Published online 5 April 2007
Published in J Environ Qual 36:621-627 (2007)
DOI: 10.2134/jeq2006.0357
© 2007 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
TECHNICAL REPORTS
Bioremediation and Biodegradation
Bacterial Reduction of Selenium in Coal Mine Tailings Pond Sediment
Tariq Siddiquea,
Joselito M. Arocenaa,b,*,
Ronald W. Thringa and
Yiqiang Zhangc
a Environmental Science and Engineering, Univ. of Northern British Columbia, 3333 University Way, Prince George, BC V2N 4Z9 Canada
b Canada Research Chair–Soil and Environmental Sciences, Univ. of Northern British Columbia, 3333 University Way, Prince George, BC V2N 4Z9 Canada
c Dep. of Environmental Sciences, Univ. of California, Riverside, CA 92521 USA. T. Siddique, present address, Dep. of Biological Sciences, University of Alberta, Edmonton, AB, Canada T6G 2E9
* Corresponding author (arocenaj{at}unbc.ca)
Received for publication September 6, 2006.
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ABSTRACT
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Sediment from a storage facility for coal tailings solids was assessed for its capacity to reduce selenium (Se) by native bacterial community. One Se6+–reducing bacterium Enterobacter hormaechei (Tar11) and four Se4+–reducing bacteria, Klebsiella pneumoniae (Tar1), Pseudomonas fluorescens (Tar3), Stenotrophomonas maltophilia (Tar6), and Enterobacter amnigenus (Tar8) were isolated from the sediment. Enterobacter hormaechei removed 96% of the added Se6+ (0.92 mg L–1) from the effluents when Se6+ was determined after 5 d of incubation. Analysis of the red precipitates showed that Se6+ reduction resulted in the formation of spherical particles (<1.0 µm) of Se0 as observed under scanning electron microscope (SEM) and confirmed by EDAX. Selenium speciation was performed to examine the fate of the added Se6+ in the sediment with or without addition of Enterobacter hormaechei cells. More than 99% of the added Se6+ (
2.5 mg L–1) was transformed in the nonsterilized sediment (without Enterobacter hormaechei cells) as well as in the sterilized (heat-killed) sediment (with Enterobacter hormaechei cells). The results of this study suggest that the lagoon sediments at the mine site harbor Se6+– and Se4+–reducing bacteria and may be important sinks for soluble Se (Se6+ and Se4+). Enterobacter hormaechei isolated from metal-contaminated sediment may have potential application in removing Se from industrial effluents.
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INTRODUCTION
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THE solubility (biological availability) and toxicity of selenium (Se) are influenced by chemical forms and oxidation states with different biological activities and physicochemical properties. The different Se species in the environment are selenate (Se6+), SeO42–; selenite (Se4+), SeO32–; elemental selenium (Se0); and organic selenide (Se2–) (Dungan and Frankenberger, 1999). The concentration, speciation, and chemical associations of Se are determined by factors including pH and redox conditions, the solubility of Se salts, biological metabolism, and chemical reaction kinetics (McNeal and Balistrieri, 1989; Masscheleyn et al., 1993). Soluble Se oxyanions (SeO42–, Se6+; SeO32–, Se4+) are of major concern because of their bioaccumulation and toxicity (Presser and Ohlendorf, 1987; Weres et al., 1989).
In wetlands, bacterially mediated oxidation–reduction reactions are the most important processes controlling Se speciation, precipitation/dissolution, sorption/desorption, methylation, and volatilization. Oremland et al. (1989) demonstrated that heterotrophic bacteria were capable of dissimilatory Se6+ reduction in anaerobic sediments. Bacterial reduction of toxic soluble Se6+ and Se4+ into nontoxic insoluble Se0 has been observed in contaminated and pristine environments (Oremland, 1994) that could be a useful strategy for bioremediation (Losi and Frankenberger, 1997a).
