TIBTEC-1255; No. of Pages 8
Opinion
Selenium biomineralization for biotechnological applications Yarlagadda V. Nancharaiah1,2 and Piet N.L. Lens1,3 1
Environmental Engineering and Water Technology Department, UNESCO-IHE Institute for Water Education, PO Box 3015, Delft DA 2601, The Netherlands 2 Biofouling and Biofilm Processes Section of Water and Steam Chemistry Division, Bhabha Atomic Research Centre, Kalpakkam, 603102, Tamil Nadu, India 3 Department of Chemistry and Bioengineering, Tampere University of Technology, PO Box 541, Tampere, Finland
Selenium (Se) is not only a strategic element in high-tech electronics and an essential trace element in living organisms, but also a potential toxin with low threshold concentrations. Environmental biotechnological applications using bacterial biomineralization have the potential not only to remove selenium from contaminated waters, but also to sequester it in a reusable form. Selenium biomineralization has been observed in phylogenetically diverse microorganisms isolated from pristine and contaminated environments, yet it is one of the most poorly understood biogeochemical processes. Microbial respiration of selenium is unique because the microbial cells are presented with both soluble (SeO42– and SeO32–) and insoluble (Se0) forms of selenium as terminal electron acceptor. Here, we highlight selenium biomineralization and the potential biotechnological uses for it in bioremediation and wastewater treatment. Selenium: a multifaceted element Selenium (Se) is a naturally occurring scarce element with significant importance in health and technological applications. It is unevenly distributed on the surface of the Earth, resulting in selenium-deficient and seleniferous geographical regions [1]. The selenium resources of the world need to be managed carefully because not only are they finite, but also the difference between the essential and toxic levels of selenium is just an order of magnitude. Anthropogenic activities, such as agricultural irrigation, coal and phosphate mining, coal combustion, and oil refining, have led to selenium pollution, resulting in fatal reproductive and teratogenic (see Glossary) defects in aquatic ecosystems, particularly in egg-laying vertebrates [2]. Apart from the well-perceived toxic impact of elevated selenium concentrations, trace amounts of selenium are equally increasingly well recognized for their beneficial role in essential metabolic functions and mitigating oxidative stress in living organisms (Box 1). Selenocysteine (Sec) has Corresponding authors: Nancharaiah, Y.V. ([email protected], [email protected]); Lens, P.N.L. ([email protected], [email protected]). Keywords: biomineralization; selenium bioreduction; selenium deficiency; selenium supplementation; selenium nanomaterials; wastewater treatment. 0167-7799/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2015.03.004
been recognized as the 21st amino acid, and a constituent of at least 25 proteins, named selenoproteins, present in all living systems, from Archaea and Bacteria, to Eukarya [3,4]. In humans, selenium takes part in several metabolic Glossary Anaerobic digestion: the process of microbial degradation of organic waste (e.g., food waste or waste biomass) coupled to the production of biogas under anaerobic conditions. The energy present in the waste is recovered as biogas (a mixture of CO2 and CH4) and microbial biomass. It is one of most efficient treatment technologies applied worldwide for the digestion of municipal sludge and food waste. Anaerobic respiration: the process in which prokaryotic organisms (i.e., bacteria and Archaea) obtain energy by transferring electrons to electron acceptors (e.g., nitrate, selenate, sulfate, etc.) for growth. In anaerobic respiration, selenium oxyanions are used as terminal electron acceptors and their reduction is linked to energy conservation and growth. Biofilms: microbial assemblages, typically comprising microbial cells in an extracellular polymeric substances matrix, often concentrated at solid–liquid interfaces. Aggregates of microbial communities that separate from the liquid by flocculation are referred to as ‘flocs’. Millimeter-scale microbial aggregates that separate from liquid as distinct particles under quiescent conditions are called ‘granular sludge’. Biofilms, flocs, and granular sludge share many similar characteristics and are all used in wastewater treatment. Biomineralization: the process in which living organisms produce minerals. The most ready examples of biominerals are magnetite crystals in bacteria, silicate shells in carbonate shells in invertebrates, and carbonates and