The ability of the rhizobacterium Azospirillum brasilense to reduce selenium(IV) to selenium(0)

Effect of selenium(+4) as selenite (Se ₃ ^2? ) on two Azospirillum brasilense strains, which occupy different ecological niches (an epiphyte Sp7 and a facultative endophyte Sp245), was studied. The cultures grown in the medium with sodium selenite exhibited intense red coloration. Transmission electron microscopy and X-ray fluorescence analysis revealed accumulation of elementary selenium within the cells of both strains as nanoparticles 50–400 nm in diameter. The ability to reduce inorganic selenium(+4) to elementary selenium (as nanoparticles) has not been previously reported for azospirilla. Our results indicate the possibility to apply Azospirillum strains as microsymbionts for phytoremediation of, and cereal cultivation on, selenium-contaminated soils. The ability of azospirilla to synthesize selenium nanoparticles may be of interest for nanobiotechnology.     

A novel method for the measurement of elemental selenium produced by bacterial reduction of selenite

The measurement of elemental selenium (Se⁰) is needed to assess the rate and magnitude of bacteria reduction of selenite or selenate. We have developed a spectrophotometric method for the measurement Se⁰ that is rapid and can be employed to measure the quantity of Se⁰ produced by bacterial cultures. This method employs the use of 1 M Na₂S to convert the insoluble elemental selenium to a red-brown solution and with this method there is a direct correlation between concentration of elemental selenium and the absorption at 500 nm. To demonstrate the utility of this assay, we have followed the reduction of selenite to Se⁰ by Moraxella bovis, and by bacterial consortia in soil and water samples.     

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.     

Reduction of selenium oxyanions by unicellular,polymorphic and filamentous fungi: Cellular location of reduced selenium and implications for tolerance

Summary The ability of several filamentous, polymorphic and unicellular fungi to reduce selenite to elemental selenium on solid medium was examined.Fusarium sp. andTrichoderma reeii were the only filamentous fungi, of those tested, which reduced selenite to elemental selenium on Czapek-Dox agar resulting in a red colouration of colonies. Other organisms (Aspergillus niger, Coriolus versicolor, Mucor SK, andRhizopus arrhizus) were able to reduce selenite only on malt extract agar. Several fungi were able to grow in the presence of sodium selenite but were apparently unable to reduce selenite to elemental selenium, indicating that other mechanisms of selenite tolerance were employed, such as reduced uptake and/or biomethylation to less toxic, volatile derivatives. Sodium selenate was more toxic toFusarium sp. than selenite, and the toxicity of both oxyanions was increased in sulphur-free medium, with this effect being more marked for selenate. Scanning electron microscopy ofAspergillus funiculosus andFusarium sp. incubated with sodium selenite showed the presence of needle-like crystals of elemental selenium on the surfaces of hyphae and conidia, while transmission electron microscopy ofA. funiculosus revealed the deposition of electron-dense granules in vacuoles of selenite-treated fungi. Several yeasts were able to grow on MYGP agar containing sodium selenate or sodium selenite at millimolar concentrations. Sone, notablyRhodotorula rubra andCandida lipolytica, and the polymorphic fungusAureobasidium pullulans were also effective at reducing selenite to elemental selenium, resulting in red-coloured colonies.Schizosaccharomyces pombe was able to grow at selenite concentrations up to 5 mmol L^–1 without any evidence of reduction, again indicating the operation of other tolerance mechanisms.     

Microbial transformations of selenite by methane-oxidizing bacteria

Methane-oxidizing bacteria are well known for their role in the global methane cycle and their potential for microbial transformation of wide range of hydrocarbon and chlorinated hydrocarbon pollution. Recently, it has also emerged that methane-oxidizing bacteria interact with inorganic pollutants in the environment. Here, we report what we believe to be the first study of the interaction of pure strains of methane-oxidizing bacteria with selenite. Results indicate that the commonly used laboratory model strains of methane-oxidizing bacteria, Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b, are both able to reduce the toxic selenite (SeO₃ ^2?) but not selenate (SeO₄ ^2?) to red spherical nanoparticulate elemental selenium (Se⁰), which was characterized via energy-dispersive X-ray spectroscopy (EDXS), X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS). The cultures also produced volatile selenium-containing species, which suggests that both strains may have an additional activity that can transform either Se⁰ or selenite into volatile methylated forms of selenium. Transmission electron microscopy (TEM) measurements and experiments with the cell fractions cytoplasm, cell wall and cell membrane show that the nanoparticles are formed mainly on the cell wall. Collectively, these results are promising for the use of methane-oxidizing bacteria for bioremediation or suggest possible uses in the production of selenium nanoparticles for biotechnology.

