**3.1. Microbial consortia**

*Lactococcus lactis* [98] and *R. capsulatus* [99, 100], considering that this Te-species is a strong competitive inhibitor of the phosphate group [19]. However, other carriers can be used to

*capsulatus* [101], as well as an ATP-dependent efflux pump responsible for the arsenite/arsenate/antimonite resistance in *E. coli* [102]. Since Te shares several chemical properties with Se, microorganisms tolerant and/or resistant toward Te-oxyanions process them exploiting similar mechanisms to those described above for Se-species. In this regard, the biomethylation of Te-oxyanions to produce dimethyl telluride and dimethyl ditelluride [56] has been observed in several bacteria able to biomethylate Se-oxyanions as well, such as *R. rubrum* G9, *R. capsulatus* [59], *P. fluorescens* K27 [103] and *D. gigas* [57]. Moreover, *P. aeruginosa* ML4262 [104], *G. stearothermophilus* V [105] and *Mycobacterium tuberculosis* [106] showed their capability of

2− presence in lower amount in the environment compared to TeO4

2−-tolerant/resistant microorganisms as ideal candidate for bioremedi-

2− oxyanions as terminal electron acceptors in the respiratory chain

rite showed toxicity 10 times higher than tellurate [40, 41], leading the experimental research to

ation purposes. Nevertheless, *B. beveridgei* [22], *B. selenitireducens*, *S. barnesii* [29] and *Shewanella frigidimarina* ER-Te-48 [28, 107] showed their ability under anaerobic growth conditions to

to sustain their growth [8]. To date, the proposed mechanisms of Te-oxyanions bioconversion in microorganisms are similar to those described for Se-species [13, 56, 88, 104, 108]. Further,

fied as responsible for the anaerobic respiration of this Te-oxyanion in *Bacillus* sp. GT-83 [113].

The majority of the investigations regarding the bioremediation of Se- and Te-contaminated environments have been focused on the exploitation of bacterial species grown as free planktonic cells [8]. However, in natural settings microorganisms are most often found in close association with surfaces and interfaces as complex communities, which are indicated as biofilms [114–116]. In bacterial biofilms, the cells are embedded and protected from the surrounding environments by the presence of a matrix defined as Extracellular Polymeric Substance, containing a high amount of water, polysaccharides, proteins, extracellular-DNA (e-DNA) and lipids [117, 118]. The communal life of bacterial cells in the form of biofilm offers them several advantages [114, 117, 119], resulting in their innate ability to populate a vast array of environments [119], including those contaminated by chalcogen-oxyanions. Thus, peculiar features of bacterial biofilms (i.e., quorum sensing signaling process, different cellular physiology, presence of the EPS and colony morphology variants) [120–124] confer them tolerance and/or resistance toward either Se- or Te-oxyanions without having specific Se- and Te- genetic resistant determinants

**2.3. Bioremediation of chalcogen-polluted environments based on bacterial biofilms**

[19]. In this regard, sulfate-reducing bacteria (SRB) within a biofilm produce sulfide (S<sup>2</sup>

2− processing in microorganisms have been ascribed to enzymatic reductions by periplasmic or cytoplasmic oxidoreductases [107, 109], such as nitrate reductases [109, 110], catalases [111] and thiol:disulfide oxidoreductase [112]. However, the function of all these enzymes for bioconverting Te-oxyanions appears to be not specific, leading to a low resistance level toward

2− [39], tellu-

), which

2− reductase has been identi-

2− uptake in microorganisms, such as the ActP monocarboxylate transporter of *R.* 

assist TeO3

122 Biosorption

Despite of TeO3

use both TeO4

TeO3

focus on the study of TeO3

biomethylating only Te-oxyanions.

2− and TeO3

Te-species in these microorganisms. To date, only one specific TeO<sup>3</sup>

In the environment, microorganisms usually thrive as communities composed by multiple species, generally referred as microbial consortia [132]. The employment of these microbial consortia in the treatment of environmental matrices contaminated with different inorganic or organic pollutants is currently a field of great interest for researchers [133]. There are significant advantages for the utilization of microbial consortia over pure cultures, such as the larger volumes of wastewaters treatable, the ability of microbial communities to adapt to diverse conditions, the presence of synergic interactions among members within the consortium and the possibility to work in non-aseptic conditions [23]. This last aspect is particularly significant, since it facilitates process control and it reduces both maintenance and operational costs [134].

In the following section, the different biological systems based on processes of biosorption and bioconversion of Se- and Te-oxyanions from contaminated matrices by using microbial consortia will be discussed.