A diverse array of microorganisms has the ability to reduce Se oxyanions (Frankenberger and Karlson, 1994; Ike et al., 2000; Siddique et al., 2005, 2006). Bacteria can also use this reaction to overcome toxic effects of Se (Moore and Kaplan, 1992). Numerous studies of Se transformations, cycling, and volatilization in aquatic and terrestrial ecosystems have been reported in recent years (Oremland et al., 1989; Oremland et al., 1990; Oremland, 1994; Frankenberger and Arshad, 2001; Zhang et al., 2004; Fujita et al., 2005; Siddique et al., 2005, 2006) but rates for these processes vary greatly depending on temperature, moisture, organic carbon content of soil/sediment, Se concentrations and chemical forms, and microbiological activity. This study was intended to assess the reduction potential of coal mine tailings pond sediment. The sediment was collected from a storage lagoon that had been receiving industrial effluents containing small amounts of various metals. We isolated and identified indigenous Se-reducing bacteria from the sediment. Selenium speciation was done to monitor the fate of added Se6+ and its transformation in the sediment by either Enterobacter hormaechei or indigenous microflora. The results of this study can contribute toward the long-range goal of developing strategies for removing Se from environment.
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MATERIALS AND METHODS
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Materials
Sodium selenate (Na2SeO4) and selenium (IV) oxide (SeO2) were purchased from Sigma-Aldrich (Oakville, ON). Selenium (IV) standard solution and sodium borohydride (NaBH4) were purchased from Fisher Scientific (Pittsburgh, PA). Effluent used in this study was a treated water from a refinery operation. Selected characteristics of the effluent and sediment used in this study are given in Table 1.
Isolation of Selenate- and Selenite-Reducing Bacteria from Sediment
The sediment was used to inoculate the enrichment cultures for bacterial isolation. Enrichment cultures were performed in 50-mL sterile Erlenmeyer flasks containing 30 mL minimal nutrient medium and 3 g of sediment. Flasks were amended with each of yeast (250 mg L–1) and tryptic soy broth (TSB; 250 mg L–1) (DifcoTM, Sparks, MD), and spiked with sterile Na2SeO4 solution (passed through a sterile 0.2 µm membrane filter, Millipore Corp, Bedford, MA) to a final concentration of 50 mg Se6+ L–1. The flasks were capped with sterile rubber stopper and incubated statically at 30°C for 3 d when a red color of Se0 appeared in the flasks. The contents of the flask were serially diluted in sterile deionized water and spread on tryptic soy agar (TSA) (Difco, Sparks, MD) plates supplemented with yeast extract (Difco, Sparks, MD) at 5 g L–1. Separate plates were prepared for screening Se6+– and Se4+–reducing bacteria. The molten medium made for the Se6+ plates was spiked with a sterile Na2SeO4 solution, while for the Se4+ plates, sterile SeO2 was added to the molten medium to give a final concentration of 50 mg L–1. The same serially diluted aliquots were plated on Se6+ and Se4+ containing TSA plates. The plates were incubated at 30°C for 2 to 3 d when several bacterial colonies with red Se0 precipitates were observed on the TSA plates. The single colonies were restreaked on TSA plates with and without Se6+ or Se4+ to ensure that the red color of the colony was not due to a bacterial pigment.
Phylogenetic Analyses of Selenate- and Selenite-Reducing Bacteria
Pure cultures of morphologically different Se6+– and Se4+–reducing bacteria isolated from the enrichment culture were identified by 16S rDNA sequence analysis. Microbial genomic DNA was extracted according to the method described by Sambrook and Russell (2001). Amplification of the partial 16S rRNA gene (rDNA) was performed with the primers 27F and 519R (Lane, 1991). Polymerase chain reaction (PCR) master mix (Promega, Madison, WI) was used according to the manufacturer's instructions. DNA was amplified using a 35-cycle PCR (initial denaturation, 95°C for 5 min; subsequent denaturation, 95°C for 1 min; annealing, 55°C for 1 min; extension, 72°C for 1 min, and final extension, 72°C for 5 min) using a PTC-100 Programmable Thermal Controller (MJ Research Inc., MA). The PCR product was analyzed on 1.5% agarose gel and purified using a Qiaex II gel kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The amplicon was sequenced using CEQ Dye Terminator Cycle Sequencing Kit on a Beckman Coulter automated DNA sequencer (Beckman Coulter, Inc. Fullerton, CA) and aligned to the database sequences using BLASTN (Altschul et al., 1997). Phylogenetic and molecular evolutionary analyses were conducted using MEGA program (Ver. 3.1) (Kumar et al., 2004) based on the sequence distance method. Distance estimation was performed by Jin and Nei (1990) model using Kimura's 2-parameter model. The Neighbor-joining method was used to infer the phylogenetic tree and tree topology was tested by bootstrap analysis.
