Appl Microbiol Biotechnol (2007) 77:713–721 DOI 10.1007/s00253-007-1183-6 ENVIRONMENTAL BIOTECHNOLOGY Influence of bicarbonate, sulfate, and electron donors on biological reduction of uranium and microbial community composition Wensui Luo & Wei-Min Wu & Tingfen Yan & Craig S. Criddle & Philip M. Jardine & Jizhong Zhou & Baohua Gu Received: 1 June 2007 /Revised: 22 August 2007 /Accepted: 26 August 2007 / Published online: 14 September 2007 # Springer-Verlag 2007 Abstract A microcosm study was performed to investigate capable of reducing U(VI) were significantly enriched by the effect of ethanol and acetate on uranium(VI) biological ethanol and acetate in low-bicarbonate buffer. Ethanol reduction and microbial community changes under various increased the population of unclassified Desulfuromonales, geochemical conditions. Each microcosm contained an while acetate increased the population of Desulfovibrio. uranium-contaminated sediment (up to 2.8 g U/kg) sus- Additionally, species in the Geobacteraceae family were pended in buffer with bicarbonate at concentrations of not enriched in high-bicarbonate buffer, but the Geothrix either 1 or 40 mM and sulfate at either 1.1 or 3.2 mM. and the unclassified Betaproteobacteria species were Ethanol or acetate was used as an electron donor. Results enriched. This study concludes that ethanol could be a indicate that ethanol yielded in significantly higher U(VI) better electron donor than acetate for reducing U(VI) under reduction rates than acetate. A low bicarbonate concentra- given experimental conditions, and electron donor and tion (1 mM) was favored for U(VI) bioreduction to occur in groundwater geochemistry alter microbial communities sediments, but high concentrations of bicarbonate (40 mM) responsible for U(VI) reduction. and sulfate (3.2 mM) decreased the reduction rates of U(VI). Microbial communities were dominated by species from the Keywords Uranium(VI) . Biological reduction . Geothrix genus and Proteobacteria phylum in all micro- Electron donor . Groundwater . Microbial community. cosms. However, species in the Geobacteraceae family 16S rRNA analysis : W. Luo T. Yan Introduction Oak Ridge Institute for Science and Education, Oak Ridge, TN 37831, USA Uranium mining and enrichment activities during the Cold W. Luo : P. M. Jardine : B. Gu (*) War produced large amounts of radioactive wastes and Environmental Sciences Division, contaminated significant volumes of soil and groundwater Oak Ridge National Laboratory, at the US Department of Energy’s (DOE) complexes Oak Ridge, TN 37831, USA e-mail: [email protected] (Hazen and Tabak 2005). Uranium commonly exists in the : environment either as oxidized, hexavalent U(VI) or as W.-M. Wu C. S. Criddle reduced, tetravalent U(IV) species, and its fate and Department of Civil and Environmental Engineering, transport are largely influenced by these oxidation states. Stanford University, Stanford, CA 94305, USA Under oxic conditions, U(VI) forms carbonate complexes ðÞ2À ðÞ4À such as UO2 CO3 2 or UO2 CO3 3 when groundwater J. Zhou contains carbonate and bicarbonate anions at pH > 6.5 Department of Botany and Microbiology, (Langmuir 1978). These U(VI) complexes are highly Institute for Environmental Genomics, University of Oklahoma, soluble and mobile in the subsurface environment. On Norman, OK 73019, USA the other hand, reduced U(IV) forms precipitates under 714 Appl Microbiol Biotechnol (2007) 77:713–721 anaerobic conditions. Therefore, in the past decade, bio- Materials and methods logically mediated reduction of U(VI) to sparingly soluble U(IV) has been extensively explored as an alternative Microcosm setup approach to remediate U(VI)-contaminated sites (Hazen and Tabak 2005). The U(VI)-contaminated sediment was collected from well Selection of electron donor source is essential for FW026 at the DOE Field Research Center at Oak Ridge, successful implementation of biological reduction of urani- TN. The sediment sample was stored in a glass bottle filled um in situ by stimulating the growth of indigenous micro- with site groundwater at 4°C for 2 months before use. A organisms. Under anaerobic conditions, metal-reducing factorial experimental design was employed for the micro- bacteria have limited ability to metabolize high-molecular- cosm study, and three factors were considered, including weight compounds such as proteins, celluloses, or long- bicarbonate (1 vs 40 mM), sulfate (1.1 vs 3.2 mM), and chain fatty acids (Kourtev et al. 2006). Low molecular electron donors (ethanol, acetate, and control). The low weight organics such as ethanol, acetate, lactate, glucose, concentration level of bicarbonate was to simulate the and formate have been selected as preferred electron