#### **3.2. Microbial consortia for Se-removal from contaminated environments**

In recent years, the utilization of biological treatments based on the exploitation of microbial consortia has become the leading approach for the removal of toxic Se-species from environmental matrices, particularly from wastewaters (i.e., mine runoff, agricultural drainage, and flue gas desulfurization wastewater from plants) [23]. This decontamination strategy has several advantages over chemical–physical remediation technologies, being: the cost-effectiveness of microbial-based remediation approach, the avoidance in employing hazardous chemicals, and the possibility to recover Se0 in a recyclable form either as precipitates or as nanostructures, which are technologically and economically more valuable [23, 135]. Since using microbial consortia under aerobic conditions has a lower efficiency of the whole system compared to the anaerobic processes, microbial communities used in these systems are mostly capable of anaerobically bioconverting Se-oxyanions to their elemental state [136]. In this regard, the dissimilatory reduction of SeO4 2− under anaerobic conditions by a microbial community was firstly reported for sediment slurries by Oremland and coworkers [89], while an anaerobic co-culture isolated from agricultural drainage water in the San Joaquin Valley in California of a not-identified Gram-positive rod-shaped bacterium and a *Pseudomonas* sp. was capable of bioconverting both SeO4 2− and SeO3 2− to Se0 [72]. Further, several anaerobic microbial consortia able to process Se-species have been found in biological wastewaters, such as activated, denitrifying, sulfate-reducing and methanogenic sludges [135]. Among them, methanogenic anaerobic granular sludges were the most effective to remove high SeO<sup>4</sup> 2− concentrations using different electron donors (e.g., methanol, ethanol, acetate, lactate, glucose) [137].

Considering the large amount of Se-oxyanions present in laden wastewaters, different technologies and reactor configurations have been developed in order to treat these environmental samples (**Figure 1**), such as the ABMet® biofilter system, the electro-biochemical reactors (EBR), the biofilm reactors (BSeR), the membrane biofilm reactors (MBfR), the upflow anaerobic sludge blanket reactors (UASB) and the sequencing batch reactors (SBR) [23]. In the following sub-sections, examples of bioreactor configurations used to bioremediate Se-contaminated waters and their operating procedures are briefly discussed.

#### *3.2.1. The ABMet® reactor system*

The ABMet® reactor is both a biological and a filtration system, in which microbial consortia are grown on porous granular activated carbon (GAC) beds, creating anoxic conditions for optimal SeO4 2− and SeO3 2− reduction [23]. The system consists of biofilter tanks where Se-oxyanions are bioconverted to their elemental state, followed by the removal of Se0 from the biofilter through a backwash cycle [138, 139]. This reactor uses a nutrient dosage tank generally containing a molasses-based solution, which acts as an electron donor sink for the microbial consortia, allowing the bioconversion of Se-oxyanions [139]. Thus, in this reactor configuration, the microbial communities require only a small amount of supplemented nutrient, decreasing the maintenance costs of the entire system [23]. Further, the GAC beds are used as substratum to sustain the bacterial growth, allowing the formation of a biofilm, which is morphologically more robust as compared to planktonic cells, resisting to the washing steps of the reactor [23]. Recently, Se-oxyanions bioconversion using anaerobic microbial communities inoculated in a ABMet® biofilter system has been observed within 16 h of empty bed contact time (EBCT) (i.e., the residence time of the water in the reactor) with a removal efficiency of 99.3% at the Duke Energy and Progress Energy in North Carolina [138]. Moreover, co-contaminants present in these wastewaters, such as NO3 − and heavy metals, along with Se-oxyanions resulted to be removed with a high efficacy by the microbial consortium grown on the ABMet® biofilter system [23].

*3.2.2. The EBR system*

low as 1°C [141].

trodes reducing inorganic compounds (e.g., SeO4

Se-wastewater treatment is also achieved by using the electro-biochemical reactor (EBR), which utilizes the ability of certain microbial consortia to accept electrons from graphite elec-

**Figure 1.** Schematic illustration of bioreactor configurations used for bioremediation of chalcogen-contaminated matrices.

tron transfer [140]. In this process, electrons obtained from the oxidation of electron donors (i.e., graphite electrodes) are transferred to the outer surface of a bacterial cell to reduce the extracellular terminal electron acceptor (i.e., Se-oxyanions) [140]. The efficiency of this system is strictly dependent on the retention times of the microbial consortia, with optimal performances between 6 to 18 h [141]. In this regard, on-site pilot scale study using an EBR system in British Columbia (Canada) for the decontamination of coal mine wastewaters from Se-oxyanions reported a decrease of their concentration from over 500–5 μg L−1 (below US discharge limits), showing its high effectiveness even with influent streams at temperature as

2− and SeO3

2−) through direct interspecies elec-

Microbial-Based Bioremediation of Selenium and Tellurium Compounds

http://dx.doi.org/10.5772/intechopen.72096

125

Microbial-Based Bioremediation of Selenium and Tellurium Compounds http://dx.doi.org/10.5772/intechopen.72096 125

**Figure 1.** Schematic illustration of bioreactor configurations used for bioremediation of chalcogen-contaminated matrices.

#### *3.2.2. The EBR system*

several advantages over chemical–physical remediation technologies, being: the cost-effectiveness of microbial-based remediation approach, the avoidance in employing hazardous

nanostructures, which are technologically and economically more valuable [23, 135]. Since using microbial consortia under aerobic conditions has a lower efficiency of the whole system compared to the anaerobic processes, microbial communities used in these systems are mostly capable of anaerobically bioconverting Se-oxyanions to their elemental state [136]. In

community was firstly reported for sediment slurries by Oremland and coworkers [89], while an anaerobic co-culture isolated from agricultural drainage water in the San Joaquin Valley in California of a not-identified Gram-positive rod-shaped bacterium and a *Pseudomonas* sp. was

bial consortia able to process Se-species have been found in biological wastewaters, such as activated, denitrifying, sulfate-reducing and methanogenic sludges [135]. Among them, meth-

tions using different electron donors (e.g., methanol, ethanol, acetate, lactate, glucose) [137]. Considering the large amount of Se-oxyanions present in laden wastewaters, different technologies and reactor configurations have been developed in order to treat these environmental samples (**Figure 1**), such as the ABMet® biofilter system, the electro-biochemical reactors (EBR), the biofilm reactors (BSeR), the membrane biofilm reactors (MBfR), the upflow anaerobic sludge blanket reactors (UASB) and the sequencing batch reactors (SBR) [23]. In the following sub-sections, examples of bioreactor configurations used to bioremediate Se-contaminated