     

Bioavailability of selenium accumulated by selenite-reducing bacteria

The bioavailability of selenium (Se) was determined in bacterial strains that reduce selenite to red elemental Se (Se^o). A laboratory strain ofBacillus subtilis and a bacterial rod isolated from soil in the vicinity of the Kesterson Reservoir, San Joaquin Valley, CA, (Microbacterium arborescens) were cultured in the presence of 1 mM sodium selenite (Na₂SeO₃). After harvest, the washed, lyophilizedB. subtilis andM. arborescens samples contained 2.62 and 4.23% total Se, respectively, which was shown to consist, within error, entirely of Se^o. These preparations were fed to chicks as supplements to a low-Se, vitamin E-free diet. Three experiments showed that the Se in both bacteria had bioavailabilities of approx 2% that of selenite. A fourth experiment revealed that gray Se^o had a bioavailability of 2% of selenite, but that the bioavailability of red Se^o depended on the way it was prepared (by reduction of selenite). When glutathione was the reductant, bioavailability resembled that of gray Se^o and bacterial Se; when ascorbate was the reductant, bioavailability was twice that level (3–4%). These findings suggest that aerobic bacteria such asB. subtilis andM. arborescens may be useful for the bioremediation of Se-contaminated sites, i.e., by converting selenite to a form of Se with very low bioavailability.     

Reduction of Selenite by Azospirillum brasilense with the Formation of Selenium Nanoparticles

The ability to reduce selenite (SeO₃ ^2?) ions with the formation of selenium nanoparticles was demonstrated in Azospirillum brasilense for the first time. The influence of selenite ions on the growth of A. brasilense Sp7 and Sp245, two widely studied wild-type strains, was investigated. Growth of cultures on both liquid and solid (2 % agar) media in the presence of SeO₃ ^2? was found to be accompanied by the appearance of the typical red colouration. By means of transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS) and X-ray fluorescence analysis (XFA), intracellular accumulation of elementary selenium in the form of nanoparticles (50 to 400 nm in diameter) was demonstrated for both strains. The proposed mechanism of selenite-to-selenium (0) reduction could involve SeO₃ ^2? in the denitrification process, which has been well studied in azospirilla, rather than a selenite detoxification strategy. The results obtained point to the possibility of using Azospirillum strains as endophytic or rhizospheric bacteria to assist phytoremediation of, and cereal cultivation on, selenium-contaminated soils. The ability of A. brasilense to synthesise selenium nanoparticles may be of interest to nanobiotechnology for “green synthesis” of bioavailable amorphous red selenium nanostructures.     

Reduction of diacetophenonyl selenide (DAPS-25 formulation) to acetophenone with the formation of selenium micro- and nanoparticles in the presence of <Emphasis Type="Italic">Saccharomyces cerevisiaе</Emphasis> culture

It was found that DAPS-25 in the presence of Saccharomyces cerevisiaе culture underwent a reduction, yielding acetophenone and elemental selenium. The latter appeared as selenium-containing spherical micro- and nanoparticles ranging from 50 to 400 nm in size. Under these conditions, sodium selenite was reduced with the formation of needle-shaped selenium particles up to 500 nm in length. The conversion speed of the formulation in contact with yeast culture fluid was independent of the nutrient medium content.     

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 (Se⁰). 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 Se⁰ 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 Se⁰. Subsequently, selenite uptake strongly increased (up to 340 mg Se per g of proteins) and Se⁰ 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, Se⁰ 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.     

