Kevin T. Finneran

Enrichment of Members of the Family Geobacteraceae Associated with Stimulation of Dissimilatory Metal Reduction in Uranium-Contaminated Aquifer Sediments

ABSTRACT Stimulating microbial reduction of soluble U(VI) to insoluble U(IV) shows promise as a strategy for immobilizing uranium in uranium-contaminated subsurface environments. In order to learn more about which microorganisms might be involved in U(VI) reduction in situ, the changes in the microbial community when U(VI) reduction was stimulated with the addition of acetate were monitored in sediments from three different uranium-contaminated sites in the floodplain of the San Juan River in Shiprock, N.Mex. In all three sediments U(VI) reduction was accompanied by concurrent Fe(III) reduction and a dramatic enrichment of microorganisms in the family Geobacteraceae, which are known U(VI)- and Fe(III)-reducing microorganisms. At the point when U(VI) reduction and Fe(III) reduction were nearing completion, Geobacteraceae accounted for ca. 40% of the 16S ribosomal DNA (rDNA) sequences recovered from the sediments with bacterial PCR primers, whereas Geobacteraceae accounted for fewer than 5% of the 16S rDNA sequences in control sediments that were not amended with acetate and in which U(VI) and Fe(III) reduction were not stimulated. Between 55 and 65% of these Geobacteraceae sequences were most similar to sequences from Desulfuromonas species, with the remainder being most closely related to Geobacter species. Quantitative analysis of Geobacteraceae sequences with most-probable-number PCR and TaqMan analyses indicated that the number of Geobacteraceae sequences increased from 2 to 4 orders of magnitude over the course of U(VI) and Fe(III) reduction in the acetate-amended sediments from the three sites. No increase in Geobacteraceae sequences was observed in control sediments. In contrast to the predominance of Geobacteraceae sequences, no sequences related to other known Fe(III)-reducing microorganisms were detected in sediments. These results compare favorably with an increasing number of studies which have demonstrated that Geobacteraceae are important components of the microbial community in a diversity of subsurface environments in which Fe(III) reduction is an important process. The combination of these results with the finding that U(VI) reduction takes place during Fe(III) reduction and prior to sulfate reduction suggests that Geobacteraceae will be responsible for much of the Fe(III) and U(VI) reduction during uranium bioremediation in these sediments.

Desulfitobacterium metallireducens sp. nov., an anaerobic bacterium that couples growth to the reduction of metals and humic acids as well as chlorinated compounds

Strain 853-15A(T) was enriched and isolated from uranium-contaminated aquifer sediment by its ability to grow under anaerobic conditions via the oxidation of lactate coupled to the reduction of anthraquinone-2,6-disulfonate (AQDS) to anthrahydroquinone-2,6-disulfonate (AHQDS). Lactate was oxidized incompletely to acetate and carbon dioxide according to the reaction CH3CHOHCOO(-)+ 2AQDS+H2O --> CH3COO(-)+ 2AHQDS+CO2. Additional electron donors utilized included formate, ethanol, butanol, butyrate, malate and pyruvate. Lactate also supported growth with Fe(III) citrate, Mn(IV) oxide, humic substances, elemental sulfur, 3-chloro-4-hydroxyphenylacetate, trichloroethylene or tetrachloroethylene serving as the electron acceptor. Growth was not observed with sulfate, sulfite, nitrate or fumarate as the terminal electron acceptor. The temperature optimum for growth was 30 degrees C, but growth was also observed at 20 and 37 degrees C. The pH optimum was approximately 7.0. The 16S rDNA sequence of strain 853-15A(T) suggested that it was most closely related to Desulfitobacterium dehalogenans and closely related to Desulfitobacterium chlororespirans and Desulfitobacterium frappieri. The phylogenetic and physiological properties exhibited by strain 853-15A(T) (= ATCC BAA-636(T)) place it within the genus Desulfitobacterium as the type strain of a novel species, Desulfitobacterium metallireducens sp. nov.