The ABMet® reactor is both a biological and a filtration system, in which microbial consortia are grown on porous granular activated carbon (GAC) beds, creating anoxic conditions for optimal

a backwash cycle [138, 139]. This reactor uses a nutrient dosage tank generally containing a molasses-based solution, which acts as an electron donor sink for the microbial consortia, allowing the bioconversion of Se-oxyanions [139]. Thus, in this reactor configuration, the microbial communities require only a small amount of supplemented nutrient, decreasing the maintenance costs of the entire system [23]. Further, the GAC beds are used as substratum to sustain the bacterial growth, allowing the formation of a biofilm, which is morphologically more robust as compared to planktonic cells, resisting to the washing steps of the reactor [23]. Recently, Se-oxyanions bioconversion using anaerobic microbial communities inoculated in a ABMet® biofilter system has been observed within 16 h of empty bed contact time (EBCT) (i.e., the residence time of the water in the reactor) with a removal efficiency of 99.3% at the Duke Energy and Progress Energy in North Carolina [138]. Moreover, co-contaminants present in these

with a high efficacy by the microbial consortium grown on the ABMet® biofilter system [23].

2− reduction [23]. The system consists of biofilter tanks where Se-oxyanions are

and heavy metals, along with Se-oxyanions resulted to be removed

2− to Se0

2− and SeO3

anogenic anaerobic granular sludges were the most effective to remove high SeO<sup>4</sup>

waters and their operating procedures are briefly discussed.

bioconverted to their elemental state, followed by the removal of Se0

in a recyclable form either as precipitates or as

2− under anaerobic conditions by a microbial

[72]. Further, several anaerobic micro-

2− concentra-

from the biofilter through

chemicals, and the possibility to recover Se0

this regard, the dissimilatory reduction of SeO4

capable of bioconverting both SeO4

*3.2.1. The ABMet® reactor system*

SeO4

124 Biosorption

2− and SeO3

wastewaters, such as NO3

−

Se-wastewater treatment is also achieved by using the electro-biochemical reactor (EBR), which utilizes the ability of certain microbial consortia to accept electrons from graphite electrodes reducing inorganic compounds (e.g., SeO4 2− and SeO3 2−) through direct interspecies electron transfer [140]. In this process, electrons obtained from the oxidation of electron donors (i.e., graphite electrodes) are transferred to the outer surface of a bacterial cell to reduce the extracellular terminal electron acceptor (i.e., Se-oxyanions) [140]. The efficiency of this system is strictly dependent on the retention times of the microbial consortia, with optimal performances between 6 to 18 h [141]. In this regard, on-site pilot scale study using an EBR system in British Columbia (Canada) for the decontamination of coal mine wastewaters from Se-oxyanions reported a decrease of their concentration from over 500–5 μg L−1 (below US discharge limits), showing its high effectiveness even with influent streams at temperature as low as 1°C [141].

#### *3.2.3. The BSeR and MBfR systems*

Reactors containing multispecies biofilms (BSeR) represent another promising approach for the treatment of Se-contaminated wastewaters. Indeed, microbial biofilms play a dominant role in the biogeochemical natural cycle of different inorganic compounds. In a recent study, a multispecies biofilm composed of strains (i.e., *Rhodococcus* sp., *Pseudomonas* sp., *Bacillus* sp. and *Arthrobacter* sp.) adapted to high concentration of SeO3 2− has been investigated for its potential in converting these oxyanions to their elemental form (Se0 ) [142]. Moreover, it has been highlighted the presence of specific biofilm regions where Se<sup>0</sup> was deposited as sub-micrometer-sized particles, associated with the microbial biomass [142]. In the BSeR methodology, bacterial biofilms are grown on granular activated carbon in anaerobic fixed-film reactors showing a high bioprocess proficiency toward both SeO<sup>4</sup> 2− and SeO3 2− [143], which resulted in the recovery of ca. 97% of Se0 from agriculture drainage wastewater (Garfield Wetlands-Kessler Springs, Utah, USA) [144].

to efficiently remove Se-oxyanions from contaminated environments, its implementation at industrial scale has not been investigated yet, likely due to the high cost of electron donors needed to the working-system, which is still prohibitive for large-scale applications [143].

