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.     

Selenium Transport in Root Systems of Tomato

Selenate and selenite transport through tomato root systemswere followed for periods up to 4 h after removal of the planttops, using ⁷⁵Se as a tracer. With selenate, ⁷⁵Se concentrations in the xylem exudate were6 to 13 times higher than in the external solution, and chromatographicanalysis showed that the selenium was transported as inorganicselenate ( ). With selenite, ⁷⁵Se concentrations in the exudate were alwayslower than in the external solution. Analyses of exudate samplesshowed that negligible amounts of selenium were transportedas inorganic selenite ( except at very high external selenite concentrations (500 ?M), when up to 7 percent was transported as selenite. Most of the selenium transportin selenite-fed plants was as selenate or as an unknown seleniumcompound, the relative proportions of these two forms varyingboth with time and with external selenite concentration. Additionof a 5-fold excess of sulphate over selenite had no detectableeffect on the concentrations of selenate in the exudate, butcaused substantial decreases in the maximum concentrations ofboth total selenium (c. 47 per cent decrease) and the unknownselenium compound (c. 69 per cent decrease). Addition of a 5-foldexcess of sulphite decreased the concentration of the unknown(c. 39 per cent) but caused a large (2.7-fold) increase in themaximum total selenium concentration in the exudate and a 7.9-foldincrease in the maximum concentration of selenate. The resultssuggest metabolic involvement in the uptake and long distancetransport of solenium supplied as selenite, despite lower ⁷⁵Seconcentrations in the xylem exudate than in the external solution.An attempt is made to incorporate the new and existing informationinto a selenium transport model.     

Reduction of Selenite and Detoxification of Elemental Selenium by the Phototrophic Bacterium Rhodospirillum rubrum

The effect of selenite on growth kinetics, the ability of cultures to reduce selenite, and the mechanism of detoxification of selenium were investigated by using Rhodospirillum rubrum. Anoxic photosynthetic cultures were able to completely reduce as much as 1.5 mM selenite, whereas in aerobic cultures a 0.5 mM selenite concentration was only reduced to about 0.375 mM. The presence of selenite in the culture medium strongly affected cell division. In the presence of a selenite concentration of 1.5 mM cultures reached final cell densities that were only about 15% of the control final cell density. The cell density remained nearly constant during the stationary phase for all of the selenite concentrations tested, showing that the cells were not severely damaged by the presence of selenite or elemental selenium. Particles containing elemental selenium were observed in the cytoplasm, which led to an increase in the buoyant density of the cells. Interestingly, the change in the buoyant density was reversed after selenite reduction was complete; the buoyant density of the cells returned to the buoyant density of the control cells. This demonstrated that R. rubrum expels elemental selenium across the plasma membrane and the cell wall. Accordingly, electron-dense particles were more numerous in the cells during the reduction phase than after the reduction phase.     

Effects of growth conditions on production of methyl selenides in cultures of Rhodobacter sphaeroides

Rhodobacter sphaeroides 2.4.1 exposed to selenate or selenite produced volatile selenium compounds. Total amounts of dimethyl selenide, dimethyl diselenide, dimethyl sulfide and dimethyl disulfide in culture medium and headspace were determined. The highest selenate volatilization occurred in the late stationary phase of growth. However, cultures deprived of light in the stationary phase of growth produced much less of the volatile organo-selenium compounds. Lower culture pHs increased the rate of selenium volatilization. Low sulfate concentration limited biomass production and selenium volatilization; high sulfate concentrations had an enhancing effect on the release of organo-selenium compounds. Cultures of R. sphaeroides reacted very differently to amendments with increasing amounts of selenate and selenite. Only small amounts of selenite were volatilized; meanwhile high amounts of methylated selenides were found in selenate-poisoned cultures. Received 03 February 1997/ Accepted in revised form 16 May 1997     

Intraspecific variation in the selenium requirement of different geographic strains of the toxic dinoflagellate Gymnodinium catenatum

