Simultaneous Reduction of Nitrate and Selenate by Cell Suspensions of Selenium-Respiring Bacteria

Washed-cell suspensions of Sulfurospirillum barnesii reduced selenate [Se(VI)] when cells were cultured with nitrate, thiosulfate, arsenate, or fumarate as the electron acceptor. When the concentration of the electron donor was limiting, Se(VI) reduction in whole cells was approximately fourfold greater in Se(VI)-grown cells than was observed in nitrate-grown cells; correspondingly, nitrate reduction was ~11-fold higher in nitrate-grown cells than in Se(VI)-grown cells. However, a simultaneous reduction of nitrate and Se(VI) was observed in both cases. At nonlimiting electron donor concentrations, nitrate-grown cells suspended with equimolar nitrate and selenate achieved a complete reductive removal of nitrogen and selenium oxyanions, with the bulk of nitrate reduction preceding that of selenate reduction. Chloramphenicol did not inhibit these reductions. The Se(VI)-respiring haloalkaliphile Bacillus arsenicoselenatis gave similar results, but its Se(VI) reductase was not constitutive in nitrate-grown cells. No reduction of Se(VI) was noted for Bacillus selenitireducens, which respires selenite. The results of kinetic experiments with cell membrane preparations of S. barnesii suggest the presence of constitutive selenate and nitrate reduction, as well as an inducible, high-affinity nitrate reductase in nitrate-grown cells which also has a low affinity for selenate. The simultaneous reduction of micromolar Se(VI) in the presence of millimolar nitrate indicates that these organisms may have a functional use in bioremediating nitrate-rich, seleniferous agricultural wastewaters. Results with ⁷⁵Se-selenate tracer show that these organisms can lower ambient Se(VI) concentrations to levels in compliance with new regulations proposed for release of selenium oxyanions into the environment.     

Structural and spectral features of selenium nanospheres produced by Se-respiring bacteria

Certain anaerobic bacteria respire toxic selenium oxyanions and in doing so produce extracellular accumulations of elemental selenium [Se(0)]. We examined three physiologically and phylogenetically diverse species of selenate- and selenite-respiring bacteria, Sulfurospirillum barnesii, Bacillus selenitireducens, and Selenihalanaerobacter shriftii, for the occurrence of this phenomenon. When grown with selenium oxyanions as the electron acceptor, all of these organisms formed extracellular granules consisting of stable, uniform nanospheres (diameter, approximately 300 nm) of Se(0) having monoclinic crystalline structures. Intracellular packets of Se(0) were also noted. The number of intracellular Se(0) packets could be reduced by first growing cells with nitrate as the electron acceptor and then adding selenite ions to washed suspensions of the nitrate-grown cells. This resulted in the formation of primarily extracellular Se nanospheres. After harvesting and cleansing of cellular debris, we observed large differences in the optical properties (UV-visible absorption and Raman spectra) of purified extracellular nanospheres produced in this manner by the three different bacterial species. The spectral properties in turn differed substantially from those of amorphous Se(0) formed by chemical oxidation of H(2)Se and of black, vitreous Se(0) formed chemically by reduction of selenite with ascorbate. The microbial synthesis of Se(0) nanospheres results in unique, complex, compacted nanostructural arrangements of Se atoms. These arrangements probably reflect a diversity of enzymes involved in the dissimilatory reduction that are subtly different in different microbes. Remarkably, these conditions cannot be achieved by current methods of chemical synthesis.     

Isolation, Growth, and Metabolism of an Obligately Anaerobic, Selenate-Respiring Bacterium, Strain SES-3