Microbial-Based Bioremediation of Selenium and Tellurium Compounds

http://dx.doi.org/10.5772/intechopen.72096

Sludge-based reactors have also been employed for the treatment of Se-contaminated wastewaters [68]. Indeed, the most implemented process for anaerobic treatment of industrial effluents is the upflow anaerobic sludge blanket (UASB) reactor, because of the accumulation of microbial biomass and suspended solid, and a dense sludge bed at the bottom of the reactor, in which Se-oxyanions bioconversion occurs [68]. In this regard, the natural aggregation of some bacteria forming flocculates or granules leads to a high retention of active anaerobic sludge even at great organic load rates [149]. Additionally, the wastewater is kept in good contact with the bacterial biomass through both the turbulence of the upflow influent flow and the biogas produced by the anaerobic microorganisms [68]. UASB reactors have been pilot-tested for Se-removal at the Adams Avenue Agricultural Drainage Research Center in San Joaquin Valley (California) [150]. The influent had a total Se content of 500 μg L−1 and the removal efficiency ranged from 58 to 90% [150]. The efficiency of UASB reactors for the removal of Se-oxyanions was tested by Lenz and coworkers in a series of studies evaluat-

2− removal from synthetic wastewater by microbial consortia under methanogenic,

and energy, methanogenic sludges are promising for Se-oxyanions

2−

127

2− removal in UASB reac-

2−-respiring microorganisms, such as

sulfate-reducing and denitrifying conditions [151–153]. Using lactate as electron donor, a

from contaminated wastewaters, with the involvement of sulfate-reducing bacteria (sulfatereducing conditions) and a selenium-respiring sub-population (methanogenic conditions) [151]. Since the use of UASB reactors under methanogenic conditions leads to the recovery of

tors as function of the temperature, observing that the maximum efficiency of removal was obtained at thermophilic conditions (55°C) [154]. Another advantage of working at this temperature is the better retention of reduced Se in the microbial biomass. Additionally, they performed a characterization of the microbial consortia through DGGE analysis, correlating

*Sulfurospirillum barnesii* and *D. indicum* [154]. UASB reactors are very promising for removing Se-oxyanions from contaminated wastewaters, however they require constant control, since any change in operation conditions may lead to an increase of the effluent Se-concentration

Se-wastewater can be processed using a sequencing batch reactor (SBR), in which the biodegradation and solid separation take place in the same reactor [23]. In this configuration, the treatment is carried out in consecutive stages in the same tank: filling, reaction, sedimentation, draw, purging and inactivity [155]. The selection and enrichment of the desired microbial

bioconversion [143]. Further, Dessì and coworkers evaluate SeO4

2− removal efficiency to the presence of SeO<sup>4</sup>

through either biomethylation or bioconversion of Se-species [23].

2− removal efficiency of 99% was obtained in both methanogenic and sulfate-reducing conditions, demonstrating that UASB reactors can be effectively applied to remove SeO<sup>4</sup>

*3.2.4. The UASB system*

ing SeO4

decontaminated water, Se0

the high SeO4

*3.2.5. The SBR system*

SeO4

Another configuration of reactor based on microbial biofilms is the membrane biofilm reactor (MBfR) [129, 130, 145, 146]. MBfR in its standard configuration consists of a bundle of bubble-less gas transfer to a membrane delivering H2 directly to the grown biofilm consisting of autohydrogenotrophic bacteria (e.g., *Cupriavidus metallidurans*) on the outer surface of the membrane [146], resulting in a higher efficiency of Se-oxyanions bioconversion as compared to other systems [143]. Although the membrane of the MBfR system can be made of either organic or inorganic materials, mostly hollow-fiber membranes are used at high gas pressures, providing a high surface-to-volume ratio [23]. Moreover, hydrophobic membranes are generally used in these systems, allowing to maintain the pores dry to achieve a fast diffusion of gas molecules [23]. In the MBfR system, the reduction of Se-oxyanions is coupled with the oxidation of H2 , acting as electron donor, which supports the growth of the autotrophic microbial consortia [129]. SeO4 2− removal in this system has been improved to 94% by changing H<sup>2</sup> pressure, with Se0 retained inside the microbial biofilm [129] in the form of crystalloid aggregates [147]. Similarly to the ABMet® system, the MBfR reactor resulted able to remove several oxidized toxic contaminants, such as chromium and arsenic, along with Se-oxyanions [23]. The microbial composition of a MBfR system exposed to different concentrations of SeO<sup>4</sup> 2− was characterized by Ontiveros-Valencia and coworkers through 16S rRNA pyrosequencing [147]. Results showed that biofilms exposed to a high load of SeO<sup>4</sup> 2− were composed principally by denitrifying bacteria belonging to the genera of *Denitratisoma* and *Dechloromonas*, which were previously reported as capable of reducing SeO4 2− [147]. Recently, Lay and coworkers developed an MBfR system in which methane gas (CH4 ) acted as electron donor instead of H2 , exploiting the microbial consortium capability to oxidize CH4 coupled with SeO4 2− reduction [148]. Particularly, the utilization of methane over H2 has the advantages of lower cost and high availability from anaerobic digestion. Once again, the final product of the process are Se0 -nanospheres, accumulated in the microbial biomass [148]. A characterization of the microbial consortium by 16S rRNA sequencing revealed the presence of a specific methanotrophic genus (*Methylomonas*) that is able to simultaneously oxidize CH4 and reduce SeO4 2−, along with methanotrophic bacteria, which, upon methane utilization, are capable of generating organic metabolites suitable as electron donors for SeO4 2−-reducing microorganisms present in the biofilm [148]. Although the MBfR system resulted to be a promising technology to efficiently remove Se-oxyanions from contaminated environments, its implementation at industrial scale has not been investigated yet, likely due to the high cost of electron donors needed to the working-system, which is still prohibitive for large-scale applications [143].