phosphate teeth and bones in vertebrates. Cadmium selenide quantum dots: nanocrystals with a diameter of up to 10 nm. These nanocrystals exhibit a relation between particle size and electronic or optical properties. When the particle diameter becomes smaller than the exciton Bhor radius, a jump in electronic properties occurs due to quantum confinement. Detoxification: the ability to remove harmful agents (e.g., drugs or carcinogens) from the body. Microbes use various detoxification strategies to convert highly reactive and potentially toxic selenium oxyanions to the less reactive and less toxic elemental selenium (Se0). Extracellular polymeric substances (EPS): an integral component of biofilms, flocs, and granular sludge. EPS primarily comprise polysaccharides, proteins, lipids, and nucleic acids. EPS components can provide reaction and nucleation sites for biomineralization of metal(loid)s. Extracellular respiration: the process of extending the respiratory chain to the cell surface and beyond for reducing solid and/or soluble electron acceptors located close to or away from the cell surface. Microorganisms use soluble electron shuttles, outer membrane structures, conductive matrix, or other mechanisms for transferring electrons from cytoplasm to cell surface and beyond. Methanogenesis: the final step in anaerobic digestion. It is mainly performed by two groups of Archaea: hydrogenotrophic methanogens and acetoclastic methanogens. Hydrogenotrophic methanogens produce methane from H2 and CO2, while acetoclastic methanogens generate methane through acetate decarboxylation pathway. Under normal conditions, acetoclastic methanogens contribute to almost 70% of methane production in anaerobic digesters treating municipal sludge. Teratogenic agents: substances that can cause disturbance in the development of an embryo or fetus and lead to birth defects. Selenium at elevated concentrations is well known to produce teratogenic defects in fish and birds.
Trends in Biotechnology xx (2015) 1–8
1
TIBTEC-1255; No. of Pages 8
Opinion
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
Box 1. Selenium deficiency and excess Selenium-deficient regions, places with low natural selenium levels, are more widespread than those of seleniferous areas [53,54]. The toxicity of selenium was recognized in 1856, when it was found to be associated with the ‘alkali disease’, now termed ‘selenosis’. The essentiality of this element was not recognized until 1957. The margin of safety between the essential and toxic levels of selenium is narrow. In humans, daily allowance and upper tolerable limits are prescribed to enjoy the health benefits of selenium and to avoid toxicity (Figure I) [4,5]. The recommended dietary allowance and upper tolerable intake levels for adults are 55 and 400 mg per day, respectively. The relation between the selenium concentration and risk is U shaped, whereby positive effects are seen only with selenium supplementation of individuals with low selenium status. Individuals with an adequate-to-high selenium status should avoid taking selenium supplements [4], given that selenium supplements often contain 50–200 mg selenium per daily dose. Similar to animals and humans, the microbial communities of anaerobic wastewater treatment systems are vulnerable to selenium toxicity [55] and deficiency [6]. The IC50 values of selenium oxyanions were below 61 mM for hydrogenotrophic methanogens [55]. For acetoclastic methanogens, the IC50 values of 83 and 55 mM for selenite and selenate, respectively, were observed [55]. Selenium supplementation of anaerobic digesters (AD) treating food waste deficient in
selenium enabled stable operation at higher organic loading rates (Figure II) [6]. Accumulation of volatile fatty acids, such as propionate, was observed in selenium-deficient anaerobic digesters. A syntrophic association between acetate-oxidizing bacteria and hydrogenotrophic methanogens is needed for stable operation of ADs. Hydrogenases and formate dehydrogenses involved in propionate and formate oxidation require selenium as a cofactor [56]. In the event of selenium deficiency, accumulation of formate, an intermediate of propionate oxidation, triggers a feed-back inhibition resulting in propionate accumulation, which may lead to process failure in anaerobic digesters (Figure III).
Anaerobic digeson (AD) of food waste deficient in selenium No selenium supplementaon
Selenium supplementaon
Process failure in ADs due to propionate accumulaon
Stable operaon, improved performance of ADs
TRENDS in Biotechnology
Figure II. Selenium supplementation of anaerobic digesters treating seleniumdeficient food wastes.
