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Published in J. Environ. Qual. 34:217-226 (2005).
© ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA

TECHNICAL REPORTS

Bioremediation and Biodegradation

Bacterial Diversity in Selenium Reduction of Agricultural Drainage Water Amended with Rice Straw

Tariq Siddiquea, Benedict C. Okekea, Yiqiang Zhanga, Muhammad Arshadb, Suk K. Hana and William T. Frankenberger, Jr.a,*

a Department of Environmental Sciences, University of California, Riverside, CA 92521
b Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad, Pakistan

* Corresponding author (william.frankenberger{at}ucr.edu)

Received for publication May 17, 2004.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Bacterial reduction of the Se oxyanions selenate [Se(VI)] and selenite [Se(IV)] to elemental selenium [Se(0)] is an important biological process in removing Se from drainage water. This study was conducted to characterize the molecular diversity of bacterial populations involved in Se reduction of drainage water amended with rice (Oryza sativa L.) straw and also to monitor the bacterial community shifts during the course of the study. Selenate was removed in the drainage water by the bacteria 5 to 6 d after addition of rice straw. Six Se(VI)- and 32 Se(IV)-reducing bacteria were isolated from rice straw containing sterilized drainage water. Three Se(VI)- and two Se(IV)-reducing bacteria were also isolated from the drainage water. Identification of Se(VI)- and Se(IV)-reducing bacteria by 16S rDNA sequence analysis showed a broad phylogenetic diversity in Se-reducing assemblages. Three major phyla (Proteobacteria, Actinobacteria, and Firmicutes) of bacterial domain with numerous classes, orders, and families constituted the Se-reducing bacterial community. We documented changes in the composition of bacterial assemblages in the drainage water amended with rice straw using polymerase chain reaction (PCR)–denaturing gradient gel electrophoresis (DGGE) of 16S rDNA. The Shannon–Weaver index (H') revealed higher bacterial diversity at Day 6 in the sterilized and Day 4 in the nonsterilized drainage water amended with rice straw. The results of this study suggest that rice straw, a good source of carbon and energy, harbors a wide range of bacteria useful in Se reduction and may be used in removing Se from drainage water.

Abbreviations: DGGE, denaturing gradient gel electrophoresis • PCR, polymerase chain reaction • Se(0), elemental selenium • Se(IV), selenite • Se(VI), selenate


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
THE United States Geological Survey produced a geology climate map that identified about 160000 square miles of lands in the western United States susceptible to irrigation-induced Se contamination (Seiler et al., 1999). Irrigation of seleniferous soils has produced subsurface drainage that contaminated wetlands and poisoned fish and migratory birds in the western United States (Lemly, 1993; Presser, 1994; Presser et al., 1994; Lemly, 1997). The National Irrigation Water Quality Program (NIWQP) conducted a detailed investigation confirming that irrigation drainage water enriched with Se had caused significant harmful effects on fish and wildlife (Seiler et al., 1999).

Selenium occurs in different oxidation states in the environment and exists primarily in two soluble forms, Se(VI) and Se(IV), in most seleniferous agricultural drainage water. Both oxyanions are toxic and can be reduced to insoluble Se(0) through bacterial mediation (Cantafio et al., 1996; Zhang and Moore, 1997; Losi and Frankenberger, 1997). Bacteria can use Se(VI) and Se(IV) as terminal electron acceptors in energy metabolism (dissimilatory reduction) or reduce and incorporate Se into organic compounds (assimilatory reduction). Methylation and subsequent volatilization of Se may be an important step in the transport of Se from contaminated terrestrial and aquatic environments (Dungan and Frankenberger, 1999). Field and laboratory studies conducted over the last 15 years have provided evidence that reduction of Se oxyanions occurs primarily via bacterial dissimilatory reduction (Stolz and Oremland, 1999). Because Se-respiring bacteria are typically heterotrophic, a source of organic carbon is required to fuel Se reduction (Cantafio et al., 1996; Losi and Frankenberger, 1997; Oremland et al., 1999). Recently, Zhang and Frankenberger (2003a) revealed that rice straw, a cheap source of carbon and nutrients, could be used to stimulate the growth and activity of natural Se-removing bacterial communities. Our previous studies (Zhang and Frankenberger, 2003a, 2003b) have also shown that rice straw is a good carrier of Se-reducing bacteria. Therefore, it is important to characterize the bacterial community active in Se reduction in drainage water amended with rice straw. The new knowledge of the diversity and composition of Se(VI)-reducing bacterial communities may be useful for understanding the influence of environmental factors on the response of ecosystems to Se contamination (Lucas and Hollibaugh, 2001).