#### *3.2.4. The UASB system*

*3.2.3. The BSeR and MBfR systems*

cess proficiency toward both SeO<sup>4</sup>

Se0

126 Biosorption

tion of H2

H2

are Se0

sure, with Se0

consortia [129]. SeO4

*Arthrobacter* sp.) adapted to high concentration of SeO3

converting these oxyanions to their elemental form (Se0

bubble-less gas transfer to a membrane delivering H2

Results showed that biofilms exposed to a high load of SeO<sup>4</sup>

, exploiting the microbial consortium capability to oxidize CH4

trophic genus (*Methylomonas*) that is able to simultaneously oxidize CH4

erating organic metabolites suitable as electron donors for SeO4

were previously reported as capable of reducing SeO4

developed an MBfR system in which methane gas (CH4

tion [148]. Particularly, the utilization of methane over H2

the presence of specific biofilm regions where Se<sup>0</sup>

Reactors containing multispecies biofilms (BSeR) represent another promising approach for the treatment of Se-contaminated wastewaters. Indeed, microbial biofilms play a dominant role in the biogeochemical natural cycle of different inorganic compounds. In a recent study, a multispecies biofilm composed of strains (i.e., *Rhodococcus* sp., *Pseudomonas* sp., *Bacillus* sp. and

ticles, associated with the microbial biomass [142]. In the BSeR methodology, bacterial biofilms are grown on granular activated carbon in anaerobic fixed-film reactors showing a high biopro-

from agriculture drainage wastewater (Garfield Wetlands-Kessler Springs, Utah, USA) [144].

Another configuration of reactor based on microbial biofilms is the membrane biofilm reactor (MBfR) [129, 130, 145, 146]. MBfR in its standard configuration consists of a bundle of

of autohydrogenotrophic bacteria (e.g., *Cupriavidus metallidurans*) on the outer surface of the membrane [146], resulting in a higher efficiency of Se-oxyanions bioconversion as compared to other systems [143]. Although the membrane of the MBfR system can be made of either organic or inorganic materials, mostly hollow-fiber membranes are used at high gas pressures, providing a high surface-to-volume ratio [23]. Moreover, hydrophobic membranes are generally used in these systems, allowing to maintain the pores dry to achieve a fast diffusion of gas molecules [23]. In the MBfR system, the reduction of Se-oxyanions is coupled with the oxida-

[147]. Similarly to the ABMet® system, the MBfR reactor resulted able to remove several oxidized toxic contaminants, such as chromium and arsenic, along with Se-oxyanions [23]. The microbial composition of a MBfR system exposed to different concentrations of SeO<sup>4</sup>

characterized by Ontiveros-Valencia and coworkers through 16S rRNA pyrosequencing [147].

by denitrifying bacteria belonging to the genera of *Denitratisoma* and *Dechloromonas*, which

and high availability from anaerobic digestion. Once again, the final product of the process

microbial consortium by 16S rRNA sequencing revealed the presence of a specific methano-

along with methanotrophic bacteria, which, upon methane utilization, are capable of gen-

present in the biofilm [148]. Although the MBfR system resulted to be a promising technology


, acting as electron donor, which supports the growth of the autotrophic microbial

2− removal in this system has been improved to 94% by changing H<sup>2</sup>

retained inside the microbial biofilm [129] in the form of crystalloid aggregates

2− and SeO3

2− has been investigated for its potential in

) [142]. Moreover, it has been highlighted

directly to the grown biofilm consisting

pres-

2− was

2− reduc-

2−,

2− were composed principally

2− [147]. Recently, Lay and coworkers

) acted as electron donor instead of

coupled with SeO4

has the advantages of lower cost

and reduce SeO4

2−-reducing microorganisms

was deposited as sub-micrometer-sized par-

2− [143], which resulted in the recovery of ca. 97% of

Sludge-based reactors have also been employed for the treatment of Se-contaminated wastewaters [68]. Indeed, the most implemented process for anaerobic treatment of industrial effluents is the upflow anaerobic sludge blanket (UASB) reactor, because of the accumulation of microbial biomass and suspended solid, and a dense sludge bed at the bottom of the reactor, in which Se-oxyanions bioconversion occurs [68]. In this regard, the natural aggregation of some bacteria forming flocculates or granules leads to a high retention of active anaerobic sludge even at great organic load rates [149]. Additionally, the wastewater is kept in good contact with the bacterial biomass through both the turbulence of the upflow influent flow and the biogas produced by the anaerobic microorganisms [68]. UASB reactors have been pilot-tested for Se-removal at the Adams Avenue Agricultural Drainage Research Center in San Joaquin Valley (California) [150]. The influent had a total Se content of 500 μg L−1 and the removal efficiency ranged from 58 to 90% [150]. The efficiency of UASB reactors for the removal of Se-oxyanions was tested by Lenz and coworkers in a series of studies evaluating SeO4 2− removal from synthetic wastewater by microbial consortia under methanogenic, sulfate-reducing and denitrifying conditions [151–153]. Using lactate as electron donor, a SeO4 2− removal efficiency of 99% was obtained in both methanogenic and sulfate-reducing conditions, demonstrating that UASB reactors can be effectively applied to remove SeO<sup>4</sup> 2− from contaminated wastewaters, with the involvement of sulfate-reducing bacteria (sulfatereducing conditions) and a selenium-respiring sub-population (methanogenic conditions) [151]. Since the use of UASB reactors under methanogenic conditions leads to the recovery of decontaminated water, Se0 and energy, methanogenic sludges are promising for Se-oxyanions bioconversion [143]. Further, Dessì and coworkers evaluate SeO4 2− removal in UASB reactors as function of the temperature, observing that the maximum efficiency of removal was obtained at thermophilic conditions (55°C) [154]. Another advantage of working at this temperature is the better retention of reduced Se in the microbial biomass. Additionally, they performed a characterization of the microbial consortia through DGGE analysis, correlating the high SeO4 2− removal efficiency to the presence of SeO<sup>4</sup> 2−-respiring microorganisms, such as *Sulfurospirillum barnesii* and *D. indicum* [154]. UASB reactors are very promising for removing Se-oxyanions from contaminated wastewaters, however they require constant control, since any change in operation conditions may lead to an increase of the effluent Se-concentration through either biomethylation or bioconversion of Se-species [23].