Selenate Reduction in Effluent and Scanning Electron Microscope (SEM)/EDAX Analyses of Elemental Selenium
Isolate Tar11 (Enterobacter hormaechei) was pre-grown in a 1% TSB solution amended with 10 mg Se6+ L–1 and incubated (30°C) overnight. The solution was centrifuged using a Beckman Coulter Avanti-j centrifuge at 5000 rpm for 20 min. To remove the residual TSB and Se6+, bacterial cells were washed three times with 30 mL of the sterile nutrient medium as described above by centrifugation. Washed cells were resuspended in the same sterile nutrient medium for use in Se6+ reduction studies in the effluent and sediment.
Selenate reduction in the industrial effluent was conducted (in triplicate) in 50-mL Erlenmeyer flasks containing 40 mL of the effluent. The flasks were autoclaved at 121°C for 15 min before use. The effluent was inoculated with Enterobacter hormaechei by adding 75 µL of the cell culture (OD600 12.4). The flasks were spiked with Se6+ (1 mg L–1) and amended with TSB (500 mg L–1). The flasks were then capped with sterile rubber stoppers and incubated statically at 30°C. Samples drawn from the flasks after 5 d of incubation were analyzed on an atomic absorption spectrometer (AAS) for Se. For SEM analysis, cells were grown with Se6+ (20 mg L–1) and TSB (1000 mg L–1). Red precipitates were collected from the flasks and washed to remove the cellular materials using the method described by Oremland et al. (2004). Samples of the red precipitates were mounted on an aluminum stubs and coated with Au for 90 s before microscopic observations. A Philips XLS 30 SEM equipped with an energy dispersive analyzer was used. The SEM was operated between 20 and 30 keV. Semi-quantitative (±5%) elemental composition of precipitates was estimated using EDAX software that corrects energy spectrum based on Z (atomic number), A (absorption), and F (fluorescence) factors.
Selenate Reduction in Sediment
Selenate reduction in the sediment was studied in 50-mL Erlenmeyer flasks (in triplicate). The flasks were autoclaved before use and divided into four batches. One batch contained 30 mL of sterilized effluent and 15 g of sterilized (heat-killed) sediment (moisture content, 36%) while the other contained nonsterilized effluent and nonsterilized (live) sediment. Both batches received 75 µL of the cell culture as an inoculum. The other two batches, which had the same contents as the first and second batches, were not inoculated with Enterobacter hormaechei. The flasks were spiked with Se6+ (2 mg L–1) and amended with TSB (1000 mg L–1). After capping with sterile rubber stoppers, the flasks were incubated statically at 30°C for 5 d. Samples were collected daily for Se speciation in the sediment during the course of the study.
Selenium Species Analyses
Selenium speciation in the sediment was performed using hydride generation atomic absorption spectrometry (HGAAS) with the method developed by Zhang et al. (1999a, b). Sediment samples collected during the experiment were centrifuged to separate the sediment particles, and the aqueous phase was used for Se speciation. Selenite was directly determined in the sample buffered to pH 7. Total soluble Se in the sample was monitored by oxidizing all Se to Se6+ by K2S2O8 and then reducing to Se4+ with 6 N HCl and determined by HGAAS. Organic Se2– in the sample was oxidized to Se4+ by K2S2O8 which was indicated by the precipitation of Mn oxides formed from the oxidation of added Mn2+. The organic Se2– concentration was then calculated as the difference between Se4+ in the sample treated with K2S2O8 in the presence of Mn2+and Se4+ determined in another subsample. The Se6+ concentration was calculated by subtracting Se4+ and organic Se2– values from the total soluble Se concentration. Accumulated Se in the sediment was calculated as the difference between added Se6+ and total soluble Se.
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RESULTS
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Isolation and Identification of Selenate- and Selenite-Reducing Bacteria from Sediment
Five different bacterial strains capable of reducing Se6+ and Se4+ were isolated from the sediment-enrichment culture and subjected to 16s rDNA gene sequence analyses for identification. One bacterium (Tar11) reduced Se6+ while the other four bacteria (Tar1, Tar3, Tar6, and Tar8) were able to reduce Se4+ to Se0. Molecular taxonomy results indicate that strain Tar11 was 99% homologous with Enterobacter hormaechei (Table 2). The other Se4+–reducing bacteria were classified under the order Enterobacteriales, Pseudomonadales, and Xanthomonadales. Comparison of the nucleotide query sequences against a nucleotide sequence database recognized these Se4+–reducing bacteria as Klebsiella pneumoniae (Tar1), Pseudomonas fluorescens (Tar3), Stenotrophomonas maltophilia (Tar6), and Enterobacter amnigenus (Tar8) (Table 2). The phylogenetic relationship of the isolated Se6+– and Se4+–reducing bacteria is presented in Fig. 1.