Molecular biological techniques provide new tools to analyze bacterial assemblages. The main objective of this study was to characterize the molecular diversity of the bacterial populations involved in Se reduction in drainage water amended with rice straw. A second objective was to monitor the bacterial community dynamics during Se(VI) reduction. Sterilized and nonsterilized drainage waters were used with the rice straw to assess the contribution of the bacterial community associated with the drainage water in Se reduction. Selenium-reducing isolates were identified by 16S rDNA sequencing. We used PCR amplification of 16S rDNA and DGGE to analyze shifts in diversity and structure of bacterial communities in agricultural drainage water spiked with Se(VI) and amended with rice straw. Selenium reduction activities were monitored by measuring Se(VI) and Se(IV) in the drainage water amended with rice straw.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Sodium selenate (Na2SeO4) and selenium(IV) oxide (SeO2) were purchased from Sigma (St. Louis, MO) and Aldrich (Milwaukee, WI), respectively. Selenite standard solution (1000 mg L–1) and sodium borohydride (NaBH4) were purchased from Fisher Scientific (Pittsburgh, PA). Air-dried rice straw was obtained from the Broadview Water District, CA, and used without post-treatment. The rice straw contained 0.412 mg kg–1 of total Se and 0.346 mg kg–1 of soluble Se. The total Se in rice straw was determined with the acid digestion method described by Banuelos and Meek (1990) and distilled water was used to extract the soluble Se. Natural drainage water used in this study was collected from the Westlands Water District, San Joaquin Valley, California, a severely Se-contaminated area, and stored at 4°C before use. Characteristics of the drainage water are given in Table 1.


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  		Table 1. Characteristics of the drainage water used in the experiment.

 
Enrichment Culture for Selenate Reduction
To study Se(VI) reduction to Se(0) in the drainage water in the presence of rice straw, batch experiments were conducted in 250-mL Erlenmeyer flasks. The flasks were autoclaved before use at 121°C for 15 min and divided into two batches. One batch contained 125 mL of autoclaved drainage water and 1.25 g of nonsterilized rice straw, while the other contained nonsterilized drainage water and nonsterilized rice straw. The flasks were spiked with sterile Na2SeO4 solution (passed through a sterile 0.2-µm membrane filter) to give a final concentration of 2000 µg L–1 of Se(VI). The flasks were then capped with sterile rubber stoppers and incubated at room temperature (21 ± 1°C). This experiment was performed in triplicate. In a parallel experiment, a set of six flasks, prepared as described above, was used to determine the redox potential (Eh) in sterilized and nonsterilized drainage water amended with rice straw. Each flask was sampled (3 mL) daily for the duration of experiment (8 d).

Redox Potential Measurement
A 720A pH/ISE meter (Thermo Orion, Beverly, MA) was used to measure Eh in the experimental flasks. Redox potential was measured with an Accumet combination platinum electrode (Ag/AgCl) (Fisher Scientific). The measured potential (Eh,measured) was converted to potential in the rice straw solution (Eh,actual) relative to a standard H electrode as follows (Jayaweera and Biggar, 1996): Eh,actual = Eh,measured + 224.4 mV. The Eh electrode was tested by immersion in pH 4 and 7 buffer solutions saturated with quinhydrone (Q2H2) before each day's measurement.