#### *3.2.5. The SBR system*

Se-wastewater can be processed using a sequencing batch reactor (SBR), in which the biodegradation and solid separation take place in the same reactor [23]. In this configuration, the treatment is carried out in consecutive stages in the same tank: filling, reaction, sedimentation, draw, purging and inactivity [155]. The selection and enrichment of the desired microbial consortia is achieved by the alternation of anaerobic and aerobic phases, which results in the complete integration of both oxic and anoxic conditions in the same reactor [69, 155]. The SRB systems have been mostly used in the treatment of textile wastewater, thanks to their efficiency in removing dyes [69]. Further, this system has been employed for Se-laden wastewater treatment by Rege and coworkers, which used a denitrifying bacterial consortium for the reduction of both SeO3 2− and SeO4 2− with acetate as electron donor, observing a lag phase of 150 h and a SeO3 2− reduction rate higher than SeO4 2− [156]. In other studies, SBR reactors have been used for the remediation of SeO4 2− specifically inoculating the bacterial strains *Thauera selenatis* [157] and *Bacillus* sp. SF-1 [158]. However, SeO3 2− accumulation in the reactor over the time exerted to a toxic effect toward the bacteria present in the system [158]. More recently, Mal and coworkers studied the potential of SeO4 2− removal in the presence of NH4 + in an SBR inoculated with an activated sludge collected from a wastewater treatment plant [159]. In this study, the microbial consortium removed up to 100% of SeO<sup>4</sup> 2− and 95% of ammonium through partial nitrification as well as nitrification/denitrification, with alternating between anaerobic and aerobic phases [159]. The efficiency of the system was improved by prolonging the anaerobic phase from 3 to 4.5 h. Interestingly, the effluent presented low concentrations of both volatile and elemental Se, suggesting that most part of biogenic Se0 formed by the microbial consortium was retained in the activated sludge [159].

Even if the performances of the bioreactor configurations described above are promising, there are still challenges for the utilization of these approaches to remediate Se-laden wastewater, such as the presence of co-contaminations with different types of metals, the discharge limits for the effluent, and the disposal of the concentrated selenium solids [23, 143]. The bioremediation of Se-contaminated soils has been less explored than wastewater treatment. In this regard, a study by Prakash and coworkers, analyzing the capability of a microbial consortium, composed by aerobic rhizo-bacteria belonging to *Bacillus* genus, to remove SeO4 2− and SeO3 2− contamination from soils amended with different concentrations of these oxyanions [160]. The study revealed higher rate of removal for SeO3 2− as compared to SeO4 2−, due to the greater bioavailability in the soils of SeO3 2− [160]. Moreover, microbial consortia can play a major role in assisting hyperaccumulator plants in phytoremediation approaches by enhancing both plant growth and Se-accumulation (**Figure 2**) [161, 162].

Lake (California) [22]. Thus, the identified slurries were exposed under anaerobic conditions

**Figure 2.** Schematic illustration of a phytoremediation system for the treatment of Se-wastewater through a synergistic cooperation of a Se-hyperaccumulator plant and selenite/selenate bioconverting bacteria of the rhizosphere [162].

they were incubated at 28°C for 30 days [22]. During the timeframe of microbial consortium's growth, a progressively blackening of the cultures has been observed, which indicated both

More recently, Ramos-Ruiz and coworkers analyzed an anaerobic mixed microbial culture in a methanogenic granular sludge obtained from a wastewater treatment plant at Mahou's (beer brewery in Spain) [163]. In this regard, the granular sludge was chosen over planktonic cells considering that the latter one should be exposed more directly to the toxic Te-species [163]. As a result, the anaerobic sludge was able to catalyze the reduction of both TeO4

2− added to the system at a concentration of 20 mg L−1, showing a rate of TeO3

Te-oxyanions bioconversion by the anaerobic sludge, the formation of intra and extracellular Te-nanoprecipitates has been detected through electron microscopy [163]. Interestingly, the microbial consortium did not show any lag phase when exposed to Te-oxyanions even in the case of a sludge originated from wastewater not contaminated with Te-species [163]. In order

duced by SRB microorganisms generally present in microbial consortia, all the experiments have been performed in a (S)-free medium. Furthermore, the authors observed an increase

with riboflavin and lawsone causing the highest effect [163]. Finally, the addition of these redox mediators increased the percentage of extracellular Te-nanoprecipitates, determining a

Te-oxyanions bioreduction and the simultaneous accumulation of Te0

to avoid the possibility of an abiotic bioreduction of TeO4

change in the shape of the nanomaterials produced [163].

by electron microscopy observations of the solid phase of the slurries [22].

