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Selenium biomineralization for biotechnological applications
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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.
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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
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Figure II. Selenium supplementation of anaerobic digesters treating seleniumdeficient food wastes.
Se deficiency
Food waste
Se excess <55 µg/day
d ffood waste Hydrolyzed
>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
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Feedback inhibion
Formate Se
Formate dehydrogenase
CO2 + H2 Syntrophic acetate oxidizers Hydrogenotrophic methanogens
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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
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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.
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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 (<50 nm) than those found in the cytoplasm [20]. Most often, selenospheres found in the extracellular environment are abundant. By contrast, only a few fairly large selenospheres, sometimes almost the diameter of the bacterial cell itself, are found in the cytoplasm. Reduction of selenate to selenite occurs primarily in the periplasm and is catalyzed by Ser (Figure 1). By contrast, reduction of SeO32– is mostly driven by detoxification mechanisms that operate in the cytoplasm; thus, formation of Se0 atoms and subsequent selenosphere assembly occur in the cytoplasm. To avoid accumulation of selenospheres and associated necrosis, the cells must have a mechanism to export these intracellularly formed selenospheres. An export system was proposed for the
SeO42– OM
SeO42–
Q-pool
SeO32–
SeO32–
FccA e–
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cytC4 e–
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SerA SerB SerC
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?
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?
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SeO32–
SeO32–
Gram-negave bacteria
CysT CysW CysA
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SefA
Cytoplasm
Gram-posive bacteria TRENDS in Biotechnology
Figure 1. Schematic diagram showing the various cellular processes involved in the microbial reduction of selenium oxyanions through anaerobic respiration and detoxification, and selenium nanosphere assembly and export [17–19,22]. Reduction of selenate is catalyzed by either a soluble or membrane-bound selenate reductase (SER). Abbreviations: CM, cytoplasmic membrane; OM, outer membrane; SefA, Selenium factor A protein; SerABC, selenate reductase; SrdBCA, selenate reductase; cytC4, cytochrome C4; CymA, c-type cytochrome; FccA, fumarate reductase; CysTWA, sulphate permease; Q-pool, quinone pool.
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Opinion transport of selenospheres formed in the cytoplasm of Thauera selenatis [22,23]. Selenium factor A (SefA) is a 95-kDa protein associated with the assembly and secretion of the selenospheres. The function of SefA in selenosphere assembly is comparable to that of the well-studied protein Mms6, which controls the size and morphology of magnetite crystals inside magnetotactic bacteria [8]. However, the big question remains: how are these large selenospheres formed in the cytoplasm excreted to the extracellular medium (Box 2)? It has been postulated that the SefA selenospheres are translocated directly from the cytoplasm of T. selenatis [22,23], because of the lack of evidence of outer membrane distortion or bulging, and the absence of periplasmic material with the secreted selenospheres. However, the extent of SeO32– reduction that occurs either in the periplasm or on the cell membrane via detoxification and respiration is unknown. Biogenic synthesis of selenium nanomaterials The use of microorganisms in the green synthesis of nanomaterials is attractive mainly because microorganisms are inexpensive catalysts, precursor materials are usually sourced from inexpensive raw materials and waste streams, the synthesis occurs at near neutral pH, ambient temperature, and pressure, and the use of hazardous reducing agents is avoided [24–26]. Thus, selenium-reducing bacteria have an as yet unexploited potential for the biosynthesis of both elemental selenium and metal selenide nanomaterials under ambient conditions using selenium precursors from inexpensive raw materials or waste streams [27,28]. The potential for bionanofabrication is well documented, but so far few studies have demonstrated the functionality of biogenic selenium nanomaterials and addressed the scale-up issues. Nanoscale elemental selenium Despite the differences in mechanisms of biomineralization, microbially produced Se0 atoms always form spherical nanospheres with a diameter of up to 400 nm, rather than large crystals. In general, smaller-diameter Se particles are observed during the early phases of inoculation
Box 2. Outstanding questions What governs the size of selenospheres formed by microorganisms? Is selenosphere assembly controlled by biomolecules? How are selenospheres formed in the cytoplasm exported? Are membrane vesicles involved in shedding mineral deposits? Do we have enough understanding of the origin and biochemical nature of organic layer associated with selenium biominerals? Are elemental selenium particles reduced through extracellular respiration by microorganisms? What are the effects of selenium biominerals on microbial communities and on long-term stability of bioreduction? What is the effect of engineered selenium nanomaterials on the structure and function of microbial communities? How toxic are the effluents of selenium treatment systems? Is biomineralization from selenium wastewaters the answer to meet selenium scarcity? Is the production of metal selenides, such as quantum dots, scalable using microorganisms? 4
