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.     

High-level production of animal-free recombinant transferrin from Saccharomyces cerevisiae

Background

Microorganisms that are exposed to pollutants in the environment, such as metals/metalloids, have a remarkable ability to fight the metal stress by various mechanisms. These metal-microbe interactions have already found an important role in biotechnological applications. It is only recently that microorganisms have been explored as potential biofactories for synthesis of metal/metalloid nanoparticles. Biosynthesis of selenium (Se⁰) nanospheres in aerobic conditions by a bacterial strain isolated from the coalmine soil is reported in the present study.

Results

The strain CM100B, identified as Bacillus cereus by morphological, biochemical and 16S rRNA gene sequencing [GenBank:GU551935.1] was studied for its ability to generate selenium nanoparticles (SNs) by transformation of toxic selenite (SeO₃ ^2-) anions into red elemental selenium (Se⁰) under aerobic conditions. Also, the ability of the strain to tolerate high levels of toxic selenite ions was studied by challenging the microbe with different concentrations of sodium selenite (0.5 mM-10 mM). ESEM, AFM and SEM studies revealed the spherical Se⁰ nanospheres adhering to bacterial biomass as well as present as free particles. The TEM microscopy showed the accumulation of spherical nanostructures as intracellular and extracellular deposits. The deposits were identified as element selenium by EDX analysis. This is also indicated by the red coloration of the culture broth that starts within 2-3 h of exposure to selenite oxyions. Selenium nanoparticles (SNs) were further characterized by UV-Visible spectroscopy, TEM and zeta potential measurement. The size of nanospheres was in the range of 150-200 nm with high negative charge of -46.86 mV.

Conclusions

This bacterial isolate has the potential to be used as a bionanofactory for the synthesis of stable, nearly monodisperse Se⁰ nanoparticles as well as for detoxification of the toxic selenite anions in the environment. A hypothetical mechanism for the biogenesis of selenium nanoparticles (SNs) involving membrane associated reductase enzyme(s) that reduces selenite (SeO₃ ^2-) to Se⁰ through electron shuttle enzymatic metal reduction process has been proposed.     

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.     

Variation in selenium tolerance and accumulation among 19 Arabidopsis thaliana accessions

Selenium (Se) is an essential element for many organisms but also toxic at higher levels. The objective of this study was to identify accessions from the model species Arabidopsis thaliana that differ in Se tolerance and accumulation. Nineteen Arabidopsis accessions were grown from seed on agar medium with or without selenate (50 microM) or selenite (20 microM), followed by analysis of Se tolerance and accumulation. Tissue sulfur levels were also compared. The Se Tolerance Index (root length+Se/root length control) varied among the accessions from 0.11 to 0.44 for selenite and from 0.05 to 0.24 for selenate. When treated with selenite, the accessions differed by two-fold in shoot Se concentration (up to 250 mgkg(-1)) and three-fold in root Se concentration (up to 1000 mgkg(-1)). Selenium accumulation from selenate varied 1.7-fold in shoot (up to 1000 mgkg(-1)) and two-fold in root (up to 650 mgkg(-1)). Across all accessions, a strong correlation was observed between Se and S concentration in both shoot and root under selenate treatment, and in roots of selenite-treated plants. Shoot Se accumulation from selenate and selenite were also correlated. There was no correlation between Se tolerance and accumulation, either for selenate or selenite. The F(1) offspring from a cross between the extreme selenate-sensitive Dijon G and the extreme selenate-tolerant Estland accessions showed intermediate selenate tolerance. In contrast, the F(1) offspring from a cross between selenite-sensitive and -tolerant accessions (Dijon GxCol-PRL) were selenite tolerant. The results from this study give new insight into the mechanisms of plant selenium (Se) tolerance and accumulation, which may help develop better plants for selenium phytoremediation or as fortified foods.     

