reduced anthrahydroquinone-2,6-disulfonate (AH2QDS), (ii) resting Geobacter metallireducens strain GS-15, and (iii) a combined system containing AQDS and GS-15. Approximately 97.0% of the adsorbed RDX was transformed in each of these experimental systems by 90 h. Chemically reduced AQDS (AH2QDS) transformed 99.2% of adsorbed RDX; formaldehyde was produced rapidly and was stoichiometric (3 mol HCHO per mol RDX). Geobacter metallireducens also reduced RDX with and without AQDS present. This is the first study to demonstrate biological transformation of RDX adsorbed to GAC. Formaldehyde increased and then decreased in biological systems, suggesting a previously unreported capacity for G. metallireducens to oxidize formaldehyde, which was confirmed with resting cell suspensions. These data suggest the masses of GAC waste currently produced by activated carbon at RDX remediation sites can be minimized, decreasing the carbon footprint of the treatment technology. Alternatively, this strategy may be used to develop a Bio-GAC system for ex situ RDX treatment.

Low and high acetate amendments are equally as effective at promoting complete dechlorination of trichloroethylene (TCE).

Experiments with trichloroethylene-contaminated aquifer material demonstrated that TCE, cis-DCE, and VC were completely degraded with concurrent Fe(III) or Fe(III) and sulfate reduction when acetate was amended at stoichiometric concentration; competing TEAPs did not inhibit ethene production. Adding 10× more acetate did not increase the rate or extent of TCE reduction, but only increased methane production. Enrichment cultures demonstrated that ~90 µM TCE or ~22 µM VC was degraded primarily to ethene within 20 days with concurrent Fe(III) or Fe(III) + sulfate reduction. The dechlorination rates were comparable between the low and high acetate concentrations (0.36 vs 0.34 day(-1), respectively), with a slightly slower rate in the 10× acetate amended incubations. Methane accumulated to 13.5 (±0.5) µmol/tube in the TCE-degrading incubations with 10× acetate, and only 1.4 (±0.1) µmol/tube with low acetate concentration. Methane accumulated to 16 (±1.5) µmol/tube in VC-degrading enrichment with 10× acetate and 2 (±0.1) µmol/tube with stoichiometric acetate. The estimated fraction of electrons distributed to methanogenesis increased substantially when excessive acetate was added. Quantitative PCR analysis indicated that 10× acetate did not enhance Dehalococcoides biomass but rather increased the methanogen abundance by nearly one order of magnitude compared to that with stoichiometric acetate. The data suggest that adding low levels of substrate may be equally if not more effective as high concentrations, without producing excessive methane. This has implications for field remediation efforts, in that adding excess electron donor may not benefit the reactions of interest, which in turn will increase treatment costs without direct benefit to the stakeholders.

Potential natural attenuation of petroleum hydrocarbons in fuel contaminated soils: Focusing on anaerobic fuel biodegradation involving microbial Fe(III) reduction.

Liquid fossil fuels, collectively known as total petroleum hydrocarbons (TPHs), are highly toxic and frequently leak into subsurface environments due to anthropogenic activities. As an in-situ biological remedial option for TPH contamination, aerobic TPH biodegradation is limited due to oxygen's low solubility in water, and because it is consumed quickly by aerobic bacteria. Thus, we investigated the potential of anaerobic TPH degradation by indigenous fermenting bacteria and Fe(III)-reducing bacteria. Twenty 6-10 m soil cores were collected from a closed military base subject to ongoing TPH contamination since the 1980s. Physicochemical and microbial properties were determined at 0.5-m intervals in each core. To assess the relationship between TPH degradation and microbial Fe(III) reduction, soil samples were grouped into high-TPH (>500 mg kg-1) and high-Fe(II) (>450 mg kg-1), high-TPH and low-Fe(II), low-TPH and high-Fe(II), and low-TPH and low-Fe(II) groups. Alpha diversity was significantly lower in high-TPH groups than in low-TPH groups, suggesting that high TPH concentrations exerted a strong selective pressure on bacterial communities. In the high-TPH and low-Fe(II) group, fermenting bacteria, including Microgenomatia and Chlamydiae, were more abundant, suggesting that TPH biodegradation occurred via fermentation. In the high-TPH and high-Fe(II) group, Fe(III)-reducing bacteria, including Geobacter and Zoogloea, were more abundant, suggesting that microbial Fe(III) reduction enhances TPH biodegradation. In contrast, the fermenting and/or Fe(III)-reducing bacteria were not statistically abundant in the low-TPH groups.