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[29]. Phenazine-1-carboxylic acid, a metabolite produced by Pseudomonas aeruginosa JS-11, enabled SeO32– reduction and formation of monodisperse selenospheres with an average size of 21 nm [30]. The average diameter and polydispersity index of selenium particles increased during prolonged incubation in bacterial cultures [29,31,32]. Se0 is known to exist in three structural arrangements: infinite a-helical chains, and six- and eight-member rings [9]. These are the nucleating centers for the newly formed Se0 atoms. Large selenospheres are formed though Ostwald ripening, wherein the growth occurs through newly formed selenium atoms through subsequent reduction of selenium oxyanions and selenium atoms released through disassembly of other particles following the Gibbs-Thompson law and aggregation [33]. The size of the Se0 particles grows until capping agents, such as proteins, polysaccharides, phospholipids, or extracellular polymeric substances (EPS), inhibit further growth and aggregation. The polydispersity and large particle size occur because of the large number of nucleating sites and limited availability of capping agents. In some cases, transformation of selenospheres to trigonal selenium (t-Se) nanorods was observed, probably through the disassembly and assembly of Se0 atoms due to destabilization of the capping agent [34]. The precise controlling of shape and size is possible only if capping or stabilizing agents are available in sufficient amounts. Similar to biologically produced elemental sulfur [35], biogenic selenospheres are always associated with an organic layer that changes the physicochemical characteristics of solid selenium and influences their fate in the environment [36]. The colloidal stability of the biogenic selenospheres is conferred by the organic layer, which gives a net negative charge and prevents aggregation and sedimentation of selenium particles [37,38]. Attempts are being made to trace the biochemical nature and origin of the organic layer associated with biogenic selenospheres. In vitro experiments have shown that Se0 nanoparticles can be stabilized against crystallization by proteins [32,39] or EPS [36,39]. Lenz et al. [39] identified high-affinity proteins associated with Se0 nanospheres formed by selenium-respiring bacteria. The components of the organic layer could originate from the enzymes, outer membrane, or EPS components, which are involved in the reduction selenium oxyanions, assembly, or export of selenospheres. The transformation of microbially produced selenospheres into other Se0 forms (e.g., nanowires) has been demonstrated in vitro [40]. Selenospheres formed by Shewanella sp. HN-41 were incubated along with cells in dimethyl sulfoxide to transform spheres into long, thin Se0 nanoribbons and nanowires. This shows that removal of the stabilization caused by the organic layer enables the growth of selenospheres into other Se0 crystal types, which opens doors for new technological applications. Nanoscale metal selenides The bioreduction of selenium oxyanions beyond selenospheres and up to HSe– has so far been noticed only in sediments [41], and a few axenic bacterial cultures [42]. The metal-reducing bacterium Veillonella atypica has been
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used to reduce selenite up to HSe– for producing 30-nm zinc selenide (ZnSe) and cadmium selenide (CdSe) particles [42]. The use of biogenic HSe– enabled a controlled reaction rate during metal selenide synthesis, which resulted in a narrow size distribution of CdSe nanocrystals (diameter <8 nm) compared with chemical synthesis [27]. Formation of CdSe nanocrystals with a diameter of 8–11 nm was observed when growing Escherichia coli cells incubated with SeO32– and CdCl2 [43]. In these cells, a reaction between an organo selenium compound (R-Se) and Cd2+ was proposed for the formation of CdSe nuclei. The further growth of metal selenide nuclei will lead to the formation of metal selenide colloids. The nature of proteins or other biomolecules in achieving the narrow size distribution by controlling the nucleation and crystal growth rates are not known. The bacterial production of metal selenides offers a green process that not only avoids the use of toxic and expensive chemical precursors, but also introduces the use of a novel selenium and heavy metal recovery process from waste streams. However, bioengineering the bioreduction of selenium oxyanions beyond elemental selenium appears
to be challenging. Unlike Se oxyanions, Se0 is a solid electron acceptor and its reduction via anaerobic respiration might require the involvement of electron shuttles or outer membrane components. The rate of bacterial reduction of selenium oxyanions to HSe– needs to be improved several-fold to make it a viable and scalable bioprocess for metal selenide synthesis. Bioremediation and wastewater treatment Selenium wastewaters must be treated to abate selenium pollution and to comply with discharge limits. According to the US Environmental Protection Agency (EPA), the selenium content of wastewaters must be brought down to below 5 mg l–1 before discharging to the environment. There is a strong debate about whether this limit is safe enough for protecting ecosystem well-being, mainly because selenium is known to transfer across trophic levels and accumulate in biota. Stringent discharge limits for selenium wastewaters generated in coal-fired power plants are foreseen in the near future, demanding the development of efficient selenium treatment technologies [2].
Suspended growth Algal pond [microalgae/cyanobacteria]
Reducon pond [anaerobic bacteria]
Floc
FBR Fluidized bed reactor
Nutrients Sand
Biological selenium removal systems
Carrier material [Biofilm]
Biofilm
Wash waste supernatant
Wash waste tank
Plug flow reactor
PFR
Backwash tank
Molasses Biofilm
Solids
Backwash
Granular acvated carbon
MBfR H2
Biofilm
Aached growth
Resistor
Anode Biofilm
CO2
H+
Cathode
BES
Moxd
Membrane
Organic maer
Anode
Hollow fiber
Mred
UASB No carrier material [Parculate Biofilm]
Nutrient Anaerobic granule TRENDS in Biotechnology
Figure 2. Bioreactor configurations and biomass retention principles of biological selenium removal systems [10,13,48,50,52,57]. Red arrows indicate an inlet, whereas green arrows indicate an outlet. Abbreviations: BES, bioelectrochemical system; FBR, fluidized bed reactor; MBfR, membrane biofilm reactor; Moxd, metal(loid) oxidized; Mred, metal(loid) reduced; PFR, plug flow reactor; UASB, upflow anaerobic sludge blanket reactor.