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Table 2. Identification of selenate (Se6+)- and selenite (Se4+)-reducing bacteria isolated from the coal mine tailings sediment.
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Fig. 1. Neighbor-joining tree showing the phylogenetic relationship among Se-reducing bacterial strains isolated from the coal mine fine tailings and identified by 16S rRNA gene sequence. Fragments of 500 bp are used to construct the tree. Bootstrap values are indicated at each node. The tree is rooted with Thermotoga maritima as the outgroup.
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Selenate Reduction in Effluent and Scanning Electron Microscope Analysis of Elemental Selenium Produced during Selenate Reduction by Enterobacter hormaechei
Among the bacterial strains, Enterobacter hormaechei (Tar11) was used for further studies as this strain exhibited the ability to reduce Se6+ to Se0 during the enrichment and isolation process. When grown in the effluent, Enterobacter hormaechei transformed the added Se6+ (0.92 mg L–1) to Se0 and only 0.04 mg L–1 of Se6+ was detected when the culture medium in the flask was analyzed after 5 d of incubation. Red precipitates of elemental Se0 accumulated at the bottom of the flasks (set for SEM studies) were collected for SEM analysis. Cells of Enterobacter hormaechei grown on Se6+ produced many spherical particles of Se0 having different sizes in nanometer range (arrows in Fig. 2 show the Se0 particles). Though effort was made to remove the cells, Se0 spheres were seen with unrecovered cellular material. The EDAX spectrum (not shown) revealed that the spherical particles were entirely composed of Se.
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Fig. 2. Formation of elemental Se (Se0) by Enterobacter hormaechei. Scanning electron microscope (SEM) image shows Se0 spheres of different sizes formed by the bacterium during Se6+ reduction and indicated by arrows. Scale bar = 1.0 µm
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Selenate Reduction in Sediment
The fate of the added Se6+ to the sterilized (heat-killed) and the nonsterilized (live) sediments was studied with and without addition of the isolated Se6+–reducing bacterium Enterobacter hormaechei. The change in the concentration of Se6+ in the sediment during the course of the experiment is presented in Fig. 3A. Rapid reduction of Se6+ occurred in the nonsterilized sediment inoculated with Enterobacter hormaechei and Se6+ concentration dropped from 2.48 mg L–1 (Day 0) to 0.083 mg L–1 (Day 2) as compared with 0.19 mg L–1 (Day 2) but without any inoculation. Only 0.005 to 0.007 mg L–1 of Se6+ was recorded in the nonsterilized sediments at Day 5. Enterobacter hormaechei also induced a progressive decrease of Se6+ in the sterilized sediment but at a slower rate as 0.587 and 0.048 mg L–1 of Se6+ was measured in the flasks at Day 2 and Day 3, respectively. Selenate was almost totally recovered from the sterilized sediment (control) devoid of any viable cells which indicated that Se6+ was not adsorbed on the sediment.
Enterobacter hormaechei quickly reduced the total soluble Se (Fig. 3B) in the nonsterilized sediment and only 0.246 mg L–1 of total soluble Se was detected at Day 2. The concentration of total soluble Se decreased to 0.41 mg L–1 (Day 2 analysis) in the nonsterilized sediment which was not bioaugmented with Enterobacter hormaechei. In the sterilized sediment inoculated with Enterobacter hormaechei, slow removal of total soluble Se was observed until Day 5. No significant change in the total soluble Se was noted in the sterilized sediment where no bacterial cells were added.