A gram-negative, strictly anaerobic, motile vibrio was isolated from a selenate-respiring enrichment culture. The isolate, designated strain SES-3, grew by coupling the oxidation of lactate to acetate plus CO₂ with the concomitant reduction of selenate to selenite or of nitrate to ammonium. No growth was observed on sulfate or selenite, but cell suspensions readily reduced selenite to elemental selenium (Se⁰). Hence, SES-3 can carry out a complete reduction of selenate to Se⁰. Washed cell suspensions of selenate-grown cells did not reduce nitrate, and nitrate-grown cells did not reduce selenate, indicating that these reductions are achieved by separate inducible enzyme systems. However, both nitrate-grown and selenate-grown cells have a constitutive ability to reduce selenite or nitrite. The oxidation of [¹⁴C]lactate to ¹⁴CO₂ coupled to the reduction of selenate or nitrate by cell suspensions was inhibited by CCCP (carbonyl cyanide m-chlorophenylhydrazone), cyanide, and azide. High concentrations of selenite (5 mM) were readily reduced to Se⁰ by selenate-grown cells, but selenite appeared to block the synthesis of pyruvate dehydrogenase. Tracer experiments with [⁷⁵Se]selenite indicated that cell suspensions could achieve a rapid and quantitative reduction of selenite to Se⁰. This reduction was totally inhibited by sulfite, partially inhibited by selenate or nitrite, but unaffected by sulfate or nitrate. Cell suspensions could reduce thiosulfate, but not sulfite, to sulfide. These results suggest that reduction of selenite to Se⁰ may proceed, in part, by some of the components of a dissimilatory system for sulfur oxyanions.     

Structural and Spectral Features of Selenium Nanospheres Produced by Se-Respiring Bacteria

Certain anaerobic bacteria respire toxic selenium oxyanions and in doing so produce extracellular accumulations of elemental selenium [Se(0)]. We examined three physiologically and phylogenetically diverse species of selenate- and selenite-respiring bacteria, Sulfurospirillum barnesii, Bacillus selenitireducens, and Selenihalanaerobacter shriftii, for the occurrence of this phenomenon. When grown with selenium oxyanions as the electron acceptor, all of these organisms formed extracellular granules consisting of stable, uniform nanospheres (diameter, ~300 nm) of Se(0) having monoclinic crystalline structures. Intracellular packets of Se(0) were also noted. The number of intracellular Se(0) packets could be reduced by first growing cells with nitrate as the electron acceptor and then adding selenite ions to washed suspensions of the nitrate-grown cells. This resulted in the formation of primarily extracellular Se nanospheres. After harvesting and cleansing of cellular debris, we observed large differences in the optical properties (UV-visible absorption and Raman spectra) of purified extracellular nanospheres produced in this manner by the three different bacterial species. The spectral properties in turn differed substantially from those of amorphous Se(0) formed by chemical oxidation of H₂Se and of black, vitreous Se(0) formed chemically by reduction of selenite with ascorbate. The microbial synthesis of Se(0) nanospheres results in unique, complex, compacted nanostructural arrangements of Se atoms. These arrangements probably reflect a diversity of enzymes involved in the dissimilatory reduction that are subtly different in different microbes. Remarkably, these conditions cannot be achieved by current methods of chemical synthesis.     

细菌还原氧化态硒产生红色单质硒的研究进展

硒是一种生命必需的微量元素,但高浓度时毒性较强且会造成环境污染。许多细菌可以将亚硒酸盐(SeO32-)或硒酸盐(SeO42-)等毒性较高的氧化态硒还原为毒性较小的红色单质硒(Se°),形成硒-蛋白复合物,它们对于获得最佳补硒方式和治理硒环境污染具有应用潜力。近年来,关于这一生物还原过程,人们进行了大量的研究,包括碳源、氧气、元素硫、谷胱甘肽以及一些氧化还原酶和膜转运蛋白等在内的多种物质都被发现可能影响或参与了细菌对硒的代谢。综述了细菌进行生物还原氧化态硒的影响因素及不同细菌产生红色单质硒机理的研究进展。     