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Biological selenium removal methods are attractive because of the wastewater characteristics (e.g., dilute and large volume) as well as low capital and operational costs. Most technologies are based on bacterial metabolism, although fungal systems can also be considered for low pH wastewaters [44]. Furthermore, photobioreactors are emerging for the inclusion of microalgae as part of integrated algal–bacterial selenium removal systems [45], wherein selenium oxyanions are removed by biovolatalization and bioreduction [45]. Various biotechnologies using microbial reduction have been developed for removing selenium oxyanions in conjunction with water reuse and selenium recovery. Selenate reduction has been investigated under methanogenic [46,47], sulfate reducing [48], denitrifying [49], and hydrogenotrophic [50] conditions. Different bioreactor configurations have been adopted, such as upflow anaerobic sludge blanket reactors, fluidized bed reactors, plug flow reactors, membrane biofilm reactors, and bioelectrochemical systems (Figure 2) for retaining selenium-reducing microorganisms as flocs, granular sludge, or biofilms. Some of these technologies have already been applied at full scale for selenium removal from wastewater. Although the microbial reduction is proven for converting soluble selenium oxyanions into elemental selenium, the treatment remains challenging because of the stringent discharge limits, occurrence of co-contaminants (e.g., metals and competing electron acceptors), and the handling of the concentrated selenium solids to avoid re-release and hazardous waste classification. Biological treatment relies on the enrichment and retention of microorganisms that convert soluble selenium oxyanions into the less-toxic elemental selenium,
Selenium-containing wastewater
Agriculture drainage waters
Microbial selenium transformaons Selenate (aq) [Se (VI)]
Selenite (aq) [Se(IV)]
which is decanted from the wastewater by gravity settling or filtration. Given its colloidal nature [36], a significant fraction of the bioreduced selenium can still be present in the bioreactor effluent, necessitating a post treatment step, such as coagulation [38] or electrocoagulation [51]. The performance of selenium-removing bioreactors is governed by the fraction of the bioreduced selenium that has colloidal properties. The fate of this fraction impacts the treatment efficiency, thereby compromising the selenium discharge limit. Bioelectrochemical systems (BES) are emerging as attractive options especially for recovering metals from wastewaters. In BES, an organic electron donor is oxidized in the anode chamber using anode-respiring bacteria and the electrons are used for reductive precipitation of metal(loid)s in the cathode chamber via direct cathodes or biocathodes with or without external power supply [52]. It will be worth investigating these novel systems for selenium removal and recovery. The biological treatment methods may be combined with physicochemical post-treatment (e.g., ultrafiltration and coagulation) to achieve the discharge limits. However, this increases the overall treatment cost, although the effluent discharge will comply with the regulatory guidelines, thus avoiding penalties due to selenium pollution. In addition, selenium recovery from wastewater is desirable to offset the treatment costs and to make this valuable scarce element available for reuse (Figure 3). There is a need to test and evaluate the recovered selenium as a resource for applications in the formulation of food supplements, antimicrobials, crop fortification agents, and as base material for selenium materials (Figure 3).
Bioreactor
Selenium (s) (Se0) Recovery for reuse
Selenomethionineenriched yeast, microalgae
Leachates from contaminated soils
Applicaons • Anmicrobials • Ferlizers • Semiconductors • Sensors
Heavy metals (Cd2+, Zn2+, Pb2+)
Selenide (aq) [Se(-II)] Metal selenides (CdSe, ZnSe, PbSe) Applicaons • Electronics • Imaging • Photovoltaics TRENDS in Biotechnology
Figure 3. Schematic showing the possible applications of selenium biotechnology and selenium recovery options as well as reuse applications for the recovered selenium.
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Opinion Concluding remarks and future perspectives Biomineralization combines the treatment of selenium wastewaters with the potential to remove, recover, and reuse selenium in the form of selenium biominerals. Microbial reduction is a proven technology for converting soluble selenium oxyanions to insoluble forms to remove the pollution from the water. Treatment of selenium wastewaters is nevertheless challenging because the bioreduced selenium forms a colloidal suspension for which additional separation steps are required. When retained, the bioreduced selenium is a valuable resource as starting material for selenium materials, antimicrobial formulations, or as precursor of crop fortification agents and supplements. To enable rapid progress in the application of microbial nanofactories and biomimetic approaches for functionalized selenium nanomaterials along with potential recovery strategies for meeting selenium scarcity, the following aspects need to be addressed: (i) elucidating the biochemical pathways involved in selenosphere production as a function of its location. For full-scale applications in bioreactors, extracellular formation of large, well-settling Se0 or metal selenide particles is desired; (ii) identification of the proteins or other biomolecules that govern the size and surface properties of biogenic selenium in bacterial cultures and complex microbial communities. Once known, intelligent biomineralization systems can be developed by varying the biomolecule concentration to induce or stop the crystallization process; and (iii) development of integrated treatment strategies for the treatment of selenium wastewaters and selenium recovery, including a physicochemical post-treatment step subsequent to biological reduction. Acknowledgments We thank our past and present coworkers of UNESCO-IHE, Wageningen University (the Netherlands), and Bhabha Atomic Research Centre (India) as well as our national and international collaborators. We also thank our national and international granting agencies, in particular the BioMatch project (project 103922), funded by an EU Marie Curie International Incoming Fellowship.
