**2.1. Bioremediation of Se-polluted environments using bacterial pure cultures as planktonic cells**

In the last 30 years, Se-oxyanions sequestration by microorganisms has been investigated as a potential strategy for the decontamination of Se-polluted environments. Indeed, several bacterial strains have been described for their ability to uptake SeO4 2− and/or SeO3 2− using several processes, such as the sulfate transporter in *E. coli* [51], the sulfate permease in *Salmonella typhimurium* [52], the sulfite uptake system in *Clostridium pasteurianum* [53], the polyol ABC transporter in *R. sphaeroides* [54]. Thus, once inside the bacterial cell, the sequestered Se-oxyanions are usually incorporated into Se-amino acids (i.e., seleno-cysteine and -methionine) to biosynthesize selenoproteins [55].

An alternative Se-bioremediation approach is based on the bacteria's ability to biomethylate Se-oxyanions, resulting in the production of Se-methyl derivates (i.e., dimethyl selenide, dimethyl selenyl sulfide, dimethyl diselenide), as in the case of *Aeromonas* sp. VS6, *Citrobacter freundii* KS8 and *P. fluorescens* K27 [56], *Clostridium collagenovorans*, *Desulfovibrio gigas* and *Desulfovibrio vulgaris* [57], *Enterobacter cloacae* SLS1a-1 [58], *R. sphaeroides* and *R. rubrum* S1 [59]. Se-oxyanions biomethylation is achieved in microorganisms through the Challenger mechanism [56], which consists of several reduction-methylation steps that change Se-redox state from either VI or IV to II [60].

Recently, the exploitation of microorganisms able to bioconvert Se-oxyanions to Se0 has emer ged as a cost-effective *green* alternative strategy for the decontamination of Se-polluted environments, with a particular focus on surface waters and wastewaters. To date, Se-bioremediation approaches exploit bacterial strains capable of reducing SeO4 2− and SeO3 2− [23] either to conserve energy [61–63] or to detoxify their environmental niches [23]. Since Se-oxyanions bio-reduction under anoxic conditions is more characterized as compared to the aerobic mode, mainly anaerobic bacterial strains have been used for Se-decontamination purposes [23]. However, studies evaluating either SeO4 2− or SeO3 2− bioconversion by aerobic or microaerophilic microorganisms have also been conducted [61, 64–67], highlighting some disadvantages of these experimental conditions: a competition between the dissolved oxygen and the Se-oxyanion as terminal electron acceptor [68, 69], and the additional energetic cost to aerate a bioreactor [23]. Regardless, aerobic bacterial strains have been explored as pure cultures at laboratory scale for Se-bioremediation purposes, yet little work about the use of these microorganisms for largescale applications have been conducted [23].

**2. Bioremediation of chalcogen-contaminated environments**

and Te0

120 Biosorption

with NO3

**planktonic cells**

selenoproteins [55].

either VI or IV to II [60].

studies evaluating either SeO4

The exploitation of microorganisms for the decontamination of Se- and/or Te-polluted environments is based on the capability of several bacterial strains to sequester, bioconvert or biomethylate chalcogen-oxyanions [19]. Se- or Te-species sequestration is achieved by microorganisms through either their uptake in the bacterial cell or the interaction with charged surface biomolecules [19], while the bioconversion of these oxyanions in bacteria leads to their reduction to Se0

 in the form of metalloid precipitates [19]. Further, some microorganisms can biomethylate Se or Te-oxyanions, producing volatile methyl derivatives, which can react in the atmosphere

radicals, ozone and atmospheric particles, increasing their residence times [19, 50].

2− and/or SeO3

2− and SeO3

2− bioconversion by aerobic or microaerophilic micro-

2− using several pro-

has emer

2− [23] either to conserve

In the last 30 years, Se-oxyanions sequestration by microorganisms has been investigated as a potential strategy for the decontamination of Se-polluted environments. Indeed, several bacte-

cesses, such as the sulfate transporter in *E. coli* [51], the sulfate permease in *Salmonella typhimurium* [52], the sulfite uptake system in *Clostridium pasteurianum* [53], the polyol ABC transporter in *R. sphaeroides* [54]. Thus, once inside the bacterial cell, the sequestered Se-oxyanions are usually incorporated into Se-amino acids (i.e., seleno-cysteine and -methionine) to biosynthesize