Microbial community composition during anaerobic mineralization of tert-butyl alcohol (TBA) in fuel-contaminated aquifer material.

Anaerobic mineralization of tert-butyl alcohol (TBA) and methyl tert-butyl ether (MTBE) were studied in sediment incubations prepared with fuel-contaminated aquifer material. Microbial community compositions in all incubations were characterized by amplified ribosomal DNA restriction analysis (ARDRA). The aquifer material mineralized 42.3±9.9% of [U-(14)C]-TBA to 14CO2 without electron acceptor amendment. Fe(III), sulfate, and Fe(III) plus anthraquinone-2,6-disulfonate addition also promoted U-[14C]-TBA mineralization at levels similar to those of the unamended controls. Nitrate actually inhibited TBA mineralization relative to unamended controls. In contrast to TBA, [U-(14)C]-MTBE was not significantly mineralized in 400 days regardless of electron acceptor amendment. Microbial community analysis indicated that the abundance of one dominant clone group correlated closely with anaerobic TBA mineralization. The clone was phylogenetically distinct from known aerobic TBA-degrading microorganisms, Fe(III)- or sulfate-reducing bacteria. It was most closely associated with organisms belonging to the alphaproteobacteria. Microbial communities were different in MTBE and TBA amended incubations. Shannon indices and Simpson indices (statistical community comparison tools) both demonstrated that microbial community diversity decreased in incubations actively mineralizing TBA, with distinct "dominant" clones developing. These data contribute to our understanding of anaerobic microbial transformation of fuel oxygenates in contaminated aquifer material and the organisms that may catalyze the reactions.

Influence of ferric iron on complete dechlorination of trichloroethylene (TCE) to ethene: Fe(III) reduction does not always inhibit complete dechlorination.

The effects of Fe(III) reduction on TCE, cis-DCE, and VC dechlorination were studied in both contaminated aquifer material and enrichment cultures. The results from sediment batch experiments demonstrated that Fe(III) reduction did not inhibit complete dechlorination. TCE was reduced concurrently with Fe(III) in the first 40 days of the incubations. While all incubations (plus and minus Fe(III)) generated approximately the same mass of ethene within the experimental time frame, Fe(III) speciation (ferrihydrite versus Fe(III)-NTA) had an impact on daughter product distribution and dechlorination kinetics. 16S rRNA gene clone library sequencing identified Dehalococcoides and Geobacteraceae as dominant populations, which included G. lovleyi like organisms. Quantitative PCR targeting 16S rRNA genes and Reductive Dehalogenase genes (tceA, bvcA, vcrA) indicated that Dehalococcoides and Geobacteraceae were enriched concurrently in the TCE-degrading, Fe(III)-reducing sediments. Enrichment cultures demonstrated that soluble Fe(III) had a greater impact on cis-DCE and VC reduction than solid-phase Fe(III). Geobacteraceae and Dehalococcoides were also coenriched in the liquid cultures, and the Dehalococcoides abundance in the presence of Fe(III) was not significantly different from those in the cultures without Fe(III). Hydrogen reached steady-state concentrations most amenable to complete dechlorination very quickly when Fe(III) was present in the culture, suggesting that Fe(III) reduction may actually help dechlorination. This was contrasted to hydrogen levels in nitrate-amended enrichments, in which hydrogen concentration was too low for any chlororespiration.

Microbially mediated abiotic transformation of the antimicrobial agent sulfamethoxazole under iron-reducing soil conditions.