References 1 Vriens, B. et al. (2014) Natural wetland emissions of methylated trace elements. Nat. Commun. 5, 3035 2 Lemly, A.D. (2014) An urgent need for an EPA standard for disposal of coal ash. Environ. Pollut. 191, 253–255 3 Papp, L.V. et al. (2007) From selenium to selenoproteins: synthesis, identity, and their role in human health. Antioxid. Redox Signal. 9, 775–806 4 Rayman, M.P. (2012) Selenium and human health. Lancet 379, 1256– 1268 5 Duntas, L.H. and Benvenga, S. (2014) Selenium: an element for life. Endocrine 48, 756–775 6 Banks, C.J. et al. (2012) Trace element requirements for stable food waste digestion at elevated ammonia concentrations. Bioresour. Technol. 104, 127–135 7 Boyd, R. (2011) Selenium stories. Nat. Chem. 3, 570 8 Rahn-Lee, L. and Komeili, A. (2013) The magnetosome model: insights into the mechanisms of bacterial biomineralization. Front. Microbiol. 4, 352 9 Oremland, R.S. et al. (2004) Structural and spectral features of selenium nanospheres produced by Se-respiring bacteria. Appl. Environ. Microbiol. 70, 52–60 10 Lenz, M. and Lens, P.N. (2009) The essential toxic: the changing perception of selenium in environmental sciences. Sci. Total Environ. 407, 3620–3633
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11 Stolz, J.F. et al. (2006) Arsenic and selenium in microbial metabolism. Annu. Rev. Microbiol. 60, 107–130 12 Williams, K.H. et al. (2013) Field evidence of selenium bioreduction in a uranium-contaminated aquifer. Environ. Microbiol. Rep. 5, 444–452 13 Nancharaiah, Y.V. and Lens, P.N.L. (2015) The ecology and biotechnology of selenium-respiring bacteria. Microbiol. Mol. Biol. Rev. 79, 61–80 14 Zannoni, D. et al. (2008) The bacterial response to the chalcogen metalloids Se and Te. Adv. Microb. Physiol. 53, 1–71 15 Macy, J.M. et al. (1989) Selenate reduction by a Pseudomonas species: a new mode of anaerobic respiration. FEMS Microbiol. Lett. 61, 195–198 16 Oremland, R.S. et al. (1989) Selenate reduction to elemental selenium by anaerobic bacteria in sediments and culture: biogeochemical significance of a novel, sulfate-independent respiration. Appl. Environ. Microbiol. 55, 2333–2343 17 Kuroda, M. et al. (2011) Molecular cloning and characterization of the srdBCA operon, encoding the respiratory selenate reductase complex, from the selenate-reducing bacterium Bacillus selenatarsenatis SF-1. J. Bacteriol. 193, 2141–2148 18 Ridley, H. et al. (2006) Resolution of distinct membrane-bound enzymes from Enterobacter cloaceae SLD1a-1 that are responsible for selective reduction of nitrate and selenate oxyanions. Appl. Environ. Microb. 72, 5173–5180 19 Schro¨der, I. et al. (1997) Purification and characterization of the selenate reductase from Thauera selenatis. J. Biol. Chem. 272, 23765–23768 20 Li, D.B. et al. (2014) Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm. Sci. Rep. 4, 3735 21 Pittman, M.S. et al. (2005) A bacterial glutathione transporter (Escherichia coli CydDC) exports reductant to the periplasm. J. Biol. Chem. 280, 32254–32261 22 Debieux, C.M. et al. (2011) A bacterial process for selenium nanosphere assembly. Proc. Natl. Acad. Sci. U.S.A. 108, 13480–13485 23 Butler, C.S. et al. (2012) Biomineralization of selenium by the selenaterespiring bacterium Thauera selenatis. Biochem. Soc. Trans. 40, 1239– 1243 24 Faramarzi, M.A. and Sadighi, A. (2013) Insights into biogenic and chemical production of inorganic nanomaterials and nanostructures. Adv. Colloid Interface Sci. 189–190, 1–20 25 Husen, A. and Siddiqi, K.S. (2014) Plants and microbes assisted selenium nanoparticles: characterization and application. J. Nanobiotechnol. 12, 28 26 LIoyd, J.R. et al. (2011) Biotechnological synthesis of functional nanomaterials. Curr. Opin. Biotechnol. 22, 509–515 27 Fellowes, J.W. et al. (2013) Ex situ formation of metal selenide quantum dots using bacterially derived selenide precursors. Nanotechnology 24, 145603 28 Park, B. et al. (2013) Microbially induced synthesis of cubic and hexagonal selenium nanoparticles. J. Nanosci. Nanotechnol. 13, 1854–1857 29 Lampis, S. et al. (2014) Delayed formation of zero-valent selenium nanoparticles by Bacillus mycoides SelTE01 as a consequence of selenite reduction under aerobic conditions. Microb. Cell Fact. 13, 35 30 Dwivedi, S. et al. (2013) Biometic synthesis of selenium nanospheres by bacterial strain JS-11 and its role as a biosensor for nanotoxicity assessment: a novel Se-bioassay. PLoS ONE 8, e57404 31 Tam, K. et al. (2010) Growth mechanism of amorphous selenium nanoparticles synthesized by Shewanella sp. HN-41. Biosci. Biotechnol. Biochem. 