The requirement for selenium (IV) was assessed in five strainsof the toxic dinoflagellate Gymnodinium catenatum Graham, representingthree populations from Tasmania (Australia), as well as oneeach from Japan and Spain. Strains were grown in nutrient-enrichedsea water medium with 10^–9 M selenium added as selenite(H₂SeO₃), or with no added selenium, and monitored for growthand cell yield. Strains exhibited different selenium (Se) requirements,as evidenced by (i) a decrease in exponential growth rate (10–20%)and cell yield (up to 80%) (Japanese strain); (ii) a decreasein cell yield only (Tasmania Derwent Estuary 1987, TasmaniaHuon Estuary and Spanish strains); and (iii) no decrease ingrowth or cell yield (Tasmania Derwent Estuary 1993 strain).Variation in the response to Se deficiency was greatest betweenthe two strains isolated from the Derwent Estuary, Tasmania,in different years (1987 and 1993) and less between Tasmanianstrains from different localities (Huon and Derwent Estuariesare 50 km apart) or between Tasmanian and Japanese or Spanishstrains. Strain variability in micro-nutrient responses suchas described here may provide a partial explanation for differentbloom patterns exhibited by the same dinoflagellate taxon underapparently similar environmental conditions.     

Selenite-stress selected mutant strains of probiotic bacteria for Se source production

Selenium deficiency is a major health problem worldwide for about 1 billion people. Bacterial cells usually possess low tolerance to selenite stress and also low ability to reduce high concentrations of toxic selenite. Here, high tolerance to selenite and selenium bioaccumulation capability were developed in mutated clones of probiotic and starter bacteria including Enterococcus faecium, Bifidobacterium animalis ssp. lactis, Lactobacillus casei and Lactococcus lactis ssp. lactis by food-level strain development process and clone selection. All mutant clones possessed increased glutathione concentration and glutathione reductase activity. The selenite treatment increased further these values in L. casei mutant strain pointing at a different selenite reduction pathway and/or stress response in this organism. Considerable conversion of selenite to cell bound selenium forms with a concomitant high biomass production was detected in E. faecium and B. animalis ssp. lactis cultures. Possible application of these strains as food and feed supplements is under investigation.     

THE EFFECT OF SELENITE ON THE PHYSIOLOGICAL AND MORPHOLOGICAL PROPERTIES OF THE BLUE-GREEN ALGA PHORMIDIUM LURIDUM VAR. OLIVACEA1

Phormidium luridum cultures were treated with sodium selenite in concentrations ranging from 10^?6 M to 10^?2 M. In contrast to the increasing culture turbidity of control and 10^?6 M selenite cultures, the turbidity of the other selenite cultures declined in proportion to time and selenite concentration. Chlorophyll extraction revealed similar results. Photosynthetic activity was inhibited within 6 hr in all cultures except control and 10^?6 M selenite. Phormidium at concentrations greater than 10^?6 M selenite showed a gradual loss of the bright green color and turned semitransparent. Cell-associated granules of reduced selenium were observed at higher selenite concentrations. Other structural changes observed were the presence of intracellular and intercellular spaces, spheroplast formation, and gradual cell lysis. Protein analyses of total cell samples and supernatant fractions confirmed cellular breakdown of selenite-treated algal cells.     

Effects of Sodium Selenite on the Growth of Bracken Fern Root Callus

Sodium selenite promotes the growth of bracken fern root callusgrowth over a narrow, low range of concentrations, while higherconcentrations are strongly inhibitory. Pteridium aquilinum, bracken fern, sodium selenite, selenium, callus     

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.     

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.     

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%).     

Reduction of selenite and detoxification of elemental selenium by the phototrophic bacterium Rhodospirillum rubrum.