Large quantities of antimicrobial agents used in livestock production are released to soils by land application of manure, but only limited information is available on mechanisms that contribute to antimicrobial fate in soils under variable biogeochemical conditions. Dissipation of the sulfonamide antimicrobial sulfamethoxazole was examined in soil microcosms incubated under different terminal electron-accepting conditions (aerobic, nitrate-reducing, Fe(III)-reducing, and sulfate-reducing). Somewhat unexpectedly, sulfamethoxazole dissipation was fastest under Fe(III)-reducing conditions, with concentrations decreasing by >95% within 1 day. The rapid transformation was attributed to abiotic reactions between sulfamethoxazole and Fe(II) generated by microbial reduction of Fe(III) soil minerals. Separate experiments demonstrated that sulfamethoxazole was abiotically transformed in Fe(II)-amended aqueous suspensions of goethite (α-FeOOH((s))), and observed rate constants varied with the extent of Fe(II) sorption to goethite. Sulfamethoxazole transformation is initiated by a 1-electron reductive cleavage of the N-O bond in the isoxazole ring substituent, and observed products are consistent with Fe(II)-mediated reduction and isomerization processes. These findings reveal potentially important, but previously unrecognized, pathways that may contribute to the fate of sulfamethoxazole and related chemicals in reducing soil environments.

Diversity and composition of soil Acidobacteria and Proteobacteria communities as a bacterial indicator of past land-use change from forest to farmland.

The land-use change from natural to managed farmland ecosystems can undergo perturbations and significantly impact soil environment and communities. To understand how anthropogenic land-use alteration determines in-depth relationships among soil environmental factors and soil bacterial communities, high-resolution characterization was performed using soil samples (27 spots × 3 depths; top 10-20 cm, middle 90-100 cm, bottom 180-190 cm) from a natural forest and a 50 year-old farmland. The soil bacterial community abundance (number of OTU's per sample) and diversity (Faith's phylogenetic diversity) was significantly higher in the top layer of farmland soil than in forest soil. However, the differences in bacterial community abundance between farmland and forest decreased with depth, suggesting that the effect of fertilization was limited to top and middle layers. The phyla Acidobacteria and Proteobacteria were distributed distinctively during the land-use change. The subgroups Gp1-3 of Acidobacteria were more abundant in the forest samples (pH 3.5-5), while Gp4-7 and Gp10 were predominant in the farmland (pH 4.5-9.5). Members belonging to α-Proteobacteria and Xanthomonadales in γ-Proteobacteria were dominant in the forest, whereas ß-, δ-, and γ-Proteobacteria were relatively abundant in the farmland. Both multivariate and correlation network analyses revealed that Acidobacteria and Proteobacteria communities were significantly affected by soil pH, as well as toxic metals from pesticides (Zn, Cr, Ni, Cu, Cd, As) and terminal electron acceptors (NO3, bioavailable Fe(III), SO4). In line with the long history of anthropogenic fertilization, the farmland site showed high abundance of membrane and ATP-binding cassette transporter genes, suggesting the key for uptake of nutrients and for protection against toxic metals and environmental stresses. This study provides new insights into the use of both Acidobacteria and Proteobacteria community structures as a bacterial indicator for land-use change.

Electron shuttle-stimulated RDX mineralization and biological production of 4-nitro-2,4-diazabutanal (NDAB) in RDX-contaminated aquifer material.

The potential for extracellular electron shuttles to stimulate RDX biodegradation was investigated with RDX-contaminated aquifer material. Electron shuttling compounds including anthraquinone-2,6-disulfonate (AQDS) and soluble humic substances stimulated RDX mineralization in aquifer sediment. RDX mass-loss was similar in electron shuttle amended and donor-alone treatments; however, the concentrations of nitroso metabolites, in particular TNX, and ring cleavage products (e.g., HCHO, MEDINA, NDAB, and NH(4) (+)) were different in shuttle-amended incubations. Nitroso metabolites accumulated in the absence of electron shuttles (i.e., acetate alone). Most notably, 40-50% of [(14)C]-RDX was mineralized to (14)CO(2) in shuttle-amended incubations. Mineralization in acetate amended or unamended incubations was less than 12% within the same time frame. The primary differences in the presence of electron shuttles were the increased production of NDAB and formaldehyde. NDAB did not further degrade, but formaldehyde was not present at final time points, suggesting that it was the mineralization precursor for Fe(III)-reducing microorganisms. RDX was reduced concurrently with Fe(III) reduction rather than nitrate or sulfate reduction. Amplified 16S rDNA restriction analysis (ARDRA) indicated that unique Fe(III)-reducing microbial communities (ß- and γ-proteobacteria) predominated in shuttle-amended incubations. These results demonstrate that indigenous Fe(III)-reducing microorganisms in RDX-contaminated environments utilize extracellular electron shuttles to enhance RDX mineralization. Electron shuttle-mediated RDX mineralization may become an effective in situ option for contaminated environments.