Microorganisms Associated with Uranium Bioremediation in a High-Salinity Subsurface Sediment

ABSTRACT Although stimulation of dissimilatory metal reduction to promote the reductive precipitation of uranium has been shown to successfully remove uranium from some aquifer sediments, the organisms in the family Geobacteraceae that have been found to be associated with metal reduction in previous studies are not known to grow at the high salinities found in some uranium-contaminated groundwaters. Studies with a highly saline uranium-contaminated aquifer sediment demonstrated that the addition of acetate could stimulate the removal of U(VI) from the groundwater. This removal was associated with an enrichment in microorganisms most closely related to Pseudomonas and Desulfosporosinus species.

Microbially Mediated Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5- Triazine by Extracellular Electron Shuttling Compounds

ABSTRACT The potential for humic substances to stimulate the reduction of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was investigated. This study describes a novel approach for the remediation of RDX-contaminated environments using microbially mediated electron shuttling. Incubations without cells demonstrated that reduced AQDS transfers electrons directly to RDX, which was reduced without significant accumulation of the nitroso intermediates. Three times as much reduced AQDS (molar basis) was needed to completely reduce RDX. The rate and extent of RDX reduction differed greatly among electron shuttle/acceptor amendments for resting cell suspensions of Geobacter metallireducens and G. sulfurreducens with acetate as the sole electron donor. AQDS and purified humic substances stimulated the fastest rate of RDX reduction. The nitroso metabolites did not significantly accumulate in the presence of AQDS or humic substances. RDX reduction in the presence of poorly crystalline Fe(III) was relatively slow and metabolites transiently accumulated. However, adding humic substances or AQDS to Fe(III)-containing incubations increased the reduction rates. Cells of G. metallireducens alone reduced RDX; however, the rate of RDX reduction was slow relative to AQDS-amended incubations. These data suggest that extracellular electron shuttle-mediated RDX transformation is not organism specific but rather is catalyzed by multiple Fe(III)- and humic-reducing species. Electron shuttle-mediated RDX reduction may eventually become a rapid and effective cleanup strategy in both Fe(III)-rich and Fe(III)-poor environments.

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.

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.

Influence of reduced electron shuttling compounds on biological H2 production in the fermentative pure culture Clostridium beijerinckii.

Several reports suggest that extracellular electron shuttles influence fermentative metabolism in a beneficial manner for bioremediation and biotechnology strategies. The focus of this research was to characterize the effects of reduced electron shuttling molecules on fermentative H2 production. Reduced electron shuttles may provide reducing equivalents to generate H2, which influences alternate cellular processes. Electron shuttling compounds cycle between reduced-oxidized states and influence fermentative physiology. Clostridium beijerinckii fermentation was altered using a physiological approach that resulted in H2 production with the reduced extracellular electron shuttle anthrahydroquinone-2,6,-disulfonate (AH2QDS) and biologically reduced humic substances as the primary electron donors. Cells were suspended in a buffer with an excess of the biological electron transfer molecule NAD+, with AH2QDS (100–1000 μM) or biologically reduced humic substances (0.01–0.025 g/L) as the sole electron source. Increasing concentrations of AH2QDS and reduced humics increased H2 production, while H2 production was suppressed by Fe(III) hydroxides, which outcompeted the cells for electrons from the reduced shuttles, suggesting that the shuttles are in fact electron donors for H2 production. Oxidized AQDS/humics did not increase H2 production. Organic acid production shifted toward butyric acid in the presence of reduced electron shuttles, particularly with growing cells. Growth and hydrogen production rates in growing cells were initially faster in the presence of the reduced electron shuttles; however, the final biomass yield was inversely proportional to the starting AH2QDS concentration, which suggests that reduced shuttles may compete with anabolic cell processes for available energetic resources or that the shift to excess butyrate becomes toxic to the cells.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) Biodegradation Kinetics Amongst Several Fe(III)-Reducing Genera