Selenium Species Analyses
The method developed by Zhang and Frankenberger (2003a) was used for Se speciation. Selenite concentrations in all prepared solutions were analyzed by hydride generation atomic absorption spectrometry (HGAAS) (Zhang et al., 1999a, 1999b). In brief, samples collected from the flasks containing drainage water and rice straw were centrifuged 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 Se(VI) by K2S2O8 and then reduced to Se(IV) with 6 M HCl and determined by HGAAS. Organic Se(–II) in the sample was oxidized to Se(IV) by K2S2O8, which was indicated by the precipitation of Mn oxides formed from the oxidation of added Mn2+. The organic Se(–II) concentration was then calculated as the difference between Se(IV) in the sample treated with K2S2O8 in the presence of Mn2+ and Se(IV) determined in another subsample. The Se(VI) concentration was calculated by subtracting Se(IV) and organic Se(–II) values from the total soluble Se concentration. Elemental selenium in the sample was calculated as the difference between added Se(VI) and total soluble Se.

Screening and Isolation of Selenate- and Selenite-Reducing Bacteria
Samples collected during the experiment from rice straw–amended sterilized and nonsterilized drainage water were serially diluted and plated on Tryptic Soy Agar (Difco, Sparks, MD) plates supplemented with yeast extract (Difco) at 5 g L–1. Drainage water without any rice straw was also plated to isolate Se(VI)- and Se(IV)-reducing bacteria. Separate plates were prepared for screening Se(VI)- and Se(IV)-reducing bacteria. The molten medium made for the Se(VI) plates was spiked with a sterile Na2SeO4 solution to a final concentration of 50 mg Se(VI) L–1. For the Se(IV) plates, sterile SeO2 was added to the molten medium to give a final concentration of 50 mg Se(IV) L–1. Stock solutions of Na2SeO4 and SeO2 were filtered with a sterile 0.2-µm membrane filter (Millipore, Billerica, MA). The same serially diluted aliquots were plated on Se(VI) and Se(IV) containing Tryptic Soy Agar plates. The plates were incubated at 30°C for 2 to 3 d. Bacterial colonies that turned red on accumulation of Se(0) were isolated and purified by further streaking on fresh plates.

Identification of Selenate- and Selenite-Reducing Bacteria
The bacterial isolates were identified by 16S rDNA sequence analysis. Bacterial colonies were suspended in nuclease-free water, and DNA was extracted from the suspension according to the method described by Ausubel et al. (1992). Colonies of the isolate, suspended in a mixture of TE buffer, SDS (10%; w/v), and proteinase K, were incubated for 1 h at 37°C. Sodium chloride (5 M) was added to the samples and mixed thoroughly. Then CTAB–NaCl solution (4.1 g of NaCl and 10 g of CTAB [N-cetyl-N,N,N-trimethylammoniumbromide] in 100 mL of pre-warmed distilled water) was added and samples were incubated for 10 min at 65°C. The solution was extracted by adding 780 µL of chloroform-isoamyl alcohol (24:1) and centrifuging (5 min) to separate aqueous and organic phases. The aqueous phase containing the DNA was further extracted with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) to remove impurities. DNA present in the aqueous phase was precipitated with 0.6 volumes of isopropanol and the DNA precipitate was washed with 70% (v/v) ethanol. The DNA pellet was dried using a SpeedVac Concentrator AES 1000 (Savant Instruments, Farmingdale, NY) and resuspended in nuclease-free water. Universal bacterial primers corresponding to E. coli positions 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 519R (5'-GWATTACCGCGGCKGCTG-3') were used for amplification of 16S rDNA by PCR. A sequence of >400 base pairs is desirable to ensure reliable phylogenetic positioning; however, it is possible to use partial sequences to identify organisms or to assign groups, as long as the database contains sequences of close relatives (Ludwig et al., 1998). A PCR master mix (Catalog no. M7502; Promega, Madison, WI) was used according to the manufacturer's instructions. Genomic DNA (1 µL) was the template. 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, Waltham, 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. Briefly, the gel slice was suspended in buffer QxI, and the QiaexII suspension was added and mixed by vortexing. The suspension was incubated at 50°C for 10 min, centrifuged for 30 s, and the pellet was washed twice with buffer QxI and buffer PE. After air-drying, the DNA was eluted with nuclease-free water. DNA cycle sequencing (30 cycles using 27F primer) was done with the ABI Prism BigDye terminator kit (Version 3.1; PerkinElmer Applied Biosystems, Foster City, CA) and an Applied Biosystems ABI 3100 genetic analyzer. Analysis of DNA sequences and homology searches were completed with a MEGABLAST (Altschul et al., 1997) using the BLAST algorithm for the comparison of a nucleotide query sequence against a nucleotide sequence database (blastn).