74, 696–700 32 Dobias, J. et al. (2011) Role of proteins in controlling selenium nanoparticle size. Nanotechnology 22, 195605 33 Kaur, G. et al. (2009) Biomineralization of fine selenium crystalline rods and amorphous spheres. J. Phys. Chem. C 113, 13670–13676 34 Zhang, W. et al. (2011) Biosynthesis and structural characteristics of selenium nanoparticles by Pseudomonas alcaliphila. Colloid Surf. B: Biointerfaces 88, 196–201 35 Janssen, A.J.H. et al. (1999) Removal of hydrogen sulphide from wastewater and waste gases by biological conversion to elemental sulfur: colloidal and interfacial aspects of biologically produced sulphur particles. Colloid. Surface. A 151, 389–397 36 Jain, R. et al. (2015) Extracellular polymeric substances govern the surface charge of biogenic elemental selenium nanoparticles. Environ. Sci. Technol. 49, 1713–1720 7
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Opinion 37 Buchs, B. et al. (2013) Colloidal properties of nanoparticular biogenic selenium govern environmental fate and bioremediation effectiveness. Environ. Sci. Technol. 47, 2401–2407 38 Staicu, L.C. et al. (2014) Electrocoagulation of colloidal biogenic selenium. Environ. Sci. Pollut. Res. Int. 22, 3127–3137 39 Lenz, M. et al. (2011) Shedding light on selenium biomineralization: proteins associated with bionanominerals. Appl. Environ. Microb. 77, 4676–4680 40 Ho, C.T. et al. (2010) Shewanella-mediated synthesis of selenium nanowires and nanoribbons. J. Mater. Chem. 20, 5899–5905 41 Herbel, M.J. et al. (2003) Reduction of elemental selenium to selenide: experiments with anoxic sediments and bacteria that respire Se oxyanions. Geomicrobiol. J. 20, 587–602 42 Pearce, C.I. et al. (2008) Microbial manufacture of chalcogenide-based nanoparticles via the reduction of selenite using Veillonella atypica: an in situ EXAFS study. Nanotechnology 19, 155603 43 Yan, Z. et al. (2014) Green biosynthesis of biocompatible CdSe quantum dots in living Escherichia coli cells. Mater. Res. Express 1, 015401 44 Espinosa-Ortiz, E.J. et al. (2014) Effects of selenium oxyanions on the white-rod fungus Phanerochaete chrysosporium. Appl. Microbiol. Biotechnol. 99, 2405–2418 45 Huang, J.C. et al. (2013) Development of a constructed wetland water treatment system for selenium removal: incorporation of an algal treatment component. Environ. Sci. Technol. 47, 10518–10525 46 Astrantinei, V. et al. (2006) Bioconversion of selenate in methanogenic anaerobic granular sludge. J. Environ. Qual. 35, 1873–1883 47 Lenz, M. et al. (2008) Biological alkylation and colloid formation of selenium in methanogenic UASB reactors. J. Environ. Qual. 37, 1691–1700
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48 Lenz, M. et al. (2008) Selenate removal in methanogenic and sulfatereducing upflow anaerobic sludge bed reactors. Water Res. 42, 2184– 2194 49 Lenz, M. et al. (2009) Bioaugmentation of UASB reactors with immobilized Sulfurospirillum barnesii for simultaneous selenate and nitrate removal. Appl. Microbiol. Biotechnol. 83, 377–388 50 Van Ginkel, S.W. et al. (2011) The removal of selenate to low ppb levels from flue gas desulfurization brine using the H2-based membrane biofilm reactor (MBfR). Bioresour. Technol. 102, 6360– 6364 51 Staicu, L.C. et al. (2015) Removal of colloidal biogenic selenium from wastewater. Chemosphere 125, 130–138 52 Wang, H. and Ren, Z.J. (2014) Bioelectrochemical metal recovery from wastewater: a review. Water Res. 66, 219–232 53 Winkel, L.H.E. et al. (2012) Environmental selenium research: from microscopic processes to global understanding. Environ. Sci. Technol. 46, 571–579 54 Blazina, T. et al. (2014) Terrestrial selenium distribution in China is potentially linked to monsoonal climate. Nat. Commun. 5, 3717 55 Lenz, M. et al. (2008) Selenium oxyanion inhibition of hydrogenotrophic and acetoclastic methanogenesis. Chemosphere 73, 383–388 56 Worm, P. et al. (2011) Transcription of fdh and hyd in Syntrophobacter spp. and Methanospirillum spp. as a diagnostic tool for monitoring anaerobic sludge deprived of molybdenum, tungsten and selenium. Environ. Microbiol. 13, 1228–1235 57 Lai, C.Y. et al. (2014) Nitrate shaped the selenate-reducing microbial community in a hydrogen-based biofilm reactor. Environ. Sci. Technol. 48, 3395–3402
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.
