394 (LEN-1122-315836) Optimization of bioremediation processes for selenium contaminated wastewaters using anaerobic granular sludge.
Start time: 1:50 PM
Lenz, M¹, Gmerek, A¹, Lens, P.N.L.¹, ¹ Department of Environmental Technology, Wageningen University
One component in the "cocktail" of environmental hazardous substances contained in mine drainage is selenium (Simmons et al., 2002). The importance of selenium in environmental research is related to the fact that it shows only a marginal line between the nutritious optimum (as an essential element) and toxic effects upon exposure. In order to treat metal contaminated wastewaters, different physical/chemical and biological processes have been investigated. During the process of dissimilatory metal reduction, bacteria conserve energy by transferring electrons originating from the degradation of organic compounds, H2 or elemental sulfur to soluble oxy-anions of metals, which form a solid phase and thus can be removed from polluted waters This process can be used in order to convert selenate and selenite to elemental selenium. Selenium conversions are influenced by sulfate, but literature gives contradictory information about these influences. Hockin et al. (2003) describe the reduction of selenite to elemental selenium as a combination of a biological and chemical process (via biogenic sulfide). Bebien et al. (2002) state that uptake of selenate uses sulfate-permease. Both findings described make the stimulation of selenium oxyanion conversion possible by stimulation of sulfate reducing bacteria (SRB). On the other hand selenate is described to be a specific inhibitor for SRBs. Consequently, an intensive investigation of the influence of sulfate on selenium conversions is necessary. Therefore anaerobic granular sludge originating from an Upflow Anaerobic Sludge Blanket (UASB) Reactor was tested towards its removal capacity for selenium. In this study, optimization of the process by stimulating the activity of SRB was investigated by testing different feeding patterns for sulfate. Special attention was given to the influence of the redox-potential. Selenate reduction was slightly higher, when sulfate reduction in the granules was stimulated. Furthermore, it was shown that 0.5 mM selenate is indeed highly toxic to sulfate reduction.

395 (GRO-1122-316718) PASSIVE TREATMENT OF ACID DRAINAGE WATERS DURING COLD WINTER MONTHS.
Start time: 2:10 PM
Groudev, S¹, Nicolova, M¹, Georgiev, P¹, Spasova, I¹, Diels, L², Tabak, H³, ¹ University of Mining and Geology, Sofia, Bulgaria² VITO, Mol, Belgium³ U.S. Environmental Protection Agency, Cincinnati, Ohio, USA
Acid drainage waters were treated under field conditions during different climatic seasons by means of different passive systems (natural and constructed wetlands, alkalizing drains, permeable reactive barriers and rock filters) used separately or in different combinations. The waters had pH in the range of about 2.5 - 3.5 and contained radionuclides (mainly uranium and radium), heavy metals (copper, zinc, cadmium, lead, nickel, cobalt), arsenic and sulphates in concentrations usually much higher than the relevant permissible levels for waters intended for use in the agriculture and/or industry. The passive treatment of these waters was efficient but in most cases markedly depended on the temperature. The best results during the cold winter months were achieved by a multibarrier consisting of an alkalizing limestone drain and a unit for microbial dissimilatory sulphate reduction and sorption of pollutants on the dead plant biomass (plant compost, hay, straw) present in this unit. The multibarrier was located in a light building with an internal air temperature higher than 5 °C even during the coldest days. Under such conditions, the sorption of pollutants was essential and the activity of the sulphate-reducing bacteria and the other metabolically interdependent microorganisms was still quite good.

396 (BLO-1118-275804) Performance of Permeable Reactive Barriers for Treatment of Mine Drainage: Measurement and Modelling.
Start time: 2:30 PM
Blowes, D¹, Ptacek, C¹, Bain, J¹, Mayer, K², Benner, S³, Jambor, J^{1, 2}, Gould, W^{1, 4}, ¹ Department of Earth Sciences, University of Waterloo, Waterloo, ON, Canada² 2. Department of Earth and Ocean Sciences, University of British Columbia, Vancouver, BC, Canada³ Department of Geosciences, Boise State University, Boise, ID, USA⁴ Environmental Laboratory, CANMET, Ottawa, ON, Canada
Over the past decade, permeable reactive barrier (PRB) systems have been implemented to prevent and treat acidic drainage discharged from mine wastes. During this period, detailed sampling of PRB systems, including hydrogeological, geochemical, mineralogical and microbiological characterization, has been conducted. The results derived from these studies have been used in conjunction with sophisticated computer models to understand the interaction between physical, chemical and biological processes within PRB systems. These studies have been augmented with laboratory studies to assess the potential benefits of alternative barrier materials, and field studies to evaluate alternative PRB installation techniques. Combined, these studies provide an understanding of the potential applications and limitations of PRB systems for treating mine drainage water.