A phylogenetic tree was constructed by TreeconW program (Version 1.3) (Van de Peer and De Wachter, 1997) based on the sequence distance method. Sequences were aligned by ClustalW. Distance estimation was performed with the Jin and Nei (1990) model using Kimura's two parameter model. The neighbor-joining method was used to infer the phylogenetic tree. Neighbor-joining tree topology was tested by bootstrap analysis with 100 replicates.

Polymerase Chain Reaction–Denaturing Gradient Gel Electrophoresis Analysis
Genomic DNA was extracted from the samples (replications were pooled) as described above. Polymerase chain reaction was used to amplify the variable Region 3 (V3) of the 16S rRNA gene from the genomic DNA by using the GC-clamp primer 338F (5'-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGACTCCTACGGGAGGCA-3') and 518R (5'-ATTACCGCGGCTGCTGG-3'). A PCR master mix (Catalog no. M7502; Promega) was used according to the manufacturer's instruction. The PCR conditions were the same as described above except for the number of cycles (30 cycles for DGGE). Denaturing gradient gel electrophoresis was performed using the Bio-Rad (Hercules, CA) Dcode System. The PCR product generated by the 338F-GC and 518R primers were loaded onto 6% (w/v) polyacrylamide gels in 1x TAE (20 mM Tris, 10 mM acetate, 0.5 mM EDTA pH 7.4), 1-mm thick and 16- x 16-cm in size. The polyacrylamide gel was made with a denaturing gradient ranging from 40 to 60% (where 100% denaturant contains 7 M urea and 40% formamide) by Gradient Maker (Bio-Rad). The electrophoresis was run for 8 h at 100V or 4 h at 200V in 1x TAE buffer at a constant temperature of 60°C. The gel plate was cooled in ice water for 5 to 10 min and then was stained for 10 to 20 min in 200 mL of 1x TAE buffer containing 100 µg mL–1 of ethidium bromide. The stained gel was placed on a UV trans-illuminator and digitized using a CCD system (Bio-Rad).

Denaturing Gradient Gel Electrophoresis Pattern Profile Analysis
The processing of the DGGE gels was done with the Gelcompar II software Version 3.0 (Applied Maths, 2002). A dendrogram was obtained by UPGMA clustering of DGGE patterns. Similarity is expressed as a percentage value of the Pearson correlation coefficient (Pearson, 1926) based on the band intensity and location, and results are expressed in a distance matrix. Bacterial diversity was determined by the Shannon index (H'). The Shannon index was derived from the following equation H' = –{Sigma}Pi log Pi (Eichner et al., 1999). The term Pi was calculated as follows: Pi = ni/N, where ni is the band intensity for individual bands and N is the sum of the intensities of bands in a lane.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Selenate Reduction in Drainage Water Amended with Rice Straw
Variation in the concentration of different Se species in the drainage water amended with rice straw over 8 d of incubation is presented in Fig. 1 . In the nonsterilized drainage water amended with rice straw, Se(VI) concentration decreased from 1923 µg L–1 at Day 0 to 7.5 µg L–1 at Day 7 with an abrupt decrease during Days 3 and 5. No Se(VI) could be detected at Day 8. Selenite concentrations increased to 567 µg L–1 on Day 3, but declined rapidly. With the decrease in Se(VI), a simultaneous increase in Se(0) was observed. A similar trend in variation of the concentration of Se(VI) and Se(0) was observed in the sterilized drainage water amended with rice straw (Fig. 1). In the sterilized drainage water amended with rice straw, no Se(IV) peak appeared and the lag phase was comparatively longer compared with the nonsterilized drainage water (2 d for nonsterilized and 4 d for sterilized drainage water). The decreasing slope of Se(VI) was sharper in sterilized than in nonsterilized drainage water.