2− with either lactate or H2

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129

2− one in all conditions tested [163]. As a consequence of

2− and/or TeO3

2− reduction rates after the amendment of different redox mediators,

as electron donors, and

precipitates, as proven

2− by biogenic S2− pro-

2− and

2− reduc-

of growth to different concentrations of TeO<sup>3</sup>

tion seven-fold higher than TeO4

2− and TeO3

TeO3

of both TeO4

## **3.3. Microbial consortia for Te-removal from contaminated environments**

Since Te-biogeochemistry is still poorly understood [34], to date few examples of microbial consortia employed for the bioconversion of Te-oxyanions into their elemental state (Te0 ) are available in the literature [8]. Further, although Te-species are toxic for living organisms at very low concentrations [6], evaluating the actual amount of Te-contaminants present in environmental samples is challenging, due to their low general availability on Earth [34]. Indeed, even if TeO4 2−- and/or TeO3 2−- reducing bacteria are frequently isolated from natural microbial communities adapted to the stress exerted by Te-oxyanions [28, 107], the application of microbial consortia for their removal from contaminated matrices is still in its infancy.

One of the first studies regarding bioremediation of Te-contaminated environments was carried out by Baesman and coworkers, which isolated sediment slurries resistant to TeO3 2− at Mono

consortia is achieved by the alternation of anaerobic and aerobic phases, which results in the complete integration of both oxic and anoxic conditions in the same reactor [69, 155]. The SRB systems have been mostly used in the treatment of textile wastewater, thanks to their efficiency in removing dyes [69]. Further, this system has been employed for Se-laden wastewater treatment by Rege and coworkers, which used a denitrifying bacterial consortium for

time exerted to a toxic effect toward the bacteria present in the system [158]. More recently,

inoculated with an activated sludge collected from a wastewater treatment plant [159]. In

through partial nitrification as well as nitrification/denitrification, with alternating between anaerobic and aerobic phases [159]. The efficiency of the system was improved by prolonging the anaerobic phase from 3 to 4.5 h. Interestingly, the effluent presented low concentrations of

Even if the performances of the bioreactor configurations described above are promising, there are still challenges for the utilization of these approaches to remediate Se-laden wastewater, such as the presence of co-contaminations with different types of metals, the discharge limits for the effluent, and the disposal of the concentrated selenium solids [23, 143]. The bioremediation of Se-contaminated soils has been less explored than wastewater treatment. In this regard, a study by Prakash and coworkers, analyzing the capability of a microbial consortium, composed by aerobic rhizo-bacteria belonging to *Bacillus* genus, to remove SeO4

2− contamination from soils amended with different concentrations of these oxyanions

major role in assisting hyperaccumulator plants in phytoremediation approaches by enhanc-

Since Te-biogeochemistry is still poorly understood [34], to date few examples of microbial consortia employed for the bioconversion of Te-oxyanions into their elemental state (Te0

available in the literature [8]. Further, although Te-species are toxic for living organisms at very low concentrations [6], evaluating the actual amount of Te-contaminants present in environmental samples is challenging, due to their low general availability on Earth [34]. Indeed,

communities adapted to the stress exerted by Te-oxyanions [28, 107], the application of micro-

One of the first studies regarding bioremediation of Te-contaminated environments was carried

bial consortia for their removal from contaminated matrices is still in its infancy.

out by Baesman and coworkers, which isolated sediment slurries resistant to TeO3

2− with acetate as electron donor, observing a lag phase of

2− specifically inoculating the bacterial strains *Thauera* 

2− removal in the presence of NH4

2− as compared to SeO4

2− [160]. Moreover, microbial consortia can play a

2−- reducing bacteria are frequently isolated from natural microbial

2− [156]. In other studies, SBR reactors have

2− accumulation in the reactor over the

+

formed by the micro-

2− and 95% of ammonium

in an SBR

2− and

) are

2− at Mono

2−, due to the

the reduction of both SeO3

been used for the remediation of SeO4

150 h and a SeO3

128 Biosorption

SeO3

even if TeO4

2− and SeO4

*selenatis* [157] and *Bacillus* sp. SF-1 [158]. However, SeO3

bial consortium was retained in the activated sludge [159].

[160]. The study revealed higher rate of removal for SeO3

ing both plant growth and Se-accumulation (**Figure 2**) [161, 162].

**3.3. Microbial consortia for Te-removal from contaminated environments**

greater bioavailability in the soils of SeO3

2−- and/or TeO3

Mal and coworkers studied the potential of SeO4

2− reduction rate higher than SeO4

this study, the microbial consortium removed up to 100% of SeO<sup>4</sup>

both volatile and elemental Se, suggesting that most part of biogenic Se0

**Figure 2.** Schematic illustration of a phytoremediation system for the treatment of Se-wastewater through a synergistic cooperation of a Se-hyperaccumulator plant and selenite/selenate bioconverting bacteria of the rhizosphere [162].

Lake (California) [22]. Thus, the identified slurries were exposed under anaerobic conditions of growth to different concentrations of TeO<sup>3</sup> 2− with either lactate or H2 as electron donors, and they were incubated at 28°C for 30 days [22]. During the timeframe of microbial consortium's growth, a progressively blackening of the cultures has been observed, which indicated both Te-oxyanions bioreduction and the simultaneous accumulation of Te0 precipitates, as proven by electron microscopy observations of the solid phase of the slurries [22].