397 (SAN-1118-441262) Toxicity of lead on sulfate-reducing bacteria in the presence of goethite and quartz.
Start time: 2:50 PM
Sani, R¹, Peyton, B¹, Dohnalkova, A², ¹ Washington State University, Pullman, WA, USA² Pacific Northwest National Laboratory, Richland, WA, USA
Lead (Pb) and Pb-compounds are potentially mobile within ground waters and threaten down-gradient water resources at many US Department of Energy sites. The effective manipulation of indigenous bacterial communities to stimulate in situ activity in the presence of toxic heavy metals requires knowledge of the toxic effects of contaminants on subsurface bacteria. Goethite, quartz, and Pb were treated with the sulfate-reducing bacterium, Desulfovibrio desulfuricans G20, in a medium specifically designed in our laboratory to assess metal toxicity. Results showed that G20 first removed Pb from solutions, and then growth began resulting in visible black precipitates of Pb and iron sulfides. In the presence of goethite and quartz, Pb increased the lag time of G20 from two days in Pb-free treatments to five days in the presence of 26 M soluble Pb. In the absence of goethite and quartz, however, with 26 M soluble Pb, no measurable growth was observed. Analysis of thin sections of G20 treated with 10 M Pb using high resolution transmission electron microscopy showed cells with a heavy periplasmic precipitation of Pb-containing material. In fact, more than 50% cells had lead sulfide precipitates in the cytoplasmic space. Under the same conditions in the presence of goethite, however, no cells were detected that had lead sulfide precipitates in the cytoplasm. Selected area electron diffraction pattern and crystallographic analysis of transmission electron microscope lattice fringe images confirmed the structure of lead sulfide precipitates in the cytoplasmic spaces was identical to galena. Our results suggest that 1) at equivalent aqueous concentrations, the apparent Pb toxicity with and without goethite was not equivalent, i.e., addition of minerals in medium resulted in a decrease in Pb toxicity to G20; 2) once growth began, even after 5 days of lag period, cultures ultimately attained the same total cell protein yield as they did in Pb- and mineral-free treatments suggesting that observed toxic effects were reversible or non-permanent; and 3) the observed reduction in toxicity of Pb to G20 with and without minerals may reflect the differential bioavailability of Pb in the medium, since, in the presence of goethite, Pb did not enter to the cells i.e. Pb did not exert any toxicity.

(58154) break.
Start time: 3:10 PM

398 (JEN-1117-753919) The Effect of Water Saturation on Carbon Addition for Pyrite Oxidation Inhibition.
Start time: 3:50 PM
Jenkins, J¹, Silverstein, J¹, ¹ University of Colorado Boulder, Civil Environmental Engineering, Boulder, CO, USA
A strategy for remediation of acid mine drainage (AMD) sources is initiation of a shift in microbial populations from dominance of pyrite oxidizing bacteria to iron and sulfur reducing microorganisms by the addition of biodegradable organic matter. However, hydrologic conditions in waste rock formations are expected to play a major role in determining substrate availability, redox conditions affecting iron and sulfur transformations, as well as contaminant transport. Experiments were conducted using two 37.8-liter tanks packed with 0.1 to 10-cm rock particles obtained from a mine waste pile near Leadville, CO resulting in 50% bulk porosity in the tanks. Drainage water was recirculated through the tanks with addition of fresh deionized water to obtain a 9-day average hydraulic residence time. One tank was run under saturated conditions and the second was run with trickle flow to achieve 16% saturation. The headspace of both tanks was sparged with air continuously. Production of soluble ferric iron (Fe(III)) and sulfate in the unsaturated tank was significantly higher than in the saturated system. The unsaturated tank generated 16 g/L ferric iron and pH of 1.5 compared with 0.1 g/L ferric and pH 2.5 in the saturated tank. After acid generating conditions were established, glucose was added to the tanks at a rate of 36 g/day as C. The effect of water saturation and carbon addition in inducing a microbial population shift to iron and sulfur reduction was evaluated by water chemistry monitoring, including pH, iron speciation, sulfate, and redox, and as well as microbial population monitoring. Most Probable Number (MPN) technique for autotrophs and heterotrophs, Fluorescent In-Situ Hybridization with labeled small sub-unit rRNA probes for indicator genera: Acidithiobacillus, Acidiphilium, and Leptospirillum, and DNA extraction and sequencing were used to evaluate the bacterial populations. Results indicate that drainage flow controls the availability of organic matter and oxygen, and in turn bacterial activity and production of acid mine drainage.