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  		Fig. 1. Changes in the concentration of Se species in nonsterilized and sterilized drainage water amended with rice straw during 8 d of incubation.

 
The redox potential (Eh) in the drainage water amended with rice straw also decreased rapidly (Fig. 2) . In the nonsterilized drainage water amended with rice straw, Eh dropped from 408 to –180 mV during the first 3 d and then slightly increased, stabilizing between –8 and –13 mV during rest of the incubation period. The Eh decreased more in the sterilized drainage water amended with rice straw. An Eh of –226 mV was measured at Day 3, which increased and stabilized between –27 and –25 mV during 6 to 8 d.



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  		Fig. 2. Changes in the redox potential (Eh) in nonsterilized and sterilized drainage water amended with rice straw during 8 d of incubation.

 
Diversity of Selenate- and Selenite-Reducing Isolates
A total of 43 Se(VI)- and Se(IV)-reducing bacteria (from multiple sampling periods) were isolated from the agricultural drainage water (sterilized and nonsterilized) amended with rice straw and drainage water alone (Tables 2, 3, and 4). Six bacteria isolated from the drainage water amended with rice straw could reduce Se(VI) to Se(0). Thirty-two Se(IV)-reducing bacteria were selected on the basis of a very strong red color due to Se accumulation. In the drainage water, three Se(VI)- and two Se(IV)-reducing bacteria were found. To understand the diversity of the bacterial community involved in Se(VI) reduction in the drainage water amended with rice straw, 43 isolates from Se(VI) and Se(IV) enriched on Tryptic Soy Agar plates were identified by 16S rDNA sequence analysis (Tables 2, 3, and 4). Figure 3 displays a great diversity of bacteria that were active in Se(VI) and Se(IV) reduction in the drainage water amended with rice straw. All the Se(VI)- and Se(IV)-reducing bacteria represented three phyla (Proteobacteria, Actinobacteria, and Firmicutes) of bacterial domain. In general, 29 bacteria were Gram negative and 11 were Gram positive. Among the Gram-negative bacteria, 21 bacteria belonged to {gamma}-Proteobacteria, 1 to ß-Proteobacteria, and 7 to {alpha}-Proteobacteria. {gamma}-Proteobacteria consisted of Pseudomonadaceae, Xanthomonadaceae, Alteromonadaceae, Aeromonadaceae, and Enterobacteriaceae families of bacteria with different genera. Only one bacterium was classified under ß-Proteobacteria in the family Alcaligenacae and genus Achromobacter. Seven bacteria were categorized under {alpha}-Proteobacteria in the families Methylobacteriaceae, Sphingomonadaceae, Rhodobacteriaceae, and Caulobacteriaceae. Two bacteria belonged to the phylum Actinobacteria with the family of Micrococcineae and genus Arthrobacter. The rest of the bacteria (9) were from the phylum Firmicutes and were further grouped under families Staphylococcaceae, Paenibacillaceae, and Planococcaceae.