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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
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Process failure in ADs due to propionate accumulaon
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Figure II. Selenium supplementation of anaerobic digesters treating seleniumdeficient food wastes.
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d ffood waste Hydrolyzed
>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
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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
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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.
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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 (<50 nm) than those found in the cytoplasm [20]. Most often, selenospheres found in the extracellular environment are abundant. By contrast, only a few fairly large selenospheres, sometimes almost the diameter of the bacterial cell itself, are found in the cytoplasm. Reduction of selenate to selenite occurs primarily in the periplasm and is catalyzed by Ser (Figure 1). By contrast, reduction of SeO32– is mostly driven by detoxification mechanisms that operate in the cytoplasm; thus, formation of Se0 atoms and subsequent selenosphere assembly occur in the cytoplasm. To avoid accumulation of selenospheres and associated necrosis, the cells must have a mechanism to export these intracellularly formed selenospheres. An export system was proposed for the
SeO42– OM
SeO42–
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cytC4 e–
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SeO32–
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Figure 1. Schematic diagram showing the various cellular processes involved in the microbial reduction of selenium oxyanions through anaerobic respiration and detoxification, and selenium nanosphere assembly and export [17–19,22]. Reduction of selenate is catalyzed by either a soluble or membrane-bound selenate reductase (SER). Abbreviations: CM, cytoplasmic membrane; OM, outer membrane; SefA, Selenium factor A protein; SerABC, selenate reductase; SrdBCA, selenate reductase; cytC4, cytochrome C4; CymA, c-type cytochrome; FccA, fumarate reductase; CysTWA, sulphate permease; Q-pool, quinone pool.
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Opinion transport of selenospheres formed in the cytoplasm of Thauera selenatis [22,23]. Selenium factor A (SefA) is a 95-kDa protein associated with the assembly and secretion of the selenospheres. The function of SefA in selenosphere assembly is comparable to that of the well-studied protein Mms6, which controls the size and morphology of magnetite crystals inside magnetotactic bacteria [8]. However, the big question remains: how are these large selenospheres formed in the cytoplasm excreted to the extracellular medium (Box 2)? It has been postulated that the SefA selenospheres are translocated directly from the cytoplasm of T. selenatis [22,23], because of the lack of evidence of outer membrane distortion or bulging, and the absence of periplasmic material with the secreted selenospheres. However, the extent of SeO32– reduction that occurs either in the periplasm or on the cell membrane via detoxification and respiration is unknown. Biogenic synthesis of selenium nanomaterials The use of microorganisms in the green synthesis of nanomaterials is attractive mainly because microorganisms are inexpensive catalysts, precursor materials are usually sourced from inexpensive raw materials and waste streams, the synthesis occurs at near neutral pH, ambient temperature, and pressure, and the use of hazardous reducing agents is avoided [24–26]. Thus, selenium-reducing bacteria have an as yet unexploited potential for the biosynthesis of both elemental selenium and metal selenide nanomaterials under ambient conditions using selenium precursors from inexpensive raw materials or waste streams [27,28]. The potential for bionanofabrication is well documented, but so far few studies have demonstrated the functionality of biogenic selenium nanomaterials and addressed the scale-up issues. Nanoscale elemental selenium Despite the differences in mechanisms of biomineralization, microbially produced Se0 atoms always form spherical nanospheres with a diameter of up to 400 nm, rather than large crystals. In general, smaller-diameter Se particles are observed during the early phases of inoculation
Box 2. Outstanding questions What governs the size of selenospheres formed by microorganisms? Is selenosphere assembly controlled by biomolecules? How are selenospheres formed in the cytoplasm exported? Are membrane vesicles involved in shedding mineral deposits? Do we have enough understanding of the origin and biochemical nature of organic layer associated with selenium biominerals? Are elemental selenium particles reduced through extracellular respiration by microorganisms? What are the effects of selenium biominerals on microbial communities and on long-term stability of bioreduction? What is the effect of engineered selenium nanomaterials on the structure and function of microbial communities? How toxic are the effluents of selenium treatment systems? Is biomineralization from selenium wastewaters the answer to meet selenium scarcity? Is the production of metal selenides, such as quantum dots, scalable using microorganisms? 4