399 (ZAL-1116-456067) Modular Field-Bioreactor for Acid Mine Drainage Treatment.
Start time: 4:10 PM
Zaluski, M¹, Figueroa, L², Joyce, H¹, Bless, D³, Bolis, J², ¹ MSE-Technology Applications, Butte, MT, USA² Colorado School of Mines, Golden, CO, USA³ US EPA, Office of Research and Development, Cincinnati, OH, USA
The presentation focuses on the improvements to engineered features of a passive technology that has been used for remediation of acid rock drainage (ARD). This passive remedial technology, a sulfate-reducing bacteria (SRB) bioreactor, takes advantage of the ability of SRB that, if supplied with a source of organic carbon, can increase pH and alkalinity of the water and immobilize metals by precipitating them as metal sulfides or hydroxides. The remoteness of ARD sites and their abundance require that the design of an SRB bioreactor is simple and inexpensive. Therefore, bioreactors need to be designed to a size that allows for transportation using backcountry roads. To satisfy these requirements a design for a modular treatment system was developed using reactive cartridges (RC) that are prefabricated as 8-foot diameter vessels. The RC has been designed so it supports the prime functional aspects of a bioreactor such as high permeability, ample supply of organic carbon, ability to maintain anaerobic conditions, and capacity to accumulate precipitated metals and means for their periodical removal, as needed. A bioreactor system consisting of four RCs has been installed at the McClelland tunnel adit located east of Dumont, Colorado. The system performance, including changes in composition of organic carbon in two reactive substrates, is being monitored. The RC design was developed by the Mine Waste Technology Program (MWTP) at MSE Technology Applications (MSE), Butte, Montana, USA. The work was funded by the U.S. Environmental Protection Agency (EPA) and was jointly administered by the EPA and the U.S. Department of Energy (DOE) National Energy Technology Laboratory and performed at the Western Environmental Technology Office under DOE contract number DE-AC09-96EW96405.

400 (COL-1118-698064) Metal toxicity and bacterial sulfate reduction: what do we really know?
Start time: 4:30 PM
Colberg, P¹, Jin, S², ¹ University of Wyoming, Laramie, WY, USA² Western Research Institute, Laramie, WY, USA
Natural deposits of heavy metals typically occur as sulfide minerals that are insoluble under low redox conditions. Since reduction of sulfate to sulfide at surface temperatures and pressures occurs only via microbial processes, the mobility of heavy metals in anoxic environments is, in effect, controlled by microbial activity. The resultant sulfides are known to precipitate heavy metals, immobilizing them as sulfide minerals or as trace constituents of other metal sulfides. Because bacterial sulfate reduction in known to play a major role in the fate of organic contaminants, many of which coexist with metals, understanding interactions between heavy metals and sulfate-reducing bacteria (SRB) is imperative to advancing applications of SRB to ameliorating both metal and organic contamination problems in a variety of environmental settings. Only a small number of studies have focused on metal tolerance by SRB. We have investigated the tolerance of sulfate-reducing sediment enrichments to a range of metal concentrations. Our results demonstrate that SRB can efficiently detoxify metals; but more importantly, our work suggests that the apparent tolerance of SRB to metals is not likely based solely on their ability to release sulfides that bind metals. Clearly, more research is warranted to identify the precise mechanisms involved in metal tolerance in SRB and to elucidate differences in community composition and structure from a wider field of anaerobic settings. This presentation will both summarize and assess the findings of studies from a broad spectrum of applications that have focused on SRB and metal toxicity.