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。     

Allotropy of selenium nanoparticles: Colourful transition,synthesis, and biotechnological applications

Elemental selenium (Se⁰) nanomaterials undergo allotropic transition from thermodynamically-unstable to more stable phases. This process is significantly different when Se⁰ nanoparticles (NPs) are produced via physico-chemical and biological pathways. While the allotropic transition of physico-chemically synthesized Se⁰ is fast (minutes to hours), the biogenic Se⁰ takes months to complete. The biopolymer layer covering biogenic Se⁰ NPs might be the main factor controlling this retardation, but this still remains an open question. Phylogenetically-diverse bacteria reduce selenium oxyanions to red amorphous Se⁰ allotrope, which has low market value. Then, red Se⁰ undergoes allotropic transition to trigonal (metallic grey) allotrope, the end product having important industrial applications (e.g. semiconductors, alloys). Is it not yet clear whether biogenic Se⁰ presents any biological function, or it is mainly a detoxification and respiratory by-product. The better understanding of this transition would benefit the recovery of Se⁰ NPs from secondary resources and its targeted utilization with respect to each allotropic stage. This review article presents and critically discusses the main physico-chemical methods and biosynthetic pathways of Se⁰ (bio)mineralization. In addition, the article proposes a conceptual model for the resource recovery potential of trigonal selenium nanomaterials in the context of circular economy.     

Effects of selenium oxyanions on the white-rot fungus <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

The ability of Phanerochaete chrysosporium to reduce the oxidized forms of selenium, selenate and selenite, and their effects on the growth, substrate consumption rate, and pellet morphology of the fungus were assessed. The effect of different operational parameters (pH, glucose, and selenium concentration) on the response of P. chrysosporium to selenium oxyanions was explored as well. This fungal species showed a high sensitivity to selenium, particularly selenite, which inhibited the fungal growth and substrate consumption when supplied at 10 mg L^?1 in the growth medium, whereas selenate did not have such a strong influence on the fungus. Biological removal of selenite was achieved under semi-acidic conditions (pH 4.5) with about 40 % removal efficiency, whereas less than 10 % selenium removal was achieved for incubations with selenate. P. chrysosporium was found to be a selenium-reducing organism, capable of synthesizing elemental selenium from selenite but not from selenate. Analysis with transmission electron microscopy, electron energy loss spectroscopy, and a 3D reconstruction showed that elemental selenium was produced intracellularly as nanoparticles in the range of 30–400 nm. Furthermore, selenite influenced the pellet morphology of P. chrysosporium by reducing the size of the fungal pellets and inducing their compaction and smoothness.     

Biomimetic Synthesis of Selenium Nanospheres by Bacterial Strain JS-11 and Its Role as a Biosensor for Nanotoxicity Assessment: A Novel Se-Bioassay

Selenium nanoparticles (Se-NPs) were synthesized by green technology using the bacterial isolate Pseudomonas aeruginosa strain JS-11. The bacteria exhibited significant tolerance to selenite (SeO₃ ²⁻) up to 100 mM concentration with an EC₅₀ value of 140 mM. The spent medium (culture supernatant) contains the potential of reducing soluble and colorless SeO₃ ²⁻ to insoluble red elemental selenium (Se⁰) at 37°C. Characterization of red Se° product by use of UV-Vis spectroscopy, X-ray diffraction (XRD), atomic force microscopy (AFM) and transmission electron microscopy (TEM) with energy dispersive X-ray spectrum (EDX) analysis revealed the presence of stable, predominantly monodispersed and spherical selenium nanoparticles (Se-NPs) of an average size of 21 nm. Most likely, the metabolite phenazine-1-carboxylic acid (PCA) released by strain JS-11 in culture supernatant along with the known redox agents like NADH and NADH dependent reductases are responsible for biomimetic reduction of SeO₃ ²⁻ to Se° nanospheres. Based on the bioreduction of a colorless solution of SeO₃ ²⁻ to elemental red Se⁰, a high throughput colorimetric bioassay (Se-Assay) was developed for parallel detection and quantification of nanoparticles (NPs) cytotoxicity in a 96 well format. Thus, it has been concluded that the reducing power of the culture supernatant of strain JS-11 could be effectively exploited for developing a simple and environmental friendly method of Se-NPs synthesis. The results elucidated that the red colored Se° nanospheres may serve as a biosensor for nanotoxicity assessment, contemplating the inhibition of SeO₃ ²⁻ bioreduction process in NPs treated bacterial cell culture supernatant, as a toxicity end point.     