Reduction of Selenium Oxyanions in Wastewater Using Two Bacterial Strains

The biological reduction of selenium oxyanions is capable of reducing both selenate and selenite to insoluble elemental selenium. In this process, however, bacteria inevitably require expensive chemicals such as yeast extract in almost all cases. Therefore, the reduction of selenium oxyanions with inexpensive alcohol would be more practical. A Pseudomonas sp. strain 4C‐C isolated from a sludge in a wastewater treatment facility was able to reduce selenate to selenite using ethanol as an electron donor for its anaerobic respiration, but could not reduce selenite to elemental selenium. Paracoccus denitrificans JCM‐6892, on the other hand, was observed to be able to reduce selenite to elemental selenium in the presence of ethanol, but not selenate to selenite. Therefore, a mixture containing a suspension of Pseudomonas sp. strain 4C‐C and P. denitrificans JCM‐6892 cells allowed selenate to be reduced to insoluble elemental selenium via selenite in the presence of ethanol and was also capable of reducing nitrate to nitrogen gas. Aiming at simplicity of the recovery process of insoluble elemental selenium, a polymeric gel immobilized mixture of the two bacterial strains was examined using ethanol as an electron donor. The immobilized mixture could therefore reduce not only selenate to elemental selenium, but also nitrate to nitrogen gas in a single step. The gel that immobilized the microbial mixture changed its color during the process to bright red and no red elemental selenium was left in the wastewater. This indicates that the reduced elemental selenium was completely absorbed in the gel. This simple bacterial combination would therefore be effective in the presence of ethanol to reduce selenium oxyanions in various wastewaters containing selenium and the other oxyanions.     

The involvement of ATP sulfurylase in Se(VI) and Cr(VI) reduction processes in the fission yeast<Emphasis Type="Italic"> Schizosaccharomyces pombe</Emphasis>

The response of Schizosaccharomyces pombe towards the oxyanions selenate [Se(VI)] and dichromate [Cr(VI)] was investigated in order to establish the involvement of the yeast ATP sulfurylase in their reduction. An ATP sulfurylase-defective/selenate-resistant mutant of S. pombe (B-579 Se(R) -2) and an ATP sulfurylase-active/selenate-sensitive strain of S. pombe (B-579 Se(S)) were included in this study. The inhibitory effect of Se(VI) and Cr(VI) oxyanions on growth and bioaccumulation was measured. The sensitive strain showed natural sensitivity to selenate while the resistant mutant tolerated a 100-fold higher concentration of selenate. These results indicate that selenate toxicity to microorganisms is connected with the reduction of selenate to selenite. Both strains showed similar sensitivity to Cr(VI) and in this study there was no evidence that ATP sulfurylase participates in the reduction process of Cr(VI).     

An <Emphasis Type="Italic">Azospira oryzae</Emphasis> (syn <Emphasis Type="Italic">Dechlorosoma suillum</Emphasis>) Strain That Reduces Selenate and Selenite to Elemental Red Selenium

A bacterium that reduces the soluble selenium oxyanions, selenate and selenite, to insoluble elemental red selenium (Se⁰) was isolated from a laboratory reactor developed to remove selenate from groundwater. Gene sequence alignment of the 16S rRNA allowed identification of the isolate as Azospira oryzae. Biochemical and morphologic characterization confirm the identification. The isolate reduces selenate and selenite to Se⁰ under microaerophilic and denitrifying conditions but not under aerobic conditions. It does not use selenate or selenite as terminal eˉ donors. Se oxyanion reduction causes the formation of Se nanospheres that are 0.25 ± 0.04 μm in diameter. Nanospheres may be associated with the cells or free in the medium. The enzymatic activity associated with the reduction of selenate has a molecular mass of approximately 500 kD, and the enzymatic activity associated with the reduction of selenite has a mass of approximately 55 kD. Selenite reduction was inhibited by tungsten. The molecular masses of these activities were different from those associated with the reduction of dimethylsulfoxide, sulfate, and nitrite. This bacterium, or perhaps its enzymes or DNA, might be useful for the remediation of waters contaminated with Se oxyanions.     