condition used in our field pilot-scale test (Wu et al. 2006; donors in U(VI) bioreduction studies (Finneran et al. 2007), while the high level was to test if U(VI) reduction 2002). In field trials, acetate was successfully used as an may be accelerated through the extraction or desorption of electron donor (Anderson et al. 2003;Holmesetal.2002; solid-phase U(VI) by bicarbonate (Phillips et al. 1995; Nevin et al. 2003; Vrionis et al. 2005), while Ortiz-Bernad Ortiz-Bernad et al. 2004;ZhouandGu2005). Two et al. (2004) indicate that acetate has limited effect in concentration levels of sulfate were used to test if an stimulating the reduction of solid-phase U(VI). At the US elevated sulfate concentration may inhibit the reduction of DOE’s Oak Ridge site, we found that ethanol stimulated U(VI). Therefore, microcosms were performed in the the reduction of U(VI) at a faster rate than acetate following four buffer solutions: S1C1 contained 1.1 mM and lactate (Wu et al. 2006). These observations were at- Na2SO4 and 1 mM KHCO3 (pH = 7.7), S2C1 contained tributed to the fact that different electron donors have 3.2 mM Na2SO4 and 1 mM KHCO3 (pH = 7.8), S1C2 stimulated different microbial communities under site- contained 1.1 mM Na2SO4 and 40 mM KHCO3 (pH = 9.3), specific geochemical conditions. and S2C2 contained 3.2 mM Na2SO4 and 40 mM KHCO3 Among various factors, bicarbonate could significantly (pH = 9.4). Note that a relatively high pH observed in the impact the microbial community structure and the biolog- 40 mM bicarbonate buffer was due to degas or a loss of ical reduction of U(VI). Bicarbonate concentration levels CO2 because samples were purged with nitrogen and can lead to a change in groundwater pH and partitioning of experiments were performed in a N2-filled glove box. A U(VI) in the solid and solution phases (Phillips et al. 1995; relatively high pH (>7.5) used in these studies is also to Zhou and Gu 2005). High biocarbonate with calcium minimize the abiotic reduction of U(VI) to U(IV) by sulfide (0.45–5 mM) is also known to cause the formation of (Hua et al. 2006). Ca–uranyl-carbonate complex, which inhibits the biore- duction of U(VI) by both Fe-reducing bacteria (FeRB) and sulfate-reducing bacteria (SRB)(Brooks et al. 2003). Table 1 Geochemical properties of the FRC FW026 well sediment Sulfate is another geochemically important anion with and the microcosm solution controversial influences on the bioreduction of U(VI). Sulfate supports the growth of SRB such as Desulfovibrio Analyte Sediment (g/kg) Microcosms (mg/L) spp., which are capable of reducing U(VI) (Lovley and S1C1 S2C1 S1C2 S2C2 Phillips 1992). However, sulfate could also support the growth of sulfate reducers such as Desulfobacter spp., Fe 101.4 0.3 0.5 0.1 0.1 which are unable to reduce U(VI) but utilize acetate, Al 163.2 0.6 1.0 0.4 1.1 competing with U(VI)-reducing bacteria for electron Mg 22.8 2.2 2.5 2.3 1.2 Mn 1.6 0.7 0.7 0.3 0.1 donors (Lovley et al. 1993). Ca 5.6 16.0 18.0 10.7 3.4 In this study, factorial microcosms were designed to Si 3.8 0.9 1.0 0.6 0.6 investigate the interactions of the effects of electron donors P 5.8 0.0 0.1 0.2 0.1 (ethanol and acetate) and geochemical conditions (bicar- U 2.8 1.8 2.2 8.2 9.1 2À bonate and sulfate) on U(VI) biological reduction. The SO4 ND 107.0 255.0 143.5 307.5 associated microbial community was monitored during TOC 5.98 10.8 10.2 108.7 119.6 biostimulation using 16S ribosomal ribonucleic acid S1C1 1.1 mM Na2SO4 and 1 mM KHCO3, S2C1 3.2 mM Na2SO4 and (rRNA) cloning library methods to better understand the 1 mM KHCO3, S1C2 1.1 mM Na2SO4 and 40 mM KHCO3, S2C2 microbial processes underlying U(VI) bioreduction. 3.2 mM Na2SO4 and 40 mM KHCO3, ND not determined Appl Microbiol Biotechnol (2007) 77:713–721 715 The microcosms were established in an anaerobic glove composition, the sediment was digested with addition of bag (N2/H2 = 98:2) by adding 6 g of sediment (dry weight) concentrated nitric acid and 30% hydrogen peroxide into 160-mL serum bottles and adding 120 mL of one of the according to US EPA SW-846 Method 3050B. The TOC prepared buffer solution, which was prepurged with N2 for content was determined by combustion technique using US 1h to remove dissolved O2. Next, 110 μL of 2.2 M ethanol EPA Method 9060A. or 2.2 M Na-acetate or water (as no-electron-donor control) At various time intervals, samples in serum bottles were was added to the serum bottles. The initial concentrations taken by removing the crimp caps and rubber septa in the of ethanol and acetate were approximately 2 mM. Micro- anaerobic glove bag. Four-milliliter samples were taken cosms without addition of electron donors were used as from each bottle and centrifuged at 12,000 × g for 5 min.