Se deficiency
Food waste
Se excess 400 µg/day Syntrophic propionate oxidizers
Selenium deficiency below dietary allowance • Immune response impairment • Thyroid problems • Increased risk of cancer • Liver and pancreas cirrhosis • Cardiovascular diseases • Abnormal tooth decay • Keshan disease • Kashin-Beck disease
Selenium excess above upper tolerable limits • Skin discoloraon • Garlic odor on breath • Deformaon and loss of nails • Lack of mental alertness • Redness of skin, skin rash • Heart diseases • Selenosis
Propionate p
Acetogens
Se
Long chain volale v ffay acids (VFA)
Feedback inhibion
Formate Se
Formate dehydrogenase
CO2 + H2 Syntrophic acetate oxidizers Hydrogenotrophic methanogens
NH3
CO2 + CH4
Acetoclasc methanogens TRENDS in Biotechnology
Figure I. Effects of selenium deficiency and excess in animals and humans.
pathways, including thyroid hormone metabolism, immune responses, and antioxidant systems [5]. The maintenance of physiological selenium concentrations through an optimal diet or selenium supplements is considered a prerequisite to protect human health in selenium-deficient regions [5]. Over the past two decades, selenium research has gathered momentum within the scientific community because of the risks and environmental concern of elevated levels of selenium, its positive effect on the well-being of humans, animals and even microbial communities, and its use in photovoltaics [2,4–7]. Microbial communities have a pivotal role in the biogeochemical cycles of many elements, the containment of certain problematic elements through bioremediation, and 2
Acetate
TRENDS in Biotechnology
Figure III. Role of selenium in anaerobic wastewater treatment of food waste.
in shaping the geochemical environment [8]. Diverse groups of microorganism have the metabolic capability to turn selenium pollution and wastes into value-added materials, such as elemental selenium, metal selenide, and other bimetallic or organoselenium compounds. Compared with other bacteriogenic biominerals, such as magnetite (Fe3O4) and calcite (CaCO3), the biomineralization of selenium is not well understood, other than its potential in bioremediation and bionanofabrication [9]. The metabolic capacity of microorganisms has so far only been exploited to remove selenium oxyanions from contaminated water, with the aim of reducing environmental contamination [10,11], but little attention has been paid to the reuse of this valuable resource.
TIBTEC-1255; No. of Pages 8
Opinion
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
Bioreduction of selenium oxyanions Bacterial selenium biomineralization processes comprise two main steps: (i) reduction of selenium oxyanions and elemental selenium; and (ii) nucleation and assembly of selenium minerals, such as elemental selenium and metal selenides. The microbial selenate (SeO42–) reduction pathway transforms this oxyanion into the stable inorganic selenium [Se0] species, as given by Equation 1: 2 0 SeO2 4 ðaqÞ ! SeO3 ðaqÞ ! Se ðsÞ ! HSe ðaqÞ
[1]
Reduction of selenium oxyanions by microorganisms has been observed under aerobic, microaerophilic, and anaerobic conditions [11–14]. Anaerobic respiration of selenate by microorganisms was discovered almost two decades ago [15,16]. However, few microorganisms that use selenium (SeO42–, SeO32–, or Se0) as the terminal electron acceptor in anaerobic respiration have been isolated and studied in pure culture [11,13]. The bioreduction of SeO42– to SeO32– is primarily catalyzed by either a soluble or membrane-bound selenate reductase (Ser) (Figure 1). Ser characterized so far comprise three subunits with molybdenum as a co-factor, located either in the periplasm or on the cytoplasmic membrane [17–19]. The product of Ser, SeO32–, is always released in the periplasm or outside the cytoplasmic membrane in Gram-negative and Grampositive bacteria, respectively. Unlike selenate, the reduction of SeO32– to Se0 has been observed in a range of microorganisms under aerobic and anaerobic growth conditions. Microbial conversion of SeO32– to Se0 is widely recognized as a detoxification strategy, whereby the toxic and soluble oxyanion is converted to solid Se0. Various biomolecules facilitate the conversion of SeO32– to Se0 in microbial cells, including glutathione, glutaredoxin, and siderophores [13]. SeO32–
reduction in the cytoplasm is often driven by reduced thiols, such as glutathione and glutaredoxin, which are abundant in microorganisms. In addition, terminal reductases of anaerobic respiration, such as nitrite reductase, sulfite reductase, and fumarate reductase, catalyze SeO32– reduction [11,20]. Nevertheless, selenite reduction in the periplasm or on the cell membrane cannot be ignored because glutathione is known to be exported to the periplasm via ABC-type transporters [21] or via a newly discovered periplasmic fumarate reductase-mediated detoxification [20]. Selenosphere assembly and export out the cell The selenium atoms formed during selenite reduction nucleate to form Se0 allotropes and grow in a spherical shape. Imaging of bacterial cultures and microbial communities with electron microscopy showed selenospheres (selenium particles) in the cytoplasm, on the cell surface, and in the surrounding medium. Interestingly, the selenospheres observed on the cell surface are much smaller in diameter (