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[29]. Phenazine-1-carboxylic acid, a metabolite produced by Pseudomonas aeruginosa JS-11, enabled SeO32– reduction and formation of monodisperse selenospheres with an average size of 21 nm [30]. The average diameter and polydispersity index of selenium particles increased during prolonged incubation in bacterial cultures [29,31,32]. Se0 is known to exist in three structural arrangements: infinite a-helical chains, and six- and eight-member rings [9]. These are the nucleating centers for the newly formed Se0 atoms. Large selenospheres are formed though Ostwald ripening, wherein the growth occurs through newly formed selenium atoms through subsequent reduction of selenium oxyanions and selenium atoms released through disassembly of other particles following the Gibbs-Thompson law and aggregation [33]. The size of the Se0 particles grows until capping agents, such as proteins, polysaccharides, phospholipids, or extracellular polymeric substances (EPS), inhibit further growth and aggregation. The polydispersity and large particle size occur because of the large number of nucleating sites and limited availability of capping agents. In some cases, transformation of selenospheres to trigonal selenium (t-Se) nanorods was observed, probably through the disassembly and assembly of Se0 atoms due to destabilization of the capping agent [34]. The precise controlling of shape and size is possible only if capping or stabilizing agents are available in sufficient amounts. Similar to biologically produced elemental sulfur [35], biogenic selenospheres are always associated with an organic layer that changes the physicochemical characteristics of solid selenium and influences their fate in the environment [36]. The colloidal stability of the biogenic selenospheres is conferred by the organic layer, which gives a net negative charge and prevents aggregation and sedimentation of selenium particles [37,38]. Attempts are being made to trace the biochemical nature and origin of the organic layer associated with biogenic selenospheres. In vitro experiments have shown that Se0 nanoparticles can be stabilized against crystallization by proteins [32,39] or EPS [36,39]. Lenz et al. [39] identified high-affinity proteins associated with Se0 nanospheres formed by selenium-respiring bacteria. The components of the organic layer could originate from the enzymes, outer membrane, or EPS components, which are involved in the reduction selenium oxyanions, assembly, or export of selenospheres. The transformation of microbially produced selenospheres into other Se0 forms (e.g., nanowires) has been demonstrated in vitro [40]. Selenospheres formed by Shewanella sp. HN-41 were incubated along with cells in dimethyl sulfoxide to transform spheres into long, thin Se0 nanoribbons and nanowires. This shows that removal of the stabilization caused by the organic layer enables the growth of selenospheres into other Se0 crystal types, which opens doors for new technological applications. Nanoscale metal selenides The bioreduction of selenium oxyanions beyond selenospheres and up to HSe– has so far been noticed only in sediments [41], and a few axenic bacterial cultures [42]. The metal-reducing bacterium Veillonella atypica has been
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used to reduce selenite up to HSe– for producing 30-nm zinc selenide (ZnSe) and cadmium selenide (CdSe) particles [42]. The use of biogenic HSe– enabled a controlled reaction rate during metal selenide synthesis, which resulted in a narrow size distribution of CdSe nanocrystals (diameter <8 nm) compared with chemical synthesis [27]. Formation of CdSe nanocrystals with a diameter of 8–11 nm was observed when growing Escherichia coli cells incubated with SeO32– and CdCl2 [43]. In these cells, a reaction between an organo selenium compound (R-Se) and Cd2+ was proposed for the formation of CdSe nuclei. The further growth of metal selenide nuclei will lead to the formation of metal selenide colloids. The nature of proteins or other biomolecules in achieving the narrow size distribution by controlling the nucleation and crystal growth rates are not known. The bacterial production of metal selenides offers a green process that not only avoids the use of toxic and expensive chemical precursors, but also introduces the use of a novel selenium and heavy metal recovery process from waste streams. However, bioengineering the bioreduction of selenium oxyanions beyond elemental selenium appears
to be challenging. Unlike Se oxyanions, Se0 is a solid electron acceptor and its reduction via anaerobic respiration might require the involvement of electron shuttles or outer membrane components. The rate of bacterial reduction of selenium oxyanions to HSe– needs to be improved several-fold to make it a viable and scalable bioprocess for metal selenide synthesis. Bioremediation and wastewater treatment Selenium wastewaters must be treated to abate selenium pollution and to comply with discharge limits. According to the US Environmental Protection Agency (EPA), the selenium content of wastewaters must be brought down to below 5 mg l–1 before discharging to the environment. There is a strong debate about whether this limit is safe enough for protecting ecosystem well-being, mainly because selenium is known to transfer across trophic levels and accumulate in biota. Stringent discharge limits for selenium wastewaters generated in coal-fired power plants are foreseen in the near future, demanding the development of efficient selenium treatment technologies [2].
Suspended growth Algal pond [microalgae/cyanobacteria]
Reducon pond [anaerobic bacteria]
Floc
FBR Fluidized bed reactor
Nutrients Sand
Biological selenium removal systems
Carrier material [Biofilm]
Biofilm
Wash waste supernatant
Wash waste tank
Plug flow reactor
PFR
Backwash tank
Molasses Biofilm
Solids
Backwash
Granular acvated carbon
MBfR H2
Biofilm
Aached growth
Resistor
Anode Biofilm
CO2
H+
Cathode
BES
Moxd
Membrane
Organic maer
Anode
Hollow fiber
Mred
UASB No carrier material [Parculate Biofilm]
Nutrient Anaerobic granule TRENDS in Biotechnology
Figure 2. Bioreactor configurations and biomass retention principles of biological selenium removal systems [10,13,48,50,52,57]. Red arrows indicate an inlet, whereas green arrows indicate an outlet. Abbreviations: BES, bioelectrochemical system; FBR, fluidized bed reactor; MBfR, membrane biofilm reactor; Moxd, metal(loid) oxidized; Mred, metal(loid) reduced; PFR, plug flow reactor; UASB, upflow anaerobic sludge blanket reactor.