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  		Table 2. Identification of selenate [Se(VI)]-reducing bacteria in the drainage water amended with rice straw spiked with sodium selenate (Na2SeO4) at 2 mg L–1.

 

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  		Table 3. Identification of selenite [Se(IV)]-reducing bacteria in the drainage water amended with rice straw spiked with sodium selenate (Na2SeO4) at 2 mg L–1.

 

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  		Table 4. Identification of selenate [Se(VI)]- and selenite [Se(IV)]-reducing bacteria in the drainage water.

 


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  		Fig. 3. Phylogenetic relationship among the Se-reducing bacterial strains identified by 16S rDNA sequence. Bootstrap values greater than 50 are indicated at each node. The tree is rooted with Thermotoga maritima as the outgroup.

 
Polymerase Chain Reaction–Denaturing Gradient Gel Electrophoresis Analysis of Bacterial Communities
The V3 region of 16S rDNA was amplified from the community DNA to observe the bacterial community dynamics over time during Se(VI) reduction in the drainage water amended with rice straw. The DGGE analysis of the amplified PCR fragments (primers 327F-GC clamp and 518R) from Days 0 to 8 is shown in Fig. 4 . A visual observation of the DGGE gel banding pattern revealed a shift in the composition of the bacterial communities during the 8-d study. Different banding patterns were observed in sterilized and nonsterilized drainage water amended with rice straw. To analyze the structure of the bacterial community based on the DGGE patterns of the 16S rDNA fragments, we used a cluster analysis and the Shannon index (H').



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  		Fig. 4. 16S rDNA–based denaturing gradient gel electrophoresis (DGGE) profiles from the bacterial community in the sterilized and the nonsterilized drainage water amended with rice straw. The terms S and NS denote sterilized and nonsterilized drainage water amended with rice straw, respectively, while M1 and M2 denote markers.

 
Cluster analysis of the DGGE patterns for Days 0, 2, 4, 6, and 8 is presented in Fig. 5 . Bacterial communities present in the sterilized and nonsterilized drainage water differed at the beginning of the experiment with only 32% similarity at Day 0. Resemblance in the bacterial communities increased with time and 60% similarity (Pearson correlation table not shown) was observed between the sterilized and nonsterilized drainage water at Day 8. A major shift of the bacterial community was observed in the nonsterilized drainage water where similarity within the bacterial community increased from 32 at Day 0 to 80% at Day 8. In the sterilized drainage water, similarity within the bacterial community increased from 70 at Day 0 to 90% at Day 8. Mathematical indices were applied to measure the biodiversity and describe the assemblage of bacterial populations within the community (Table 5). The Shannon index (H') measures the diversity of the bacterial population. In the sterilized drainage water amended with rice straw, the highest value of H' (3.46) was observed at Day 6. High values of H' (3.46 and 3.53) were also recorded at Days 4 and 8, respectively, in the nonsterilized drainage water containing rice straw.



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  		Fig. 5. 16S rDNA–based denaturing gradient gel electrophoresis (DGGE) profiles with cluster analysis of the bacterial community in the sterilized and the nonsterilized drainage water amended with rice straw. The terms S and NS denote sterilized and nonsterilized drainage water amended with rice straw, respectively, while M1 and M2 denote markers.

 

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  		Table 5. Bacterial diversity indices based on denaturing gradient gel electrophoresis (DGGE) profiles.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
The bacterial reduction of Se(VI) and Se(IV), typically found as selenate and selenite oxyanions, to insoluble elemental Se(0) is a potential bioremediation strategy for cleanup of Se-contaminated water. In the present study, added Se(VI) was removed from the dissolved phase in 5 d in the nonsterilized drainage water and 6 d in the sterilized drainage water after addition of rice straw. Selenate reduction did not occur in a sterile rice straw solution during 14 d of incubation nor could adsorption of Se(VI) and Se(IV) on rice straw be observed in our previous study (Zhang and Frankenberger, 2003a). Therefore, reduction of Se(VI) to insoluble Se(0) is attributed to the Se(VI)- and Se(IV)-reducing bacterial communities present in the drainage water containing rice straw. Rapid reduction of Se(VI) to Se(0) may be promoted by organic compounds released from rice straw that serve as electron donors for bacterial metabolism (Zhang and Frankenberger, 2003a). Because the experiment was conducted in closed system, the reaction flasks became anoxic over the course of time due to bacterial metabolism. A decrease in redox potential (Eh) of the drainage water amended with rice straw favored the Se(VI) reduction. Masscheleyn and Patrick (1993) studied Se reduction with Eh and reported an Eh of –10 to –40 mV for Se(IV) to Se(0) reduction in a flooded sediment system.