An alternative Se-bioremediation approach is based on the bacteria's ability to biomethylate Se-oxyanions, resulting in the production of Se-methyl derivates (i.e., dimethyl selenide, dimethyl selenyl sulfide, dimethyl diselenide), as in the case of *Aeromonas* sp. VS6, *Citrobacter freundii* KS8 and *P. fluorescens* K27 [56], *Clostridium collagenovorans*, *Desulfovibrio gigas* and *Desulfovibrio vulgaris* [57], *Enterobacter cloacae* SLS1a-1 [58], *R. sphaeroides* and *R. rubrum* S1 [59]. Se-oxyanions biomethylation is achieved in microorganisms through the Challenger mechanism [56], which consists of several reduction-methylation steps that change Se-redox state from

Recently, the exploitation of microorganisms able to bioconvert Se-oxyanions to Se0

ged as a cost-effective *green* alternative strategy for the decontamination of Se-polluted environments, with a particular focus on surface waters and wastewaters. To date, Se-bioremediation

energy [61–63] or to detoxify their environmental niches [23]. Since Se-oxyanions bio-reduction under anoxic conditions is more characterized as compared to the aerobic mode, mainly anaerobic bacterial strains have been used for Se-decontamination purposes [23]. However,

organisms have also been conducted [61, 64–67], highlighting some disadvantages of these experimental conditions: a competition between the dissolved oxygen and the Se-oxyanion as terminal electron acceptor [68, 69], and the additional energetic cost to aerate a bioreactor [23].

**2.1. Bioremediation of Se-polluted environments using bacterial pure cultures as** 

rial strains have been described for their ability to uptake SeO4

approaches exploit bacterial strains capable of reducing SeO4

2− or SeO3

Among the microorganisms described for their tolerance toward Se-oxyanions, bacterial strains belonging to *Pseudomonas, Desulfovibrio, Thauera, Enterobacter, Wolinella* and *Bacillus* genera have been characterized for their capability to bioconvert SeO4 2− to SeO3 2− mainly under anoxic growth conditions [61, 70, 71]. Moreover, several anaerobic microorganisms have been characterized for their use of SeO4 2− as terminal electron acceptor to support their growth [26, 70–73], coupling the bioconversion of this Se-oxyanion to the oxidation of different carbon sources, such as aliphatic (pyruvate, lactate, acetate) as well as aromatic compounds (i.e., benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate) [61, 74, 75]. Nevertheless, facultative anaerobes, such as *Pseudomonas stutzeri*, showed their proficiency of bioreducing SeO<sup>4</sup> 2− solely for detoxification purposes [70].

Unlike SeO4 2−, both aerobic and anaerobic microorganisms can bioconvert the highly soluble and reactive SeO3 2− [76] into Se0 through either detoxification strategies or anaerobic respiration [77–79]. SeO3 2− detoxification occurs through several mechanisms based on Painter-type reactions [17, 80–82], where glutaredoxin/thioredoxin reductase systems [19, 83] and siderophores mediate the oxyanion reduction [19, 65]. SeO3 2− detoxification is mostly achieved by thiol molecules present in the cytoplasm of bacterial cells, such as GSHs, mycothiols (MSHs), and glutaredoxins [17, 84]. Moreover, GSHs can be exported into the periplasm of Gram-negative bacteria, leading to the bioreduction of SeO3 2− in the periplasm or at their cell membrane [85]. Secondary SeO3 2−-detoxification strategies exploited by microorganisms involved the interaction between SeO3 2− and reactive biogenic sulfide, [86, 87], as well as the exploitation of iron siderophores [19, 88]. On the other hand, SeO3 2− bioconversion during anaerobic respiration is mostly mediated by the presence of terminal nitrite, sulfite or fumarate reductases [19, 24, 61, 66, 67, 72, 89, 90], as described for *T. selenatis* AX, *Rhizobium sullae* HCNT1 and *C. pasteurianum*, to name a few [91–93]. Further, *Geobacter sulfurreducens* [94], *Shewanella oneidensis* MR-1 [90] and *Veillonella atypica* [94] showed high proficiency in bioreducing SeO<sup>3</sup> 2− to Se0 through dissimilatory reduction in anoxic conditions, while among the bacterial strains able to anaerobically bioconvert SeO4 2− into SeO3 2−, a high yield of Se0 production by further reducing SeO3 2− has been observed for *Bacillus beveridgei* [22], *D. indicum* [75], *Desulfovibrio desulfuricans* [95], *E. cloacae* SLD1a-1 [96] and *Sulfospirillum barnesii* SES-3 [25, 96]. Nevertheless, fewer bacterial species (i.e., *Bacillus selenitireducens* and *Aquificales* sp.) have been described for their ability to use SeO3 2− as terminal electron acceptor as compared to those using SeO4 2− [26, 27].