Multiple influences of nitrate on uranium solubility during bioremediation of uranium-contaminated subsurface sediments

Microbiological reduction of soluble U(VI) to insoluble U(IV) has been proposed as a remediation strategy for uranium-contaminated groundwater. Nitrate is a common co-contaminant with uranium. Nitrate inhibited U(VI) reduction in acetate-amended aquifer sediments collected from a uranium-contaminated site in New Mexico. Once nitrate was depleted, both U(VI) and Fe(III) were reduced concurrently. When nitrate was added to sediments in which U(VI) had been reduced, U(VI) reappeared in solution. Parallel studies with the dissimilatory Fe(III)-, U(VI)- and nitrate-reducing microorganism, Geobacter metallireducens, demonstrated that nitrate inhibited reduction of Fe(III) and U(VI) in cell suspensions of cells that had been grown with nitrate as the electron acceptor, but not in Fe(III)-grown cells. Suspensions of nitrate-grown G. metallireducens oxidized Fe(II) and U(IV) with nitrate as the electron acceptor. U(IV) oxidation was accelerated when Fe(II) was also added, presumably due to the Fe(III) being formed abiotically oxidizing U(IV). These studies demonstrate that although the presence of nitrate is not likely to be an impediment to the bioremediation of uranium contamination with microbial U(VI) reduction, it is necessary to reduce nitrate before U(VI) can be reduced. These results also suggest that anaerobic oxidation of U(IV) to U(VI) with nitrate serving as the electron acceptor may provide a novel strategy for solubilizing and extracting microbial U(IV) precipitates from the subsurface.     

The periplasmic nitrite reductase of Thauera selenatis may catalyze the reduction of selenite to elemental selenium

Thauera selenatis grows anaerobically with selenate, nitrate or nitrite as the terminal electron acceptor; use of selenite as an electron acceptor does not support growth. When grown with selenate, the product was selenite; very little of the selenite was further reduced to elemental selenium. When grown in the presence of both selenate and nitrate both electron acceptors were reduced concomitantly; selenite formed during selenate respiration was further reduced to elemental selenium. Mutants lacking the periplasmic nitrite reductase activity were unable to reduce either nitrite or selenite. Mutants possessing higher activity of nitrite reductase than the wild-type, reduced nitrite and selenite more rapidly than the wild-type. Apparently, the nitrite reductase (or a component of the nitrite respiratory system) is involved in catalyzing the reduction of selenite to elemental selenium while also reducing nitrite. While periplasmic cytochrome C ₅₅₁ may be a component of the nitrite respiratory system, the level of this cytochrome was essentially the same in mutant and wild-type cells grown under two different growth conditions (i.e. with either selenate or selenate plus nitrate as the terminal electron acceptors). The ability of certain other denitrifying and nitrate respiring bacteria to reduce selenite will also be described.     

Selenium (IV,VI) reduction and tolerance by fungi in an oxic environment

Microbial processes are known to mediate selenium (Se) oxidation–reduction reactions, strongly influencing Se speciation, bioavailability, and transport throughout the environment. While these processes have commonly been studied in anaerobic bacteria, the role that aerobic fungi play in Se redox reactions could be important for Se‐rich soil systems, dominated by microbial activity. We quantified fungal growth, aerobic Se(IV, VI) reduction, and Se immobilization and volatilization in the presence of six, metal‐tolerant Ascomycete fungi. We found that the removal of dissolved Se was dependent on the fungal species, Se form (i.e., selenite or selenate), and Se concentration. All six species grew and removed dissolved Se(IV) or Se(VI) from solution, with five species reducing both oxyanions to Se(0) biominerals, and all six species removing at least 15%–20% of the supplied Se via volatilization. Growth rates of all fungi, however, decreased with increasing Se(IV,VI) concentrations. All fungi removed 85%–93% of the dissolved Se(IV) within 10 d in the presence of 0.01 mm Se(IV), although only about 20%–30% Se(VI) was removed when grown with 0.01 mm Se(VI). Fungi‐produced biominerals were typically 50‐ to 300‐nm‐diameter amorphous or paracrystalline spherical Se(0) nanoparticles. Our results demonstrate that activity of common soil fungi can influence Se form and distribution, and these organisms may therefore play a role in detoxifying Se‐polluted environments.     