Microbial community analyses of three distinct, liquid cultures that degrade methyl tert-butyl ether using anaerobic metabolism.

Methyl tert-butyl ether (MTBE) is a prevalent groundwater contaminant. In this study, three distinct MTBE-degrading, anaerobic cultures were derived from MTBE-contaminated aquifer material: cultures NW1, NW2 and NW3. The electron acceptors used are anthraquinone-2,6-disulfonate (AQDS; NW1), sulfate (NW2) and fumarate (NW3), respectively. About 1-2 mM MTBE is consistently degraded within 20-30 days in each culture. The 16S rDNA-based amplified ribosomal DNA restriction analysis (ARDRA) was used to analyze the microbial community in each culture. Results indicate novel microorganisms (i.e. no closely related known genera or species) catalyze anaerobic MTBE biodegradation, and microbial diversity varied with different electron acceptors. Tert-butyl alcohol (TBA) accumulated to nearly stoichiometric levels, and these cultures will be critical to understanding the factors that influence TBA accumulation versus degradation. The cultures presented here are the first stable anaerobic MTBE-degrading cultures that have been characterized with respect to taxonomy.

Photobiological transformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) using Rhodobacter sphaeroides.

Pump-and-treat strategies for groundwater containing explosives may be necessary when the contaminated water approaches sensitive receptors. This project investigated bacterial photosynthesis as a strategy for ex situ treatment, using light as the primary energy source to facilitate RDX transformation. The objective was to characterize the ability of photosynthetic Rhodobacter sphaeroides (strain ATCC(®) 17023 ™) to transform the high-energy explosive RDX. R. sphaeroides transformed 30 µM RDX within 40 h under light conditions; RDX was not fully transformed in the dark (non-photosynthetic conditions), suggesting that photosynthetic electron transfer was the primary mechanism. Experiments with RDX demonstrated that succinate and malate were the most effective electron donors for photosynthesis, but glycerol was also utilized as a photosynthetic electron donor. RDX was transformed irrespective of the presence of carbon dioxide. The electron shuttling compound anthraquinone-2,6-disulfonate (AQDS) increased transformation kinetics in the absence of CO2, when the cells had excess NADPH that needed to be re-oxidized because there was limited CO2 for carbon fixation. When CO2 was added, the cells generated more biomass, and AQDS had no stimulatory effect. End products indicated that RDX carbon became CO2, biomass, and a soluble, uncharacterized aqueous metabolite, determined using (14)C-labeled RDX. These data are the first to suggest that photobiological explosives transformation is possible and will provide a framework for which phototrophy can be used in environmental restoration of explosives contaminated water.

Fe(III) reduction-mediated phosphate removal as vivianite (Fe3(PO4)2⋠8H2O) in septic system wastewater.

Phosphate is a water contaminant from fertilizers, soaps, and detergents that enters municipal and onsite wastewater from households, businesses, and other commercial operations. Phosphate is a limiting nutrient for algae, and is one of the molecules that promotes eutrophication of water bodies. Phosphate is especially problematic in onsite wastewater because there are few removal mechanisms under normal operating conditions; a system must be amended specifically with compounds to bond to or adsorb phosphate in the septic tank or within the leach field. Vivianite (Fe3(PO4)2⋅8H2O) is a stable mineral formed from ferrous iron and phosphate, often as the result of Fe(III) reducing microbial activity. What was unknown was the concentration of phosphate that could be removed by this process, and whether it was relevant to mixed microbial systems like septic tank wastewater. Data presented here demonstrate that significant concentrations of phosphate (12-14mM) were removed as vivianite in growing cultures of Geobacter metallireducens strain GS-15. Vivianite precipitates were identified on the cell surfaces and within multi cell clusters using TEM-EDX; the mineral phases were directly characterized using XRD. Phosphate was also removed in dilute and raw (undiluted) septic wastewater amended with different forms of Fe(III) including solid phase and soluble Fe(III). Vivianite precipitates were recovered and identified using XRD, along with siderite (ferrous carbonate), which was expected given that the systems were likely bicarbonate buffered. These data demonstrate that ferric iron amendments in septic wastewater increase phosphate removal as the mineral vivianite, and this may be a good strategy for phosphate attenuation in the septic tank portion of onsite wastewater systems.