Opinion
Selenium biomineralization for biotechnological applications Yarlagadda V. Nancharaiah1,2 and Piet N.L. Lens1,3 1
Environmental Engineering and Water Technology Department, UNESCO-IHE Institute for Water Education, PO Box 3015, Delft DA 2601, The Netherlands 2 Biofouling and Biofilm Processes Section of Water and Steam Chemistry Division, Bhabha Atomic Research Centre, Kalpakkam, 603102, Tamil Nadu, India 3 Department of Chemistry and Bioengineering, Tampere University of Technology, PO Box 541, Tampere, Finland
Selenium (Se) is not only a strategic element in high-tech electronics and an essential trace element in living organisms, but also a potential toxin with low threshold concentrations. Environmental biotechnological applications using bacterial biomineralization have the potential not only to remove selenium from contaminated waters, but also to sequester it in a reusable form. Selenium biomineralization has been observed in phylogenetically diverse microorganisms isolated from pristine and contaminated environments, yet it is one of the most poorly understood biogeochemical processes. Microbial respiration of selenium is unique because the microbial cells are presented with both soluble (SeO42– and SeO32–) and insoluble (Se0) forms of selenium as terminal electron acceptor. Here, we highlight selenium biomineralization and the potential biotechnological uses for it in bioremediation and wastewater treatment. Selenium: a multifaceted element Selenium (Se) is a naturally occurring scarce element with significant importance in health and technological applications. It is unevenly distributed on the surface of the Earth, resulting in selenium-deficient and seleniferous geographical regions [1]. The selenium resources of the world need to be managed carefully because not only are they finite, but also the difference between the essential and toxic levels of selenium is just an order of magnitude. Anthropogenic activities, such as agricultural irrigation, coal and phosphate mining, coal combustion, and oil refining, have led to selenium pollution, resulting in fatal reproductive and teratogenic (see Glossary) defects in aquatic ecosystems, particularly in egg-laying vertebrates [2]. Apart from the well-perceived toxic impact of elevated selenium concentrations, trace amounts of selenium are equally increasingly well recognized for their beneficial role in essential metabolic functions and mitigating oxidative stress in living organisms (Box 1). Selenocysteine (Sec) has Corresponding authors: Nancharaiah, Y.V. ([email protected], [email protected]); Lens, P.N.L. ([email protected], [email protected]). Keywords: biomineralization; selenium bioreduction; selenium deficiency; selenium supplementation; selenium nanomaterials; wastewater treatment. 0167-7799/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2015.03.004
been recognized as the 21st amino acid, and a constituent of at least 25 proteins, named selenoproteins, present in all living systems, from Archaea and Bacteria, to Eukarya [3,4]. In humans, selenium takes part in several metabolic Glossary Anaerobic digestion: the process of microbial degradation of organic waste (e.g., food waste or waste biomass) coupled to the production of biogas under anaerobic conditions. The energy present in the waste is recovered as biogas (a mixture of CO2 and CH4) and microbial biomass. It is one of most efficient treatment technologies applied worldwide for the digestion of municipal sludge and food waste. Anaerobic respiration: the process in which prokaryotic organisms (i.e., bacteria and Archaea) obtain energy by transferring electrons to electron acceptors (e.g., nitrate, selenate, sulfate, etc.) for growth. In anaerobic respiration, selenium oxyanions are used as terminal electron acceptors and their reduction is linked to energy conservation and growth. Biofilms: microbial assemblages, typically comprising microbial cells in an extracellular polymeric substances matrix, often concentrated at solid–liquid interfaces. Aggregates of microbial communities that separate from the liquid by flocculation are referred to as ‘flocs’. Millimeter-scale microbial aggregates that separate from liquid as distinct particles under quiescent conditions are called ‘granular sludge’. Biofilms, flocs, and granular sludge share many similar characteristics and are all used in wastewater treatment. Biomineralization: the process in which living organisms produce minerals. The most ready examples of biominerals are magnetite crystals in bacteria, silicate shells in carbonate shells in invertebrates, and carbonates and phosphate teeth and bones in vertebrates. Cadmium selenide quantum dots: nanocrystals with a diameter of up to 10 nm. These nanocrystals exhibit a relation between particle size and electronic or optical properties. When the particle diameter becomes smaller than the exciton Bhor radius, a jump in electronic properties occurs due to quantum confinement. Detoxification: the ability to remove harmful agents (e.g., drugs or carcinogens) from the body. Microbes use various detoxification strategies to convert highly reactive and potentially toxic selenium oxyanions to the less reactive and less toxic elemental selenium (Se0). Extracellular polymeric substances (EPS): an integral component of biofilms, flocs, and granular sludge. EPS primarily comprise polysaccharides, proteins, lipids, and nucleic acids. EPS components can provide reaction and nucleation sites for biomineralization of metal(loid)s. Extracellular respiration: the process of extending the respiratory chain to the cell surface and beyond for reducing solid and/or soluble electron acceptors located close to or away from the cell surface. Microorganisms use soluble electron shuttles, outer membrane structures, conductive matrix, or other mechanisms for transferring electrons from cytoplasm to cell surface and beyond. Methanogenesis: the final step in anaerobic digestion. It is mainly performed by two groups of Archaea: hydrogenotrophic methanogens and acetoclastic methanogens. Hydrogenotrophic methanogens produce methane from H2 and CO2, while acetoclastic methanogens generate methane through acetate decarboxylation pathway. Under normal conditions, acetoclastic methanogens contribute to almost 70% of methane production in anaerobic digesters treating municipal sludge. Teratogenic agents: substances that can cause disturbance in the development of an embryo or fetus and lead to birth defects. Selenium at elevated concentrations is well known to produce teratogenic defects in fish and birds.
Trends in Biotechnology xx (2015) 1–8
1
TIBTEC-1255; No. of Pages 8
Opinion
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
Box 1. Selenium deficiency and excess Selenium-deficient regions, places with low natural selenium levels, are more widespread than those of seleniferous areas [53,54]. The toxicity of selenium was recognized in 1856, when it was found to be associated with the ‘alkali disease’, now termed ‘selenosis’. The essentiality of this element was not recognized until 1957. The margin of safety between the essential and toxic levels of selenium is narrow. In humans, daily allowance and upper tolerable limits are prescribed to enjoy the health benefits of selenium and to avoid toxicity (Figure I) [4,5]. The recommended dietary allowance and upper tolerable intake levels for adults are 55 and 400 mg per day, respectively. The relation between the selenium concentration and risk is U shaped, whereby positive effects are seen only with selenium supplementation of individuals with low selenium status. Individuals with an adequate-to-high selenium status should avoid taking selenium supplements [4], given that selenium supplements often contain 50–200 mg selenium per daily dose. Similar to animals and humans, the microbial communities of anaerobic wastewater treatment systems are vulnerable to selenium toxicity [55] and deficiency [6]. The IC50 values of selenium oxyanions were below 61 mM for hydrogenotrophic methanogens [55]. For acetoclastic methanogens, the IC50 values of 83 and 55 mM for selenite and selenate, respectively, were observed [55]. Selenium supplementation of anaerobic digesters (AD) treating food waste deficient in
selenium enabled stable operation at higher organic loading rates (Figure II) [6]. Accumulation of volatile fatty acids, such as propionate, was observed in selenium-deficient anaerobic digesters. A syntrophic association between acetate-oxidizing bacteria and hydrogenotrophic methanogens is needed for stable operation of ADs. Hydrogenases and formate dehydrogenses involved in propionate and formate oxidation require selenium as a cofactor [56]. In the event of selenium deficiency, accumulation of formate, an intermediate of propionate oxidation, triggers a feed-back inhibition resulting in propionate accumulation, which may lead to process failure in anaerobic digesters (Figure III).
Anaerobic digeson (AD) of food waste deficient in selenium No selenium supplementaon
Selenium supplementaon
Process failure in ADs due to propionate accumulaon
Stable operaon, improved performance of ADs
TRENDS in Biotechnology
Figure II. Selenium supplementation of anaerobic digesters treating seleniumdeficient food wastes.