Rhizosphere-induced selenium precipitation for possible applications in phytoremediation of se polluted effluents

Two bacterial isolates were obtained in axenic culture from the rhizosphere soil of Astragalus bisulcatus, a legume able to hyperaccumulate selenium. Both strains resulted of particular interest for their high resistance to the toxic oxyanion SeO3(2-) (selenite, Se(IV)). On the basis of molecular and biochemical analyses, these two isolates were attributed to the species Bacillus mycoides and Stenotrophomonas maltophilia, respectively. Their capability in axenic culture to precipitate the soluble, bioavailable and highly toxic selenium form selenite to insoluble and relatively non-toxic Se(0) (elemental selenium) was evaluated in defined medium added with 0.2 or 0.5 mM Se(IV). Both strains showed to completely reduce 0.2 mM selenite in 120 h, while 0.5 mM Se(IV) was reduced up to 67% of the initial concentration by B. mycoides and to about 50% by S. maltophilia in 48 h. Together in a dual consortium, B. mycoides and S. maltophilia increased the kinetics of selenite reduction, thus improving the efficiency of the process. A model system for selenium rhizofiltration based on plant-rhizobacteria interactions has been proposed.     

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。     

Detection of Selenium Deposits in Escherichia coli by Electron Microscopy

Cells of Escherichia coli will reduce sodium selenite to elemental selenium. Examination by electron microscopy of E. coli cultures grown in the presence of sodium selenite show selenium deposited on the cell membrane and cell wall but not in the cytoplasm.     

Fungal transformation of selenium and tellurium located in a volcanogenic sulfide deposit

Microbial reduction of soluble selenium (Se) or tellurium (Te) species results in immobilization as elemental forms and this process has been employed in soil bioremediation. However, little is known of direct and indirect fungal interactions with Se-/Te-bearing ores. In this research, the ability of Phoma glomerata to effect transformation of selenite and tellurite was investigated including interaction with Se and Te present in sulfide ores from the Kisgruva Proterozoic volcanogenic deposit. Phoma glomerata could precipitate elemental Se and Te as nanoparticles, intracellularly and extracellularly, when grown with selenite or tellurite. The nanoparticles possessed various surface capping molecules, with formation being influenced by extracellular polymeric substances. The presence of sulfide ore also affected the production of exopolysaccharide and protein. Although differences were undetectable in gross Se and Te ore levels before and after fungal interaction using X-ray fluorescence, laser ablation inductively coupled plasma mass spectrometry of polished flat ore surfaces revealed that P. glomerata could effect changes in Se/Te distribution and concentration indicating Se/Te enrichment in the biomass. These findings provide further understanding of fungal roles in metalloid transformations and are relevant to the geomicrobiology of environmental metalloid cycling as well as informing applied approaches for Se and Te immobilization, biorecovery or bioremediation.     

还原亚硒酸盐产生红色单质硒光合细菌菌株的筛选与鉴定

从实验室保藏的光合细菌中筛选出一株对亚硒酸钠还原效率较高的菌株S3,其亚硒酸钠还原产物通过透射电子显微镜及EDX(Electron-Dispersive X-ray)分析确定为红色单质硒。菌株S3的形态学特征、生理生化特征及光合色素扫描结果与固氮红细菌(Rhodobacter azotoformans)的特征基本一致;16S rDNA序列(GenBank登录号为DQ402051)在系统发育树中与固氮红细菌同属一个类群,序列同源性为99%。根据上述结果将菌株S3鉴定为固氮红细菌。初步研究了该菌株还原亚硒酸钠的特性,首次报道固氮红细菌具有还原亚硒酸盐产生红色单质硒的能力,为今后利用微生物方法治理环境中硒污染、利用微生物方法获得活性红色单质硒以及对微生物还原亚硒酸盐产生红色单质硒的机理研究奠定了良好的基础。     

Studies on the effects of selenium on rumen microbial fermentation in vitro

The effects of selenium (Se) on ruminant microbial fermentation were investigated in vitro using rumen microflora collected from a rumen-fistulated dairy cow. First, the effects ofl-selenomethionine (SeMet; at 0.2 or 2 ppm Se) in the presence or absence of wheat bran (WB, 500 mg per incubation flask) were evaluated. Second, the effects of several forms of Se (elemental Se: 50 ppm Se; sodium selenite: 2 ppm Se; SeMet: 2 ppm Se) were compared. Results showed that the amounts of short-chain fatty acids (SCFAs) tended to be increased by SeMet treatment, whereas SeMet in the presence of WB transiently suppressed fermentation. The addition of SeMet tended to increase the production of acetate while reducing the production of butyrate with and without WB supplementation. Among the different Se compounds tested, the amounts of SCFAs were greater with SeMet treatment, which yielded a higher proportion of acetate compared to other treatments. Selenite did not influence the total SCFAs concentrations; however, it increased the relative proportion of butyrate at the expense of acetate. Elemental Se did not significantly affect fermentation. Higher bacterial Se concentrations were observed for selenite than for SeMet. It was concluded that Se supplementation can influence rumen microbial fermentation and that Se compounds differ in this regard.     