During biotransformation of added Se6+, changes in the concentrations of other important Se species were noticed. Variation in Se4+ concentration during the incubation is shown in Fig. 4A. At the onset of Se6+ reduction, Se4+ accumulated in the sterilized sediment inoculated with Enterobacter hormaechei. Concentration of Se4+ increased from 0.67 (Day 1) to 1.35 mg L–1 (Day 2) and then gradually decreased to 0.17 mg L–1 at the end of the experiment (Day 5). In the nonsterilized sediments either inoculated or not with Enterobacter hormaechei, 0.73 to 0.75 mg L–1 of Se4+ (Day 1) was measured which reduced immediately and complete disappearance was observed at Day 5. In the sterilized sediment without any bacteria (control), Se4+ was not detected during the duration of the incubation period.
Small amounts of organic Se (Se2–) were produced in the process of Se6+ reduction (Fig. 4B) and no definite trend in Se2– formation was observed during 5 d of incubation (inset, Fig. 4B). However, the formation of Se2– during biotransformation of Se6+ was significantly higher than the sterilized sediment (control). Microbial reduction of Se6+ in the sediments produced accumulated Se (Fig. 4C). An increase in the accumulated Se was measured with the corresponding decrease in the total soluble Se in the sediments. No significant amount of accumulated Se was observed in the sterilized sediment (control).
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DISCUSSION
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Soluble Se transported from ponded water to sediment undergoes sequential transformations to other Se species through biochemical reactions (Fujita et al., 2005). Transformation of Se in sediments is microbially driven (Oremland et al., 1990) and there are numerous bacteria that have the ability to reduce Se (Se6+ and Se4+) in the environment (Frankenberger and Karlson, 1994; Siddique et al., 2005). Selenate-reducing bacterial strain (Tar11) isolated from the sediment used in this study was identified as Enterobacter hormaechei based on the 16S rDNA gene sequence analysis. Enterobacter hormaechei is a Gram-negative, motile, rod-shaped facultative anaerobe found in different environmental niches. Many species belonging to the genus Enterobacter are known to be involved in Se reduction processes. Losi and Frankenberger (1997b) reported the reduction of Se6+ to Se0 by Enterobacter cloacae isolated from agricultural drainage water. Enterobacter cloacae is also known to reduce both Se oxyanions (Siddique et al., 2005). The ability of Enterobacter hormaechei to reduce Se6+ to Se0 is demonstrated here for the first time. The mechanism (either dissimilatory reduction or resistance/detoxification mechanism) by which Enterobacter hormaechei reduced Se6+ was not investigated in this study. However, using similar experimental design and conditions for isolation and Se reduction, Losi and Frankenberger (1997b) reported that Enterobacter cloacae reduced Se6+ to Se0 through dissimilatory reduction. Removal of 96 to more than 99% of the added Se6+ by Enterobacter hormaechei from the industrial effluent and sediment in a 5-d study exhibited its potential role in bioremediation. Before, this bacterium (Enterobacter hormaechei) has only been studied for clinical research in health perspectives (Hoffmann et al., 2005). Four other bacterial strains (Klebsiella pneumoniae Tar1, Pseudomonas fluorescens Tar3, Stenotrophomonas maltophilia Tar6, and Enterobacter amnigenus Tar8) isolated from the enrichment sediment culture were able to reduce Se4+ to Se0. These bacteria have already been reported as Se4+–reducers (Siddique et al., 2005). Gregorio et al. (2005) isolated Stenotrophomonas maltophilia from the rhizoshpere of a selenium hyperaccumulator plant (Astragalus bisulcatus) which completely reduced 0.5 mM Se4+ in liquid culture within 52 h. Stenotrophomonas maltophilia isolated from a seleniferous agricultural evaporation pond sediment by Dungan et al. (2003) reduced 81.2 and 99.8% of added Se6+ and Se4+ (initial concentration of 0.5 mM), respectively, to Se0 in the solution in 48 h. Ike et al. (2000) isolated a strain of Pseudomonas fluorescens from sediment which reduced Se4+ under anerobic conditions.
Formation of red precipitates by Enterobacter hormaechei grown in Se6+–spiked industrial effluents and its examination under SEM revealed the formation of spherical Se0 particles of different sizes (<1.0 µm in diam.). These results are in agreement with the findings of Oremland et al. (2004) who studied the structural and spectral features of Se nanospheres (diam. 300 nm) produced by three physiologically and phylogenetically different species of Se-respiring bacteria. They found both intracellular and extracellular formation of Se nanospheres and observed differences in the optical properties of the extracellular nanospheres produced by the different bacterial species. In our study, it is not confirmed whether Se0 particles were formed intracellulary or extracellularly because an unsuccessful effort to remove the cell biomass from the precipitates might have led to the release of intracellular precipitate particles.