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Biological selenium removal methods are attractive because of the wastewater characteristics (e.g., dilute and large volume) as well as low capital and operational costs. Most technologies are based on bacterial metabolism, although fungal systems can also be considered for low pH wastewaters [44]. Furthermore, photobioreactors are emerging for the inclusion of microalgae as part of integrated algal–bacterial selenium removal systems [45], wherein selenium oxyanions are removed by biovolatalization and bioreduction [45]. Various biotechnologies using microbial reduction have been developed for removing selenium oxyanions in conjunction with water reuse and selenium recovery. Selenate reduction has been investigated under methanogenic [46,47], sulfate reducing [48], denitrifying [49], and hydrogenotrophic [50] conditions. Different bioreactor configurations have been adopted, such as upflow anaerobic sludge blanket reactors, fluidized bed reactors, plug flow reactors, membrane biofilm reactors, and bioelectrochemical systems (Figure 2) for retaining selenium-reducing microorganisms as flocs, granular sludge, or biofilms. Some of these technologies have already been applied at full scale for selenium removal from wastewater. Although the microbial reduction is proven for converting soluble selenium oxyanions into elemental selenium, the treatment remains challenging because of the stringent discharge limits, occurrence of co-contaminants (e.g., metals and competing electron acceptors), and the handling of the concentrated selenium solids to avoid re-release and hazardous waste classification. Biological treatment relies on the enrichment and retention of microorganisms that convert soluble selenium oxyanions into the less-toxic elemental selenium,
Selenium-containing wastewater
Agriculture drainage waters
Microbial selenium transformaons Selenate (aq) [Se (VI)]
Selenite (aq) [Se(IV)]
which is decanted from the wastewater by gravity settling or filtration. Given its colloidal nature [36], a significant fraction of the bioreduced selenium can still be present in the bioreactor effluent, necessitating a post treatment step, such as coagulation [38] or electrocoagulation [51]. The performance of selenium-removing bioreactors is governed by the fraction of the bioreduced selenium that has colloidal properties. The fate of this fraction impacts the treatment efficiency, thereby compromising the selenium discharge limit. Bioelectrochemical systems (BES) are emerging as attractive options especially for recovering metals from wastewaters. In BES, an organic electron donor is oxidized in the anode chamber using anode-respiring bacteria and the electrons are used for reductive precipitation of metal(loid)s in the cathode chamber via direct cathodes or biocathodes with or without external power supply [52]. It will be worth investigating these novel systems for selenium removal and recovery. The biological treatment methods may be combined with physicochemical post-treatment (e.g., ultrafiltration and coagulation) to achieve the discharge limits. However, this increases the overall treatment cost, although the effluent discharge will comply with the regulatory guidelines, thus avoiding penalties due to selenium pollution. In addition, selenium recovery from wastewater is desirable to offset the treatment costs and to make this valuable scarce element available for reuse (Figure 3). There is a need to test and evaluate the recovered selenium as a resource for applications in the formulation of food supplements, antimicrobials, crop fortification agents, and as base material for selenium materials (Figure 3).
Bioreactor
Selenium (s) (Se0) Recovery for reuse
Selenomethionineenriched yeast, microalgae
Leachates from contaminated soils
Applicaons • Anmicrobials • Ferlizers • Semiconductors • Sensors
Heavy metals (Cd2+, Zn2+, Pb2+)
Selenide (aq) [Se(-II)] Metal selenides (CdSe, ZnSe, PbSe) Applicaons • Electronics • Imaging • Photovoltaics TRENDS in Biotechnology
Figure 3. Schematic showing the possible applications of selenium biotechnology and selenium recovery options as well as reuse applications for the recovered selenium.
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Opinion Concluding remarks and future perspectives Biomineralization combines the treatment of selenium wastewaters with the potential to remove, recover, and reuse selenium in the form of selenium biominerals. Microbial reduction is a proven technology for converting soluble selenium oxyanions to insoluble forms to remove the pollution from the water. Treatment of selenium wastewaters is nevertheless challenging because the bioreduced selenium forms a colloidal suspension for which additional separation steps are required. When retained, the bioreduced selenium is a valuable resource as starting material for selenium materials, antimicrobial formulations, or as precursor of crop fortification agents and supplements. To enable rapid progress in the application of microbial nanofactories and biomimetic approaches for functionalized selenium nanomaterials along with potential recovery strategies for meeting selenium scarcity, the following aspects need to be addressed: (i) elucidating the biochemical pathways involved in selenosphere production as a function of its location. For full-scale applications in bioreactors, extracellular formation of large, well-settling Se0 or metal selenide particles is desired; (ii) identification of the proteins or other biomolecules that govern the size and surface properties of biogenic selenium in bacterial cultures and complex microbial communities. Once known, intelligent biomineralization systems can be developed by varying the biomolecule concentration to induce or stop the crystallization process; and (iii) development of integrated treatment strategies for the treatment of selenium wastewaters and selenium recovery, including a physicochemical post-treatment step subsequent to biological reduction. Acknowledgments We thank our past and present coworkers of UNESCO-IHE, Wageningen University (the Netherlands), and Bhabha Atomic Research Centre (India) as well as our national and international collaborators. We also thank our national and international granting agencies, in particular the BioMatch project (project 103922), funded by an EU Marie Curie International Incoming Fellowship.
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