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.     

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.

     

<Emphasis Type="Italic">Ochrobactrum</Emphasis> sp. MPV1 from a dump of roasted pyrites can be exploited as bacterial catalyst for the biogenesis of selenium and tellurium nanoparticles

Background

Bacteria have developed different mechanisms for the transformation of metalloid oxyanions to non-toxic chemical forms. A number of bacterial isolates so far obtained in axenic culture has shown the ability to bioreduce selenite and tellurite to the elemental state in different conditions along with the formation of nanoparticles—both inside and outside the cells—characterized by a variety of morphological features. This reductive process can be considered of major importance for two reasons: firstly, toxic and soluble (i.e. bioavailable) compounds such as selenite and tellurite are converted to a less toxic chemical forms (i.e. zero valent state); secondly, chalcogen nanoparticles have attracted great interest due to their photoelectric and semiconducting properties. In addition, their exploitation as antimicrobial agents is currently becoming an area of intensive research in medical sciences.

Results

In the present study, the bacterial strain Ochrobactrum sp. MPV1, isolated from a dump of roasted arsenopyrites as residues of a formerly sulfuric acid production near Scarlino (Tuscany, Italy) was analyzed for its capability of efficaciously bioreducing the chalcogen oxyanions selenite (SeO₃ ^2?) and tellurite (TeO₃ ^2?) to their respective elemental forms (Se⁰ and Te⁰) in aerobic conditions, with generation of Se- and Te-nanoparticles (Se- and TeNPs). The isolate could bioconvert 2 mM SeO₃ ^2? and 0.5 mM TeO₃ ^2? to the corresponding Se⁰ and Te⁰ in 48 and 120 h, respectively. The intracellular accumulation of nanomaterials was demonstrated through electron microscopy. Moreover, several analyses were performed to shed light on the mechanisms involved in SeO₃ ^2? and TeO₃ ^2? bioreduction to their elemental states. Results obtained suggested that these oxyanions are bioconverted through two different mechanisms in Ochrobactrum sp. MPV1. Glutathione (GSH) seemed to play a key role in SeO₃ ^2? bioreduction, while TeO₃ ^2? bioconversion could be ascribed to the catalytic activity of intracellular NADH-dependent oxidoreductases. The organic coating surrounding biogenic Se- and TeNPs was also characterized through Fourier-transform infrared spectroscopy. This analysis revealed interesting differences among the NPs produced by Ochrobactrum sp. MPV1 and suggested a possible different role of phospholipids and proteins in both biosynthesis and stabilization of such chalcogen-NPs.

Conclusions

In conclusion, Ochrobactrum sp. MPV1 has demonstrated to be an ideal candidate for the bioconversion of toxic oxyanions such as selenite and tellurite to their respective elemental forms, producing intracellular Se- and TeNPs possibly exploitable in biomedical and industrial applications.

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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.     

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.     

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.     

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.     

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 organic and inorganic selenium compounds by the edible medicinal basidiomycete Lentinula edodes and the accumulation of elemental selenium nanoparticles in its mycelium

We report for the first time that the medicinal basidiomycete Lentinula edodes can reduce selenium from inorganic sodium selenite (Se^IV) and the organoselenium compound 1,5-diphenyl-3-selenopentanedione-1,5 (DAPS-25) to the elemental state, forming spherical nanoparticles. Submerged cultivation of the fungus with sodium selenite or with DAPS-25 produced an intense red coloration of L. edodes mycelial hyphae, indicating accumulation of elemental selenium (Se⁰) in a red modification. Several methods, including transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS), and X-ray fluorescence, were used to show that red Se⁰ accumulated intracellularly in the fungal hyphae as electron-dense nanoparticles with a diameter of 180.51±16.82 nm. Under designated cultivation conditions, shiitake did not reduce selenium from sodium selenate (Se^VI).     

Extracellular synthesis of cuprous selenide nanospheres by a biological‐chemical coupling reduction process in an anaerobic microbial system

Biosynthesis of metal nanoparticles represents a clean, eco‐friendly and sustainable “green chemistry” engineering. Lately, a number of metal selenides were successfully synthesized by biological methods. Here, cuprous selenide (Cu₂Se) nanospheres were prepared under mild conditions by a novel biological‐chemical coupling reduction process. The simple process takes place between EDTA‐Cu and Na₂SeO₃ in presence of an alkaline solution containing NaBH₄ and a selenite‐reducing bacteria, Pantoea agglomerans. It is noteworthy that the isolated Pantoea agglomerans and Cu⁺ ions, where the latter are obtained from reducing Cu²⁺ ions by NaBH₄, play a key role, and Cu⁺ ions not only can promote the generation of Se^2? ions as a catalyst, but also can react with Se^2? ions to form Cu₂Se. XRD pattern, SEM, and TEM images indicated that Cu₂Se nanoparticles were tetragonal crystal structure and the nanospheres diameter were about 100 nm. EDX, UV–vis, and FTIR spectra show that the biosynthesized Cu₂Se nanospheres are wrapped by protein and have a better stability. This work first proposes a new biosynthesis mechanism, and has important reference value for biological preparation of metal selenide nanomaterials. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1264–1270, 2016     

Selenium accumulation in durum wheat and spring canola as a function of amending soils with selenite, selenate and or sulphate

Aims

A comparison was performed between plant species to determine if extractable, rather than total soil Se, is more effective at predicting plant Se accumulation over a full growing season.