Cyclic nitramine explosives biodegradation was investigated among four Fe(III)- and quinone-reducing bacterial genera. This strategy is an attractive option for RDX and/or HMX contamination because of their ubiquity; however, the biotransformation kinetics among different microbial populations is not known. The organisms investigated included two species within the Geobacteraceae, and one species each within the genera Anaeromyxobacter, Desulfitobacterium, and Shewanella. All species directly reduced RDX; however, humic substances (HS) and the HS analog anthraquinone-2,6-disulfonate (AQDS) significantly increased the rate and extent of RDX reduction. Degradation kinetics varied amongst the species tested, but extracellular electron shuttle mediated degradation rates were the fastest for each organism. RDX reduction rates ranged from 7.4 to 269.3 nmol RDX hr − 1 mg cell protein − 1 when AQDS was present. HMX was reduced more slowly by G. metallireducens than RDX; however, electron shuttles also stimulated HMX degradation. These data suggest that electron shuttle mediated cyclic nitramine transformation is ubiquitous among the keystone Fe(III)-reducing microbial genera, and that bioremediation strategies predicated on their physiology may be a reasonable approach in situ for both Fe(III)-rich and Fe(III)-poor environments.

Geochemical and microbiological processes contributing to the transformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in contaminated aquifer material

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a potential human carcinogen, and its contamination of subsurface environments is a significant threat to public health. This study investigated abiotic and biological degradation of RDX in contaminated aquifer material. Anoxic batch systems were started with and without pre-aeration of aquifer material to distinguish initial biological RDX reduction from abiotic RDX reduction. Aerating the sediment eliminated chemical reductants in the native aquifer sediment, primarily Fe(II) sorbed to mineral surfaces. RDX (50 μM) was completely reduced and transformed to ring cleavage products when excess concentrations (2mM) of acetate or lactate were provided as the electron donor for aerated sediment. RDX was reduced concurrently with Fe(III) when acetate was provided, while RDX, Fe(III), and sulfate were reduced simultaneously with lactate amendment. Betaproteobacteria were the dominant microorganisms associated with RDX and Fe(III)/sulfate reduction. In particular, Rhodoferax spp. increased from 21% to 35% and from 28% to 60% after biostimulation by acetate and lactate, respectively. Rarefaction analyses demonstrated that microbial diversity decreased in electron-donor-amended systems with active RDX degradation. Although significant amounts of Fe(III) and/or sulfate were reduced after biostimulation, solid-phase reactive minerals such as magnetite or ferrous sulfides were not observed, suggesting that RDX reduction in the aquifer sediment is due to Fe(II) adsorbed to solid surfaces as a result of Fe(III)-reducing microbial activity. These results suggest that both biotic and abiotic processes play an important role in RDX reduction under in situ conditions.

Anthrahydroquinone-2,6,-disulfonate (AH2QDS) increases hydrogen molar yield and xylose utilization in growing cultures of Clostridium beijerinckii

H2 production and xylose utilization were investigated using the fermentative culture Clostridium beijerinckii NCIMB 8052. Adding anthrahydroquinone-2,6-disulfonate (AH2QDS) increased the extent of xylose utilization by 56% and hydrogen molar yield by 24–37%. Enhanced hydrogen molar yield correlated with increased xylose utilization and increases in the acetate/butyrate product ratio. An electron balance indicated that AH2QDS shifted the electrons from the butyric acid pathway (NADH-dependent pathway) to the acetic acid pathway (non-NADH-dependent pathway), putatively creating a surplus of reducing equivalents that were then available for hydrogen production. These data demonstrate that hydrogen yield and xylose utilization can be manipulated by amending redox active molecules into growing cultures. This will impact biohydrogen/biofuel production by allowing physiological manipulations of growing cells for increased (or decreased) output of selected metabolites using amendments that are not consumed during the reactions. Although the current yield increases are small, they suggest a target for cellular alterations. In addition, increased xylose utilization will be critical to the fermentation of pretreated lignocellulosic feedstocks, which may have higher xylose content.