Se deficiency
Food waste
Se excess 400 µg/day Syntrophic propionate oxidizers
Selenium deficiency below dietary allowance • Immune response impairment • Thyroid problems • Increased risk of cancer • Liver and pancreas cirrhosis • Cardiovascular diseases • Abnormal tooth decay • Keshan disease • Kashin-Beck disease
Selenium excess above upper tolerable limits • Skin discoloraon • Garlic odor on breath • Deformaon and loss of nails • Lack of mental alertness • Redness of skin, skin rash • Heart diseases • Selenosis
Propionate p
Acetogens
Se
Long chain volale v ffay acids (VFA)
Feedback inhibion
Formate Se
Formate dehydrogenase
CO2 + H2 Syntrophic acetate oxidizers Hydrogenotrophic methanogens
NH3
CO2 + CH4
Acetoclasc methanogens TRENDS in Biotechnology
Figure I. Effects of selenium deficiency and excess in animals and humans.
pathways, including thyroid hormone metabolism, immune responses, and antioxidant systems [5]. The maintenance of physiological selenium concentrations through an optimal diet or selenium supplements is considered a prerequisite to protect human health in selenium-deficient regions [5]. Over the past two decades, selenium research has gathered momentum within the scientific community because of the risks and environmental concern of elevated levels of selenium, its positive effect on the well-being of humans, animals and even microbial communities, and its use in photovoltaics [2,4–7]. Microbial communities have a pivotal role in the biogeochemical cycles of many elements, the containment of certain problematic elements through bioremediation, and 2
Acetate
TRENDS in Biotechnology
Figure III. Role of selenium in anaerobic wastewater treatment of food waste.
in shaping the geochemical environment [8]. Diverse groups of microorganism have the metabolic capability to turn selenium pollution and wastes into value-added materials, such as elemental selenium, metal selenide, and other bimetallic or organoselenium compounds. Compared with other bacteriogenic biominerals, such as magnetite (Fe3O4) and calcite (CaCO3), the biomineralization of selenium is not well understood, other than its potential in bioremediation and bionanofabrication [9]. The metabolic capacity of microorganisms has so far only been exploited to remove selenium oxyanions from contaminated water, with the aim of reducing environmental contamination [10,11], but little attention has been paid to the reuse of this valuable resource.
TIBTEC-1255; No. of Pages 8
Opinion
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
Bioreduction of selenium oxyanions Bacterial selenium biomineralization processes comprise two main steps: (i) reduction of selenium oxyanions and elemental selenium; and (ii) nucleation and assembly of selenium minerals, such as elemental selenium and metal selenides. The microbial selenate (SeO42–) reduction pathway transforms this oxyanion into the stable inorganic selenium [Se0] species, as given by Equation 1: 2 0 SeO2 4 ðaqÞ ! SeO3 ðaqÞ ! Se ðsÞ ! HSe ðaqÞ
[1]
Reduction of selenium oxyanions by microorganisms has been observed under aerobic, microaerophilic, and anaerobic conditions [11–14]. Anaerobic respiration of selenate by microorganisms was discovered almost two decades ago [15,16]. However, few microorganisms that use selenium (SeO42–, SeO32–, or Se0) as the terminal electron acceptor in anaerobic respiration have been isolated and studied in pure culture [11,13]. The bioreduction of SeO42– to SeO32– is primarily catalyzed by either a soluble or membrane-bound selenate reductase (Ser) (Figure 1). Ser characterized so far comprise three subunits with molybdenum as a co-factor, located either in the periplasm or on the cytoplasmic membrane [17–19]. The product of Ser, SeO32–, is always released in the periplasm or outside the cytoplasmic membrane in Gram-negative and Grampositive bacteria, respectively. Unlike selenate, the reduction of SeO32– to Se0 has been observed in a range of microorganisms under aerobic and anaerobic growth conditions. Microbial conversion of SeO32– to Se0 is widely recognized as a detoxification strategy, whereby the toxic and soluble oxyanion is converted to solid Se0. Various biomolecules facilitate the conversion of SeO32– to Se0 in microbial cells, including glutathione, glutaredoxin, and siderophores [13]. SeO32–
reduction in the cytoplasm is often driven by reduced thiols, such as glutathione and glutaredoxin, which are abundant in microorganisms. In addition, terminal reductases of anaerobic respiration, such as nitrite reductase, sulfite reductase, and fumarate reductase, catalyze SeO32– reduction [11,20]. Nevertheless, selenite reduction in the periplasm or on the cell membrane cannot be ignored because glutathione is known to be exported to the periplasm via ABC-type transporters [21] or via a newly discovered periplasmic fumarate reductase-mediated detoxification [20]. Selenosphere assembly and export out the cell The selenium atoms formed during selenite reduction nucleate to form Se0 allotropes and grow in a spherical shape. Imaging of bacterial cultures and microbial communities with electron microscopy showed selenospheres (selenium particles) in the cytoplasm, on the cell surface, and in the surrounding medium. Interestingly, the selenospheres observed on the cell surface are much smaller in diameter (