401 (WIL-1117-649267) A study of zinc metal toxicity on the cellulolytic bacteria in anaerobic passive treatment systems.
Start time: 4:50 PM
Ruhs, A¹, Figueroa, L¹, Wildeman, T¹, ¹ Colorado School of Mines, Golden, CO, 80401
The effective use of anaerobic passive treatment systems (APTS), such as sulfate-reducing bioreactors, to treat acid mine drainage will help to mitigate water contamination from mines located in remote areas as well as cut current treatment costs. One draw back to these systems has been the inhibition of sulfate reduction with high concentrations of metals. APTS contain a complex microbial ecosystem, and metal toxicity could be indirectly affecting sulfate-reduction by inhibiting other important microbes. If microbes such as the cellulolytic - fermenting bacteria are inhibited from producing viable substrate for the sulfate-reducing bacteria, then the rate of sulfate reduction over time in APTS will ultimately decline. We examined the toxic effect of zinc, a common metal found in acid mine drainage, on a pure culture of Cellulomonas flavigena, a cellulolytic - fermenting bacteria. Serum bottles containing C. flavigena, at two protein concentrations of 250 and 500 mg/L, were exposed to initial zinc concentrations of 0, 20, and 40 mg/L and monitored over a 9 hour period. The extent of inhibition on C. flavigena activity correlated best (r2=0.93) with the mass ratio of zinc uptake to cell protein. Final zinc concentrations ranged from 0.9 to 2.2 mg/L. Zinc uptake was operationally defined as the total zinc removed from solution and includes sorption and internalization. Initial and final dissolved zinc concentration did not correlate well with extent of inhibition. In the presence of higher biomass the relative rate of glucose utilization was 20 to 50% higher in the presence of zinc than at lower biomass concentration. The concurrent internalization of metals with sorption and precipitation processes can produce inhibition in the presence of low metal concentration. Thus low effluent metal concentration may not be indicative of the extent of inhibition experienced by the microbes. The inhibitory effect of metals on cellulolytic - fermenting bacteria is an important aspect to consider when establishing the limitations of sulfate reducing biozones.

402 (MAR-1117-844333) The Role of Enhanced Heterotrophic Growth on Microbial Pyrite Oxidation.
Start time: 5:10 PM
Marchand, E.¹, Plumb, P.¹, ¹ University of Nevada, Reno, Reno, NV, USA
Acid mine drainage (AMD) is formed by the oxidation of reduced sulfur- and iron-containing minerals (pyrite, arsenopyrite, etc.), resulting in the generation of sulfuric acid. This process is catalyzed by certain bacteria (e.g. Acidithiobacillus ferrooxidans) which utilize ferrous iron and reduced sulfur as energy sources and require oxygen to grow. The focus of this research is to assess a method for minimizing acid formation in situ by supplementing contaminated sites with a suitable organic carbon source. It is believed that this will allow naturally occurring heterotrophic bacteria to flourish and out-compete sulfur- and iron-oxidizing bacteria for dissolved oxygen, thereby resulting in a shift in the dominant microbial community. Laboratory experiments have been performed with mixed and enrichment cultures to identify the influence of active heterotrophic growth on pyrite oxidation. In studies with pure and mixed cultures of acidophilic microbes augmented with organic carbon substrate, a number of different phenomena have been observed by our research group. Some of the most important findings are as follow: (1) In studies with pure and mixed cultures of acidophilic microbes in shake flasks containing pyrite at an initial pH of 2.5, the presence and growth of aerobic, heterotrophic bacteria resulted in significantly lower rates of pyrite oxidation even though oxygen was not found to be limiting (DO > 6.0 mg/L). (2) Soluble metabolic products of enhanced heterotrophic growth were found to have a marked effect on both the iron-oxidizing microbial community and the rate of abiotic pyrite oxidation. (3) In systems where the amount of oxygen was limited, it was found that in the presence of heterotrophic bacteria, even lower oxidation rates were observed and iron reduction occurred once oxygen was consumed. With limited oxygen available, ferric iron was used for respiration and the pH increased from 2.5 to 4.0. This is significant because a shift in solution pH is one of the most important steps in remediation of AMD sites. While sulfate reduction has not been specifically addressed during this research, it is likely that in situ sulfate reduction would be possible as a subsequent biogeochemical reaction once the mineral-oxidizing reactions were interrupted.