Removal of soluble selenium by a selenate-reducing bacterium Bacillus sp. SF-1

In order to develop a biological process for removal of selenium from industrial wastewater, Bacillus sp. strain SF-1 was isolated from selenium-contaminated sediment. The bacterium reduces selenate to selenite and subsequently to nontoxic insoluble elemental selenium using lactate as an electron donor and selenate as an electron acceptor in an anaerobic condition. Elemental selenium transformed from soluble selenium was deposited both inside and outside of the cells. Since the selenate reduction rate of the strain SF-1 was higher than the selenite reduction rate, selenite was transiently accumulated. In an experiment of the repeated soluble selenium reduction by strain SF-1, 0.5 mM of selenate was sequentially treatable with a cycle of one day. Thus, our sequential system for removal of soluble selenium is very useful.     

Reduction of Selenium Oxyanions by Enterobacter cloacae SLD1a-1: Isolation and Growth of the Bacterium and Its Expulsion of Selenium Particles

A facultative bacterium capable of removing the selenium (Se) oxyanions selenate (SeO(inf4)(sup2-)) and selenite (SeO(inf3)(sup2-)) from solution culture in flasks open to the atmosphere was isolated and studied with the goal of assessing its potential for use in bioremediation of seleniferous agricultural drainage water. Elemental Se (Se(sup0)) was confirmed as a product of the reaction. The organism, identified as Enterobacter cloacae and designated strain SLD1a-1 (ATCC 700258), removed from 61.5 to 94.5% of added SeO(inf4)(sup2-) (the primary species present in agricultural drainage water) at concentrations from 13 to 1,266 (mu)M. Equimolar amounts of nitrate (NO(inf3)(sup-)), which interferes with SeO(inf4)(sup2-) reduction in some organisms, did not influence the reaction in growth experiments but had a slight inhibitory effect in a washed-cell suspension. Washed-cell suspension experiments also showed that (i) SeO(inf3)(sup2-) is a transitory intermediate in reduction of SeO(inf4)(sup2-), being produced and rapidly reduced concomitantly; (ii) NO(inf3)(sup-) is also reduced concomitantly and at a much higher rate than SeO(inf4)(sup2-); and (iii) although enzymatic, reduction of either oxyanion does not appear to be an inducible process. Transmission electron microscopy revealed that precipitate particles are <0.1 (mu)m in diameter, and these particles were observed free in the medium. Evidence indicates that SLD1a-1 uses SeO(inf4)(sup2-) as an alternate electron acceptor and that the reaction occurs via a membrane-associated reductase(s) followed by rapid expulsion of the Se particles.     

Chemical forms of selenium in the metal-resistant bacterium Ralstonia metallidurans CH34 exposed to selenite and selenate

Ralstonia metallidurans CH34, a soil bacterium resistant to a variety of metals, is known to reduce selenite to intracellular granules of elemental selenium (Se(0)). We have studied the kinetics of selenite (Se(IV)) and selenate (Se(VI)) accumulation and used X-ray absorption spectroscopy to identify the accumulated form of selenate, as well as possible chemical intermediates during the transformation of these two oxyanions. When introduced during the lag phase, the presence of selenite increased the duration of this phase, as previously observed. Selenite introduction was followed by a period of slow uptake, during which the bacteria contained Se(0) and alkyl selenide in equivalent proportions. This suggests that two reactions with similar kinetics take place: an assimilatory pathway leading to alkyl selenide and a slow detoxification pathway leading to Se(0). Subsequently, selenite uptake strongly increased (up to 340 mg Se per g of proteins) and Se(0) was the predominant transformation product, suggesting an activation of selenite transport and reduction systems after several hours of contact. Exposure to selenate did not induce an increase in the lag phase duration, and the bacteria accumulated approximately 25-fold less Se than when exposed to selenite. Se(IV) was detected as a transient species in the first 12 h after selenate introduction, Se(0) also occurred as a minor species, and the major accumulated form was alkyl selenide. Thus, in the present experimental conditions, selenate mostly follows an assimilatory pathway and the reduction pathway is not activated upon selenate exposure. These results show that R. metallidurans CH34 may be suitable for the remediation of selenite-, but not selenate-, contaminated environments.     