The synthesis of elemental selenium particles by Synechococcus leopoliensis

Exposure of Synechococcus leopoliensis to selenite in the light resulted in orange-colored granules associated with the cells. No such particles were made in dark grown cells or when selenite was replaced by selenate. Light and scanning electron microscopy revealed that the particles formed inside the cells. Furthermore, these were easily extracted and shown to be composed of selenium as determined by energy-dispersive X-ray spectroscopy. During selenium particle synthesis there was a concurrent loss of organic pigments in the cyanobacteria. Cells also become heavier as they produced and accumulated particles which were on average 220 nm in diameter and generally spherical in shape. The decline in selenite concentration in the culture media can be accounted for by the formation of cellular elemental selenium (Se(0)) during particle formation, although synthesis of small amounts of other Se compounds cannot be entirely discounted. Photosynthetic activity is required for the formation of Se(0), implicating the involvement of thylakoids. It is possible that an intimate association between the nascent particles and the thylakoids occurred. However, Se(0) granule formation did not occur peripherally between the thylakoid and the cytoplasmic membranes, but inside the thylakoid bands towards the center of the cells. It then appears that the particles are mobilized to the periphery and expelled from the cells, causing irreparable damage to the cell walls.     

The metabolism of selenite and selenomethionine in mouse fibroblasts grown in tissue culture

Recent reports have provided evidence that selenium is an essential growth factor for cells grown in tissue culture. The aim of the work reported in this paper was to evaluate mouse fibroblasts as a model for the study of selenium metabolism in mammalian cells. The results showed that transformed mouse lung fibroblasts grown in media containing 9.1% bovine serum did not show a growth response to added selenium as selenite over the range of 10–1000 ng/mL. Uptake of selenium by cells was a direct function of the selenium concentration in the medium. The rate of uptake varied with the time of exposure of the cells to the selenium, and to the form of selenium in the medium. Experiments using radioactive selenium showed that⁷⁵Se from selenite was rapidly absorbed into the cell wall, but slowly incorporated into the soluble protein fraction.⁷⁵Se from selenomethionine was more slowly absorbed into the cells, but once inside, it became rapidly incorporated into soluble cytoplasmic proteins. Cell fractionation and gel filtration procedures established that⁷⁵Se from selenite was rapidly incorporated into glutathione peroxidase (GSHpx), whereas⁷⁵Se from selenomethionine was initially incorporated into a wide spectrum of proteins and only after a longer period did the⁷⁵Se peak become associated with GSHpx. These findings suggest fundamental differences exist in the manner in which mammalian cells initially absorb and metabolize different selenium compounds.     

Physiological adaptations and tolerance towards higher concentration of selenite (Se+4) in Enterobacter sp. AR-4, Bacillus sp. AR-6 and Delftia tsuruhatensis AR-7

Environmental contamination with selenium is a major health concern. A few bacterial strains have been isolated that can transform toxic selenite to non-toxic elemental selenium only at low concentrations (0.001–150 mM) in recent past. We have previously reported isolation and characterization of few selenite-tolerant bacterial strains. These strains were found to be resistant to selenite at (300–600 mM) concentrations. In the present study we have characterized some physiological adaptations of strains Enterobacter sp. AR-4, Bacillus sp. AR-6 and Delftia tsuruhatensis AR-7 during exposure to higher concentration of selenite under aerobic and anaerobic environments. Adaptive responses are largely associated with alteration of cell morphology and change in total cellular fatty acid composition. Interestingly, electron microscopy studies revealed substantial decrease in cell size and intracellular deposition of Se⁰ crystals when reduction is carried out under aerobic conditions. On the other hand, cell size increased with adhesion of Se⁰ on cell surface during anaerobic reduction. Fatty acid composition analysis demonstrated selective increase in saturated and cyclic fatty acids and decrease in unsaturated ones during aerobic transformation. Changes observed during anaerobic transformation were in surprising contrast as indicated by total absence of saturated and cyclic fatty acids. Results presented here provide evidences for putative occurrence of two distinct mechanisms involved in tolerance towards higher concentrations of selenite utilization under aerobic and anaerobic conditions. Further, prior exposure to higher concentration of Se⁺⁴ enabled rapid adaptation indicating role of inducible system in adaptation.     