Methods

Durum wheat (Triticum turgidum L.) and spring canola (Brassica napus L.) were sown in potted soil amended with 0, 0.1, 1.0, or 5.0 mg kg^?1 Se as SeO₄ ^2? or SeO₃ ^2?. In addition, SeO₄ ^2?-amended soils were amended with 0 or 50 mg kg^?1 S as SO₄ ^2?. Soils were analyzed for extractable and total concentration of Se ([Se]). Twice during the growing season plants were harvested and tissue [Se] was determined.

Results

Plants exposed to SeO₃ ^2? accumulated the least Se. Fitted predictive models for whole plant accumulation based on extractable soil [Se] were similar to models based on total [Se] in soil (R²?=?0.73 or 0.74, respectively) and selenium speciation and soil [S] were important soil parameters to consider. As well, soil S amendments limited Se toxicity.

Conclusions

Soil quality guidelines (SQGs) based on extractable Se should be considered for risk assessment, particularly when Se speciation is unknown. Predictive models to estimate plant Se uptake should include soil S, a modifier of Se accumulation.     

Selenite Reduction by the Thioredoxin System: Kinetics and Identification of Protein-Bound Selenide

Selenite (SeO₃ ^2?) assimilation into a bacterial selenoprotein depends on thioredoxin (trx) reductase in Esherichia coli, but the molecular mechanism has not been elucidated. The mineral-oil overlay method made it possible to carry out anaerobic enzyme assay, which demonstrated an initial lag-phase followed by time-dependent steady NADPH consumption with a positive cooperativity toward selenite and trx. SDS-PAGE/autoradiography using ⁷⁵Se-labeled selenite as substrate revealed the formation of trx-bound selenium in the reaction mixture. The protein-bound selenium has metabolic significance in being stabilized in the divalent state, and it also produced the selenopersulfide (-S-SeH) form by the catalysis of E. coli trx reductase (TrxB).     

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.     

Reduction of Selenite to Red Elemental Selenium by Rhodopseudomonas palustris Strain N

The trace metal selenium is in demand for health supplements to human and animal nutrition. We studied the reduction of selenite (SeO₃ ⁻²) to red elemental selenium by Rhodopseudomonas palustris strain N. This strain was cultured in a medium containing SeO₃ ⁻² and the particles obtained from cultures were analyzed using transmission electron microscopy (TEM), energy dispersive microanalysis (EDX) and X ray diffraction analysis (XRD). Our results showed the strain N could reduce SeO₃ ⁻² to red elemental selenium. The diameters of particles were 80–200 nm. The bacteria exhibited significant tolerance to SeO₃ ⁻² up to 8.0 m mol/L concentration with an EC₅₀ value of 2.4 m mol/L. After 9 d of cultivation, the presence of SeO₃ ²⁻ up to 1.0 m mol/L resulted in 99.9% reduction of selenite, whereas 82.0% (p<0.05), 31.7% (p<0.05) and 2.4% (p<0.05) reduction of SeO₃ ⁻² was observed at 2.0, 4.0 and 8.0 m mol/L SeO₃ ²⁻ concentrations, respectively. This study indicated that red elemental selenium was synthesized by green technology using Rhodopseudomonas palustris strain N. This strain also indicated a high tolerance to SeO₃ ⁻². The finding of this work will contribute to the application of selenium to human health.     

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.     

Screening yeast cells for absorption of selenium oxyanions

Certain yeast cells on solid nutrient medium produced colonies surrounded by a light zone of selenite absorption. This screening procedure resulted in the selection of 22 strains out of 200 isolates with different Se⁴⁺-absorbing capacity ranging from 16 to 98.8 g Se⁴⁺ g^–1 l^–1 h^–1. The highest rate of Se⁴⁺ elimination from the Na₂SeO₃ solution was observed with an oval shaped, cream pigmented fermentative yeast, tentatively called Candida sp. strain MS4. This strain was isolated from wastewater and found to accumulate selenium oxyanions. Se⁴⁺ uptake involved both inactive and active phenomena. The amounts of selenium (initial concentration 2 mg Se⁴⁺ l^–1) removed from aqueous solution by inactive and active phenomena were 667 g Se⁴⁺ g^–1 l^–1, and 1580 g Se⁴⁺ g^–1 l^–1, respectively. The strain also removed selenate inactively (135 g Se⁶⁺ g^–1 l^–1).     

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.