Rice straw has been reported to be an excellent source of carbon, nutrients, and energy for Se-reducing bacteria (Jenkins et al., 1996; Villegas-Pangga et al., 2000; Zhang and Frankenberger, 2003a). This study reveals that rice straw is a carrier of numerous bacteria that can play an active role in the environmental reduction of oxidized selenium compounds. The Se(VI)- and Se(IV)-reducing bacterial communities in the rice straw containing drainage water displayed phylogenetic diversity, with sequences within three major phyla (Proteobacteria, Actinobacteria, and Firmicutes) of bacterial domain. The orders Pseudomonadales, Enterobacteriales and Bacillales dominated the Se(VI)- and Se(IV)-reducing assemblage. Pseudomonadales and Enterobacteriales constitute the class {gamma}-Proteobacteria. Eight Pseudomonas spp. were active in Se(VI) reduction in the drainage water amended with rice straw. Several Pseudomonas spp. have already been reported to be involved in selenium transformations. Ike et al. (2000) isolated two Se(VI)-reducing Pseudomonas spp. (Pseudomonas stutzeri and Pseudomonas fluorescens) from sediment containing water samples free from Se contamination. These strains reductively transformed 5 mM Se(VI) into Se(IV) under anaerobic condition. Lortie et al. (1992) reported that Pseudomonas stutzeri rapidly reduced both Se(VI) and Se(IV) ions to Se(0) under aerobic conditions between pH of 7.0 and 9.0, and at a temperature of 25 to 35°C. In our study, numerous species of the family Enterobacteriaceae belonging to different genera (Enterobacter, Escherichia, Klebsiella, Serratia, and Kluyvera) have been isolated from the drainage water amended with rice straw that are active in Se(VI) and Se(IV) reduction. Enterobacter sp. SI-15 reduced both Se(VI) and Se(IV). Enterobacter cloacae is already known for its ability to reduce both Se oxyanions. Losi and Frankenberger (1997) reported the reduction of Se oxyanions [61.5–94.5% of added Se(VI)] by Enterobacter cloacae isolated from agricultural drainage water. Babien et al. (2002) found Escherichia coli active in Se(VI) reduction. However, in our study other species of Escherichia were found in the drainage water amended with rice straw that were capable of only reducing Se(IV). Klebsiella sp. SI-51 and Serratia sp. SI-20 identified in the bacterial community were able to reduce Se(IV). These bacterial strains have not previously been reported for Se reduction. Klebsiella and Serratia species are facultative anaerobes and have been found in abundance in sewage treatment plants (Ampofo and Clerk, 2003). Aeromonas sp. SI-19, Stenotrophomonas sp. SI-54, and Shewanella sp. SA-101 identified as Se(IV)-reducing bacteria were also isolated from the drainage water amended with rice straw. Aeromonas hydrophila had previously been studied and found active in dissimilatory reduction of Se(VI) (Knight and Blakemore, 1998). Out of 24 isolated estuarine mesophilic aeromonads, five strains reduced Se(VI) (Knight and Blakemore, 1998). Recently, Dungan et al. (2003) reported that Stenotrophomonas maltophilia isolated from a seleniferous agricultural drainage pond sediment reduced Se(VI) and Se(IV) to Se(0).