Microbial reduction processes in the sediment are responsible for removing Se6+ from water (Zhang and Moore, 1997). Transformation of more than 99% of the added Se6+ in the nonsterilized uninoculated sediment during 5 d of incubation indicated the reduction potential of the sediment harboring Se6+– and Se4+–reducing bacteria. These results are in conformity with Lucas and Hollibaugh (2001) and Siddique et al. (2006) who attributed Se6+ reduction in sediments to Se-reducing microbial communities present in the sediments. No significant change in the Se6+ concentration in the sterilized sediment (control) suggested little influence of other abiotic factors on Se6+ reduction in the sediment system (White and Dubrovsky, 1994; Zhang and Moore, 1997; Frankenberger and Hanna, 1998). Addition of Enterobacter hormaechei cells to the sterilized sediment removed 99% of the added Se6+ from the sediment exhibiting remarkable Se6+ reduction ability of this strain. Almost the same rate of Se6+ reduction was observed in the nonsterilized sediment with or without inoculation with Enterobacter hormaechei. It might be due to the fact that Enterobacter hormaechei was isolated from the same sediment used in the Se transformation study. Inoculation of nonsterilized sediment with Enterobacter hormaechei did not make any difference because sediment already contained this bacterial strain.
Slow reduction (21%) in the concentration of total soluble Se in the inoculated sterilized sediment compared with 83 to 90% in the nonsterilized sediments (inoculated as well as uninoculated) at Day 2 could be better understood by following the changes in Se4+ concentrations in the sediment with time. Since total soluble Se includes all soluble Se species (Se6+, Se4+, Se2–), Se4+ accumulation during Se6+ reduction kept the concentration of total soluble Se high in the inoculated sterilized sediment which did not have any bacteria other than Enterobacter hormaechei. After the reduction of Se6+ to Se4+, Enterobacter hormaechei reduced Se4+ to Se0 and a small amount of Se4+ was recorded at Day 5. In contrast, rapid reduction of total soluble Se in the nonsterilized sediment was due to nonaccumulation of Se4+ because Se4+ was readily consumed by other Se4+–reducing bacteria present in the sediment and Se4+ reduction proceeds more rapidly than Se6+ reduction (Ike et al., 2000). Four Se4+–reducing bacteria (Klebsiella pneumoniae, Pseudomonas fluorescens, Stenotrophomonas maltophilia, and Enterobacter amnigenus) were identified in this sediment.
The decrease in soluble Se in the sediments increased accumulated Se in the sediment. We assume that Se0 comprised the major portion of accumulated Se since most bacteria reduce soluble Se to Se0 in the environment (Oremland et al., 1989). Our SEM/EDAX observations also confirmed the formation of Se0 during Se6+ reduction. Fujita et al. (2005) studied the transformation of Se in a water-sediment system and reported that Se6+ and Se4+ reduction yielded Se0 in the river sediments and indigenous microflora was responsible for reductive reactions.
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CONCLUSIONS
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Scarcity of data on Se biogeochemistry in the mine tailings pond system prompted a study to investigate the fate of Se6+ and explore the diversity of indigenous Se-reducing bacterial communities in this system. Identification of Se6+– and Se4+–reducing bacteria and transformation of the added Se6+ suggest that mine tailings have the potential to precipitate Se from effluent streams. In this study only one type of sediment was assessed; further investigation using different sediments from various locations are necessary for better understanding and prediction. Enterobacter hormaechei isolated from the sediment has the ability to reduce Se6+ to Se0 and may be exploited for the application in bioremediation of wastewaters derived from different industrial activities.
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ACKNOWLEDGMENTS
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Funding for this work was provided by NSERC Discovery Grants to Joselito M. Arocena, Ron W. Thring, and the Canada Research Chair program (JMA).
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ABSTRACT
INTRODUCTION
) Sterilized sediment (heat-killed) inoculated with Enterobacter hormaechei; (•) Nonsterilized sediment (live) inoculated with Enterobacter hormaechei; (
) Nonsterilized sediment (live) without any inoculation; (*) Sterilized sediment (heat-killed) without inoculation (control). Error bars (±) represent standard error of the mean (n = 3).