Bioeffects of selenite on the growth of Spirulina platensis and its biotransformation

The bioeffects of selenium on the growth of Spirulina platensis and the selenium distribution were investigated. S. platensis was batch cultured in Zarrouk medium containing increasing concentrations of sodium selenite. The biotransformation characteristic of selenium was analysed by the determination of the detailed selenium distribution forms. At 35 degrees C, 315.2 microEm(-2) x s(-1), sodium selenite concentrations below 400 mg x l(-1) were found to stimulate algal growth, especially in the range of 0.5-40 mg x l(-1). However, above 500 mg x l(-1) sodium selenite was toxic to this alga with the toxicity being related to the sulfite level in the medium. S. platensis was found to resist higher selenite by reducing toxic Se(IV) to nonsoluble Se(0). Selenium was accumulated efficiently in S. platensis during cultivation with accumulated selenium increasing with selenite concentration in the medium. It was demonstrated that inorganic selenite could be transformed into organic forms through binding with protein, lipids and polysaccharides and other cell components. The organic selenium accounted for 85.1% of the total accumulated selenium and was comprised of 25.2% water-soluble protein-bound, 10.6% lipids-bound and 2.1% polysaccharides-bound selenium. Among the organic fractions lipid possessed the strongest ability to accumulate Se (6.47 mg x kg(-1)). The 14.9% inorganic selenium in S. platensis was composed of Se(IV) (13.7%) and Se(VI) (1.2%).     

Accumulation of selenium in a model freshwater microbial food web.

The transfer of selenium between bacteria and the ciliated protozoan, Paramecium putrinum, was examined in laboratory cultures. The population growth of the ciliate was not inhibited in the presence of the highest concentrations of dissolved selenite or selenate tested (10(3) micrograms liter-1). Experiments with radioactive 75selenite or 75selenate indicated that accumulation of selenium by ciliates through time was low when feeding and metabolism were reduced by incubating at 0 degrees C. However, selenium accumulated in ciliate biomass during incubation with dissolved 75Se and bacteria at 24 degrees C and also when bacteria prelabeled with 75Se were offered as food in the absence of dissolved selenium. When 75Se-labeled bacterial food was diluted by the addition of nonradioactive bacteria, the amount of selenite and selenate in ciliates decreased over time, indicating depuration by the ciliates. In longer-term (> 5-day) fed-batch incubations with 75selenite-labeled bacteria, the selenium concentration in ciliates equilibrated at approximately 1.4 micrograms of Se g (dry weight)-1. The selenium content of ciliates was similar to that of their bacterial food on a dry-weight basis. These data indicate that selenium uptake by this ciliate occurred primarily during feeding and that biomagnification of selenium did not occur in this simple food chain.     

Deletion of Thioredoxin Reductase and Effects of Selenite and Selenate Toxicity in Caenorhabditis elegans