The effect of selenite on growth kinetics, the ability of cultures to reduce selenite, and the mechanism of detoxification of selenium were investigated by using Rhodospirillum rubrum. Anoxic photosynthetic cultures were able to completely reduce as much as 1. 5 mM selenite, whereas in aerobic cultures a 0.5 mM selenite concentration was only reduced to about 0.375 mM. The presence of selenite in the culture medium strongly affected cell division. In the presence of a selenite concentration of 1.5 mM cultures reached final cell densities that were only about 15% of the control final cell density. The cell density remained nearly constant during the stationary phase for all of the selenite concentrations tested, showing that the cells were not severely damaged by the presence of selenite or elemental selenium. Particles containing elemental selenium were observed in the cytoplasm, which led to an increase in the buoyant density of the cells. Interestingly, the change in the buoyant density was reversed after selenite reduction was complete; the buoyant density of the cells returned to the buoyant density of the control cells. This demonstrated that R. rubrum expels elemental selenium across the plasma membrane and the cell wall. Accordingly, electron-dense particles were more numerous in the cells during the reduction phase than after the reduction phase.     

Antagonistic effect of selenite on tumor promoter induced cell proliferation in cultures of rat tongue epithelium

In cultures of rat tongue epithelial cells, cell proliferation following incubation with different doses of the potent tumor promoter TPA has been studied by using a stathmokinetic method counting colchicine arrested metaphases. It was demonstrated that 24 h incubation with concentrations higher than 5 ng TPA/mL medium caused inhibition, whereas below 5 ng TPA/mL medium caused stimulation of the mitotic activity reaching a maximum around 30 h from the start of the incubation period. Based on the evidence of the anticarcinogenic effect of selenium in several animal models, experiments have been performed elucidating the influence of an atoxic dose (1/1.000.000M) of selenite on the observed TPA-induced cell proliferation. Our results indicate that addition to the culture medium of an atoxic dose of selenite, not affecting the mitotic activity of control cultures, inhibits the TPA-induced stimulation of cell proliferation.     

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.     

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.     

Uptake,distribution, and turnover rates of selenium in barley

The present communication elucidates initially the topographic distribution of selenium in barley grains. Then by the fluorimetric method the uptake of selenium (selenite) in 8–16 d old germinating barley was estimated. Finally by means of⁷⁵Se the anabolic and catabolic rates (turnover) of⁷⁵Se (selenite) was compared. The distribution of selenium in barley was evaluated after microdissection of barley grains. In dried grains the highest concentration was found in husk and pericarp with about 0.6 ppm Se. Then followed Scutellum with 0.4 and 0.3 ppm in embryon. The aleurone layer, embryonic leaves, and initial root did only have 0.2 ppm Se. In order to know more about the uptake and distribution of selenium in 8-d-old barley, the plants were cultivated for a further 8 d in the culture medium with variation in selenite concentration. In roots and leaves, the uptake did not arrive at saturation during the period studied since the dose-response curve increased up to 0.34 mM selenite in the medium, whereas the selenium levels were about 200 ppm in roots and 30 ppm in leaves. However, the uptake was linear, with concentration during 8 d of cultivation up to 0.84 μM selenite for grain and stem. At higher concentrations the dose-response curve diminished its slope. At 0.34 mM selenite the concentration in grain increased to 6.87 ppm and in the stem to 8.13 ppm. The uptake, distribution, and catabolic rate of selenium components in germinating barley were further evaluated by exposing the plants to 0.0492 μCi⁷⁵Se (12.6 μM selenite) for up to 4 d. Then the plants were moved to a selenium deficient medium for further 4 d. Then finally the medium was supplemented with high doses of cold selenite (0.126 mM selenite) for further 4 d. The first third period made it possible to estimate the rate of uptake. It was highest in roots (313 fmol/h/mg dw), i.e., about 10 times those of grains, stems, and leaves. The intermediate period where the barley was transferred to a selenium deficient medium made it possible to estimate the kinetics and eventual sparing mechanisms. The selenium losses were highest for leaves (39%), then followed by roots and stems (22 and 25%, respectively). The losses were lowest in grain with 9% Se losses. The losses were three times more pronounced during the first day than in the following 3 d. These data may argue that the selenium is distributed into different pools and that sparing mechanisms may be in function. The last period, i.e., the chase experiment, revealed the rate of elimination of selenium under conditions with surplus selenium. The catabolic rate was about 10 times faster in roots (169 fmol/h/mg dw) than in grains and about 8 times faster than in leaves.     