In situ bacterial selenate reduction in the agricultural drainage systems of western Nevada

Dissimilatory in situ selenate reduction to elemental selenium in sediments from irrigated agricultural drainage regions of western Nevada was measured at ambient Se oxyanion concentrations. Selenate reduction was rapid, with turnover rate constants ranging from 0.04 to 1.8 h-1 at total Se concentrations in pore water of 13 to 455 nM. Estimates of removal rates of selenium oxyanions were 14.38, and 155 mumol m-2 day-1 for South Lead Lake, Massie Slough, and Hunter Drain, respectively.     

In situ bacterial selenate reduction in the agricultural drainage systems of western Nevada.

Dissimilatory in situ selenate reduction to elemental selenium in sediments from irrigated agricultural drainage regions of western Nevada was measured at ambient Se oxyanion concentrations. Selenate reduction was rapid, with turnover rate constants ranging from 0.04 to 1.8 h-1 at total Se concentrations in pore water of 13 to 455 nM. Estimates of removal rates of selenium oxyanions were 14.38, and 155 mumol m-2 day-1 for South Lead Lake, Massie Slough, and Hunter Drain, respectively.     

Resolution of distinct membrane-bound enzymes from Enterobacter cloacae SLD1a-1 that are responsible for selective reduction of nitrate and selenate oxyanions

Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with alpha and beta subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (alphabetagamma) complex with an apparent molecular mass of approximately 600 kDa. The individual subunit sizes are approximately 100 kDa (alpha), approximately 55 kDa (beta), and approximately 36 kDa (gamma), with a predicted overall subunit composition of alpha3beta3gamma3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent Km for selenate was determined to be approximately 2 mM, with an observed Vmax of 500 nmol SeO4(2-) min(-1) mg(-1) (kcat, approximately 5.0 s(-1)). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.     

Characterization of sediment bacteria involved in selenium reduction

Bacterial reduction of selenium (Se) oxyanions (Se[VI] and Se[IV]) to elemental Se (Se[0]) is one of the major biogeochemical processes removing Se from agricultural drainage water and depositing Se in the sediment. This study was conducted to characterize Se-reducing bacterial populations in Lost Hills evaporation pond sediment and to observe their response to Se(VI) and organic C amendments. Se(VI) was removed from the dissolved phase in the sediment slurries amended with organic C with a decrease in redox potential (Eh). Se(VI) concentrations decreased from 2137 to 79 microg L-1 after 9 days of incubation in a 5% soil slurry. Upon our screening process, 9 Se(VI)- and 14 Se(IV)-reducing bacteria were isolated from sediment slurries and identified by amplification and sequencing of 16S rDNA. Bacillus strains appeared to be dominant in the bacterial assemblages active in Se(VI) and Se(IV) reduction in the sediment. Halomonas pacifica and Staphylococcus warneri were also identified as Se(IV)-reducers. Indigenous bacteria have a significant role in the biogeochemical cycling of Se and may be stimulated by addition of a suitable organic source for Se reduction. The bacterial strains isolated from salt-affected and Se-contaminated Lost Hills evaporation pond sediment may have potential application in removing Se from high salt drainage water.     

Laboratory-scale continuous reactor for soluble selenium removal using selenate-reducing bacterium,Bacillus sp. SF-1

A model continuous flow bioreactor (volume 0.5 L) was constructed for removing toxic soluble selenium (selenate/selenite) of high concentrations using a selenate-reducing bacterium, Bacillus sp. SF-1, which transforms selenate into elemental selenium via selenite for anaerobic respiration. Model wastewater contained 41.8 mg-Se/L selenate and excess lactate as the carbon and energy source; the bioreactor was operated as an anoxic, completely mixed chemostat with cell retention time between 2.2-95.2 h. At short cell retention times selenate was removed by the bioreactor, but accumulation of selenite was observed. At long cell retention times soluble selenium, both selenate and selenite, was successfully reduced into nontoxic elemental selenium. A simple mathematical model is proposed to evaluate Se reduction ability of strain SF-1. First-order kinetic constants for selenate and selenite reduction were estimated to be 2.9 x 10(-11) L/cells/h and 5.5 x 10(-13) L/cells/h, respectively. The yield of the bacterial cells by selenate reduction was estimated to be 2.2 x 10(9) cells/mg-Se.