Thioredoxin reductase-1 (TRXR-1) is the sole selenoprotein in C. elegans, and selenite is a substrate for thioredoxin reductase, so TRXR-1 may play a role in metabolism of selenium (Se) to toxic forms. To study the role of TRXR in Se toxicity, we cultured C. elegans with deletions of trxr-1, trxr-2, and both in axenic media with increasing concentrations of inorganic Se. Wild-type C. elegans cultured for 12 days in Se-deficient axenic media grow and reproduce equivalent to Se-supplemented media. Supplementation with 0–2 mM Se as selenite results in inverse, sigmoidal response curves with an LC₅₀ of 0.20 mM Se, due to impaired growth rather than reproduction. Deletion of trxr-1, trxr-2 or both does not modulate growth or Se toxicity in C. elegans grown axenically, and ⁷⁵Se labeling showed that TRXR-1 arises from the trxr-1 gene and not from bacterial genes. Se response curves for selenide (LC₅₀ 0.23 mM Se) were identical to selenite, but selenate was 1/4^th as toxic (LC₅₀ 0.95 mM Se) as selenite and not modulated by TRXR deletion. These nutritional and genetic studies in axenic media show that Se and TRXR are not essential for C. elegans, and that TRXR alone is not essential for metabolism of inorganic Se to toxic species.     

Effects of selenite and selenate on the antioxidant systems in Senecio scandens L

Abstract

Selenate and selenite are the most prevalent bioavailable selenium (Se) forms and most easily taken up by plants. Some studies indicate that they are differently absorbed and accumulated in plants and that selenium is toxic if accumulated at high concentrations. Toxicity is due to substitution of sulphur by selenium in cysteine and methionine aminoacids with alteration of the tertiary structure and catalytic activity of proteins and with inhibition of enzymes involved in chlorophyll biosynthesis. Moreover, the interaction between Se and thiol groups induces loss of efficiency of plant defence systems and increases the reactive oxygen species (ROS) production thus enhancing the oxidative stress. To further elucidate the role of Se in higher plants, in this study the antioxidative response to the phytotoxicity of selenite and selenate in Senecio scandens L. was evaluated. The data indicate that while selenite induces oxidative stress enhancing ROS production, lipid peroxidation and the oxidised forms of ascorbate and glutathione, selenate does not significantly affect the analysed pathways. This article outlines that the synergistic action of different antioxidant components is necessary to overcome the phytotoxicity of selenium in Senecio.     

The Role of Microorganisms in Transformation of Selenium in Marine Waters

The activity of microorganisms is a decisive factor in the transformation of the essential and, at the same time, toxic selenium (Se) in marine waters. This review provides an analysis of the literature data on the microbiological regulation of the state of Se in marine waters: the role of microorganisms in eliminating toxic Se from marine waters through precipitation of reduced Se forms and in the reverse process, transformation of Se into a form available to be taken up by organisms and involvement of this element in the biogeochemical cycle. The processes of transformation of the oxidized and reduced Se forms with the participation of microorganisms in marine waters are considered. It has been shown that in anaerobic conditions bacteria use the oxidized Se forms as electron acceptors (reduction). Bioavailable selenite and selenate ions are formed in the case of aerobic oxidation. Biotransformation of dissolved Se is a key mechanism for the formation of methylated gaseous Se forms in marine waters as one of the ways to remove this element from the aquatic environment.     

Heavy metal tolerance in Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is an aerobic, non-fermentative Gram-negative bacterium widespread in the environment. S. maltophilia Sm777 exhibits innate resistance to multiple antimicrobial agents. Furthermore, this bacterium tolerates high levels (0.1 to 50 mM) of various toxic metals, such as Cd, Pb, Co, Zn, Hg, Ag, selenite, tellurite and uranyl. S. maltophilia Sm777 was able to grow in the presence of 50 mM selenite and 25 mM tellurite and to reduce them to elemental selenium (Se(0)) and tellurium (Te(0)) respectively. Transmission electron microscopy and energy dispersive X-ray analysis showed cytoplasmic nanometer-sized electron-dense Se(0) granules and Te(0) crystals. Moreover, this bacterium can withstand up to 2 mM CdCl(2) and accumulate this metal up to 4% of its biomass. The analysis of soluble thiols in response to ten different metals showed eightfold increase of the intracellular pool of cysteine only in response to cadmium. Measurements by Cd K-edge EXAFS spectroscopy indicated the formation of Cd-S clusters in strain Sm777. Cysteine is likely to be involved in Cd tolerance and in CdS-clusters formation. Our data suggest that besides high tolerance to antibiotics by efflux mechanisms, S. maltophilia Sm777 has developed at least two different mechanisms to overcome metal toxicity, reduction of oxyanions to non-toxic elemental ions and detoxification of Cd into CdS.