Efficient Preparation of Selenium/Glutathione-Enriched Candida utilis and Its Biological Effects on Rats

The main purpose of this study was to prepare selenium/glutathione-enriched Candida utilis and investigate its effect on growth performance, antioxidant capacity, and immune response in rats. The preparation of the selenium/glutathione-enriched yeast was conducted using fed-batch culture for high cell density. The optimal culture conditions for increased intracellular organic selenium and glutathione contents were as follows: the concentrated medium was fed beginning at 12?h using a polynomial feeding strategy until a total glucose concentration of 150?g/l was reached, and sodium selenite was continuously added together with glucose to a total concentration of 60?mg/l. As a result, 81?% of sodium selenite was assimilated and transformed into organic selenium by C. utilis under optimal conditions, which in turn resulted in greater glutathione accumulation and lower malondialdehyde cellular content in the yeast. To investigate and compare the effects of the prepared selenized C. utilis and other dietary supplements, 40 female rats were divided into five groups of eight rats each, following a randomized block design. Experimental feeding was conducted for a period of 6?weeks. Selenium supplementation with inorganic selenium (sodium selenite) and organic selenium (selenized C. utilis) showed better results than the control and other groups supplemented with yeast with or without glutathione. The body mass of rats, selenium deposition, and oxidative enzymes activities in both serum and liver samples, and immunity responses were all significantly improved by selenium supplementation, and between the two sources, organic selenium was more effective than inorganic selenium.     

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。     

Responses of an American eel brain endothelial-like cell line to selenium deprivation and to selenite,selenate, and selenomethionine additions in different exposure media

The effect of selenium deprivation and addition on the American eel brain endothelial cell line (eelB) was studied in three exposure media: complete growth medium (L15/FBS), serum-free medium (L15), and minimal medium (L15/ex). L15/ex contains only galactose and pyruvate and allowed the deprivation of selenium on cells to be studied. In L15/ex, without any obvious source of selenium, eelB cells survived for at least 7 d, formed capillary-like structures (CLS) on Matrigel, and migrated to heal wounds. Three selenium compounds were added to cultures: selenite, selenate, and selenomethionine (SeMet). Adding selenite or selenate to eelB cell cultures for 24 h caused dose-dependent declines in cell viability, regardless of the exposure media. Although varying with exposure media and viability end point, selenite was approximately 70-fold more cytotoxic than selenate. By contrast, 24 h exposures to either dl- or l-SeMet in the three media caused little or no cytotoxicity. However for 7 d exposures in L15/ex, dl- and l-SeMet were very cytotoxic, even at the lowest tested concentration of 31 μM. By contrast in L15 and L15/FBS, cytotoxicity was only observed with 500 and 1000 μM l-SeMet. In L15/FBS, eelB continued to migrate and form CLS in the presence of SeMet but at 500 μM, cell migration appeared stimulated. As judged from a colony-forming assay over 14 d in L15/FBS, 500 and 1000 μM dl- and l-SeMet inhibited cell proliferation. Overall, the responses of eel cells to selenium depended on the selenium form, concentration, and exposure media, with responses to SeMet being most dependent on exposure media.     

Expulsion of selenium/protein nanoparticles through vesicle-like structures by Saccharomyces cerevisiae under microaerophilic environment

Nano-selenium/protein is a kind of lower toxic supplement to human. Many microorganisms can reduce selenite/selenate to intracellular or extracellular selenium nanoparticles. This study examined the influence of dissolved oxygen on the expulsion of extracellular selenium/protein produced in Saccharomyces cerevisiae. More of the added selenite was reduced to extracellular selenium nanoparticles by yeast cells only under oxygen-limited condition than under aerobic or anaerobic condition. For the first time, we evidenced that selenium/protein nanoparticles synthesized in vivo were transported out of the cells by vesicle-like structures under microaerophilic environment. The characterizations of the extracellular spherical selenium/protein nanoparticles were also examined by SEM, TEM, EDX and FTIR.