Transformation of selenate and selenite to elemental selenium byDesulfovibrio desulfuricans

Summary Desulfovibrio desulfuricans (DSM 1924) can be adapted to grow in the presence of 10 mM selenate or 0.1 mM selenite. This growth occurred in media containing formate as the electron donor and either fumarate or sulfate as the electron acceptor. As determined by electron microscopy with energy-dispersive X-ray analysis, selenate and selenite were reduced to elemental selenium which accumulated inside the cells. Selenium granules resulting from selenite metabolism were cytoplasmic while granules of selenium resulting from selenate reduction appeared to be in the periplasmic region. The accumulation of red elemental selenium in the media following stationary phase resulted from cell lysis with the liberation of selenium granules. Growth did not occur with either selenate or selenite as the electron acceptor and¹³C nuclear magnetic resonance indicated that neither selenium oxyanion interfered with fumarate respiration. At 1 M selenate and 100 M selenite, reduction byD. desulfuricans was 95% and 97%, respectively. The high level of total selenate and selenite reduced indicated the suitability ofD. desulfuricans for selenium detoxification.     

Identification and Characterization of an Aeromonas salmonicida (syn Haemophilus piscium) Strain That Reduces Selenite to Elemental Red Selenium

A bacterium that reduces toxic and mobile selenite to insoluble elemental selenium (Se⁰) was isolated from a laboratory scale permeable reactive biobarrier. Biochemical tests and 16S rRNA gene sequence alignment identified the isolate as Aeromonas salmonicida. Two colony types were isolated, one more resistant to selenite than the other. Both grew on agar plates containing 16 mM selenite, although the colony diameter was reduced to 8% of controls with the small colony type and to 18% with the large colony type. Further study was done with the large colony type. In anaerobic culture, this bacterium was able to use nitrate as a term electron acceptor but not selenate or selenite. In aerobic culture, when no nitrate was present, early log phase cells removed selenite at a rate of 2.6 ± 0.42 μmol SeO₃⁻²/mg protein/day. Reduction was retarded by 25 mM nitrate. Mutants with a diminished ability to reduce selenite to Se⁰ also had a reduced ability to reduce nitrate to nitrous oxide. This bacterium, or perhaps its enzymes or DNA, might be used to remove selenite from contaminated groundwaters.     

Reduction of Selenite to Elemental Red Selenium by <Emphasis Type="Italic">Rhizobium</Emphasis> sp. Strain B1

A bacterium that reduces the soluble and toxic selenite anion to insoluble elemental red selenium (Se⁰) was isolated from a laboratory bioreactor. Biochemical, morphological, and 16S rRNA gene sequence alignment identified the isolate as a Rhizobium sp. that is related to but is genetically divergent from R. radiobacter (syn. Agrobacterium tumefaciens) or R. rubi (syn. A. rubi). The isolate was capable of denitrification and reduced selenite to Se⁰ under aerobic and denitrifying conditions. It did not reduce selenate and did not use selenite or selenate as terminal e⁻ donors. Native gel electrophoresis revealed two bands, corresponding to molecular weights of ∼100 and ∼45 kDa, that reduced selenite. Tungsten inhibited in vivo selenite reduction, suggesting that a molybdenum-containing protein is involved in selenite reduction. This organism, or its enzymes or DNA, might be useful in bioreactors designed to remove selenite from water.     

Pseudomonas seleniipraecipitans Proteins Potentially Involved in Selenite Reduction

Pseudomonas seleniipraecipitans grows in the presence of high levels of selenite and selenate and reduces both oxyanions to elemental selenium (Se⁰), a property that may make P. seleniipraecipitans useful as an inoculant for biobarriers designed to remove selenite or selenate from ground or surface waters. An earlier study showed that P. seleniipraecipitans nitrate reductase reduced selenate to Se⁰, but failed to identify the protein(s) involved in selenite reduction. This study used ammonium sulfate precipitation, hydrophobic interaction chromatography, and native PAGE to isolate two electrophoretic gel regions, identified as bands A and B that showed selenite-reductase-activity. Proteomics was used to identify the proteins present in those regions. Glutathione reductase (GR) was detected in the A-band; based on this information, Saccharomyces cerevisiae GR, obtained from a commercial source, was evaluated and found to have selenite-reductase-activity, confirming that GR can reduce selenite to Se⁰. Proteomics was also used to detect the proteins present in the B-band and thioredoxin reductase (ThxR) was detected as a B-band protein; based on this information, E. coli ThxR, obtained from a commercial source, was evaluated and found to have selenite-reductase-activity, confirming that ThxR can reduce selenite to elemental selenium. Thus, evidence presented in this study shows that S. cerevisiae GR and E. coli ThxR can reduce SeO₃ ^2? to Se⁰ and strongly suggests that P. seleniipraecipitans GR and ThxR can also reduce SeO₃ ^2? to Se⁰.     

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.     

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.     

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。