Characterization of the isolates also indicated the presence of Roseomonas, Brevundimonas, Rhodobacter, and Sphingomonas spp. of {alpha}-Proteobacteria in the drainage water amended with rice straw. Roseomonas genomospecies and Brevundimonas sp. DW-1 reduced both Se oxyanions [Se(VI) and Se(IV)]. These strains were not known before for their ability to reduce Se. Rhodobacter sphaeroides has been reported to reduce Se(IV) (Babien et al. (2001), while Sphingomonas sp. has been characterized degrading pentachlorophenol (Habash et al., 2002). Several species of the genera Planococcus, Planomicrobium, and Paenibacillus were also found active in Se reduction in the drainage water amended with rice straw. Planomicrobium sp. SA-59 was able to reduce both Se(VI) and Se(IV). To our knowledge, Se-reducing activity has never been reported in these genera.

During our screening and isolation of Se-reducing bacteria, a few showed their abilities to reduce Se(VI), while the majority of bacteria exhibited their capabilities for Se(IV) reduction. In the biogeochemistry of selenium, various redox reactions are performed by bacteria. In the reduction of Se(VI) to Se(IV) and then to Se(0), different enzymes, energy requirements, and biological mechanisms are involved that determine the capability of an organism to reduce Se(VI) or Se(IV) to Se(0) (Rech and Macy, 1992; Tomei et al., 1995; Schroder et al., 1997; Babien et al., 2002). Doran (1982) reported isolation of a majority of Se(IV)-reducing bacteria, with fewer being able to reduce Se(VI).

Denaturing gradient gel electrophoresis profiling is a powerful tool to analyze complex bacterial communities (McCaig et al., 2001; Boon et al., 2002). Relative quantification of the banding pattern enabled the calculation of the Shannon index (H'), providing a better insight on the changes of the community structure. Sterilized and nonsterilized drainage water amended with rice straw showed different bacterial communities with only 32% similarity between the banding patterns at Day 0. The two bacterial communities clearly formed two different groups. The resemblance within the bacterial communities increased in the subsequent samplings in response to Se(VI) reduction and release of nutrients and other organic substances from rice straw. Other factors like O2 depletion, release of new compounds, and interactions among community members might have effects on the progression of the bacterial communities. Lucas and Hollibaugh (2001) reported that the addition of Se(VI) to the environment selects for Se(VI)-adapted organisms. They also attributed differences between the initial and subsequent samplings of bacterial communities to the adaptation to available carbon sources as electron donors. Although two bacterial communities (sterilized and nonsterilized drainage water) formed two different groups, there was no significant difference in their capabilities to reduce Se(VI) in the drainage water amended with rice straw. This is probably due to the addition of rice straw, which carried most of the identified Se(VI)- and Se(IV)-reducing bacteria, to the sterilized and nonsterilized drainage water, which overshadowed the contribution of fewer Se-reducing bacteria present in the drainage water. Diversity indices applied to examine the bacterial communities in our study revealed that the highest diversity in bacterial population was observed at Day 6 in the sterilized drainage water and Days 4 and 8 in the nonsterilized drainage water amended with rice straw. This may be related to the high metabolic activities of the bacterial communities coupled with Se(VI) reduction during that period.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
This work describes the role of naturally occurring bacterial communities in Se(VI) reduction. Diversity studies (DGGE and other fingerprinting tools) examine the dynamics in bacterial communities and identify new and useful Se-reducing bacterial species present in natural systems. This study revealed that there are numerous bacteria in rice straw with the capacity to transform Se oxyanions that have not previously been characterized. Rice straw, a cheap and readily available organic carbon source, is also an excellent carrier of Se(VI)- and Se(IV)-reducing bacteria and may be used in removing selenium oxyanions from drainage water in the field.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 


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T. Siddique, J. M. Arocena, R. W. Thring, and Y. Zhang
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