More recently, Ramos-Ruiz and coworkers analyzed an anaerobic mixed microbial culture in a methanogenic granular sludge obtained from a wastewater treatment plant at Mahou's (beer brewery in Spain) [163]. In this regard, the granular sludge was chosen over planktonic cells considering that the latter one should be exposed more directly to the toxic Te-species [163]. As a result, the anaerobic sludge was able to catalyze the reduction of both TeO4 2− and TeO3 2− added to the system at a concentration of 20 mg L−1, showing a rate of TeO3 2− reduction seven-fold higher than TeO4 2− one in all conditions tested [163]. As a consequence of Te-oxyanions bioconversion by the anaerobic sludge, the formation of intra and extracellular Te-nanoprecipitates has been detected through electron microscopy [163]. Interestingly, the microbial consortium did not show any lag phase when exposed to Te-oxyanions even in the case of a sludge originated from wastewater not contaminated with Te-species [163]. In order to avoid the possibility of an abiotic bioreduction of TeO4 2− and/or TeO3 2− by biogenic S2− produced by SRB microorganisms generally present in microbial consortia, all the experiments have been performed in a (S)-free medium. Furthermore, the authors observed an increase of both TeO4 2− and TeO3 2− reduction rates after the amendment of different redox mediators, with riboflavin and lawsone causing the highest effect [163]. Finally, the addition of these redox mediators increased the percentage of extracellular Te-nanoprecipitates, determining a change in the shape of the nanomaterials produced [163].

A following study by the same research group evaluated the feasibility to use UASB reactors for the bioconversion of TeO3 2− to Te-nanoprecipitates using a methanogenic microbial consortium in granular sludge, and the subsequent separation of the nanomaterials from the water effluent [164]. In this study, ethanol was added to the system as exogenous source of electrondonating substrate, while riboflavin was supplied as redox mediator during the biological process [164]. UASB reactors were operated with hydraulic retention time of 14 h at 28°C and supplemented with up to 20 mg L−1 of TeO3 2− [164]. Similarly to the above-mentioned study [164], the presence of riboflavin as redox mediator enhanced the efficiency of TeO<sup>3</sup> 2− bioconversion, lowering the toxicity of this oxyanion toward the microbial consortium. Moreover, a continuous removal of TeO3 2− by the anaerobic microbial consortium was observed in the UASB reactor, showing a bioreduction efficiency ranging from 83%, when riboflavin was absent, to 99.5%, when riboflavin was added to the system [164].

whose process occurs at standard conditions (i.e., near neutral pH, controlled temperature and pressure), and, more importantly, avoiding the use of harsh reducing agents as well as

Microbial-Based Bioremediation of Selenium and Tellurium Compounds

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Considering the peculiar photoconductive, semiconductive and optical properties of Se, the use of Se-based nanomaterials has been investigated in a wide range of applications, such as in the production of new optical devices, photovoltaic solar cells, photographic exposure meters and rectifiers and photo-assisted fuel cells [172–175]. Moreover, Se-nanostructures resulted to act as good catalyst for both the chelation of mercury ions (Hg2+) present as contaminants in different polluted environments [176], and the degradation of several toxic chemical com-

matrices [178]. Similarly, Te is a narrow band-gap *p*-type semiconductor, which is featured by high photoconductivity, piezoelectricity and thermoelectricity [168, 169]. These versatile properties led to the exploitation of Te-nanomaterials as optoelectronic, piezoelectric and thermoelectric devices, infrared detectors and gas sensors [179, 180], to name a few. Further, since these chalcogen-nanostructures showed great adsorptive ability, biological reactivity and antioxidant functions, their use in biomedicine have been recently explored [8, 170, 181]. Both Se- and Te-nanomaterials resulted efficient tools in protecting living organisms from DNA oxidation [181], as well as promising antimicrobial and anticancer agents [182–187]. In this regard, several Se-nanostructures produced by different microorganisms have been tested for their antimicrobial efficacy, highlighting their ability to prevent the growth of pathogenic bacteria (i.e., *E. coli*, *P. aeruginosa*, *S. aureus*) either in the form of planktonic cells or as biofilms [182, 183, 186, 187]. Particularly, biogenic Se-nanomaterials resulted to be more efficient as compared to those synthesized by mean of chemical processes, showing a strong inhibitory effect of pathogenic bacterial growth at lower concentrations [183]. Moreover, studies carried out to evaluate the cytotoxicity of biogenic Se-nanostructures toward human cell lines (i.e., fibroblasts and dendritic cells) revealed their high biocompatibility [187], which is a fundamental feature for their possible biomedical applications. Although Te-nanostructures produced by microorganisms are less studied for biomedical applications than those containing Se, recently the potential of such nanomaterials as antimicrobials has been assessed [186], showing their good efficacy in inhibiting pathogens growth. Further, a promising technological application of biogenic Te-based nanostructures regards the production of quantum dots (QDs), which are semiconductors nanocrystals featured by unique electronic and optical

Bioremediation strategies of Se- and Te-polluted environments based on the ability of microorganisms to bioprocess these toxic oxyanion species is an environmental-sustainable choice to reclaim contaminated soils, groundwater, surface water bodies and sediments. The primary microbial process after biosorption is the bioreduction of chalcogen-oxyanions into their less

materials, which can be recovered from the biomasses and used for technological purposes.

and Te0

) generating, as end-products nanoscale

O2

in different

131

the production of toxic wastes deriving from the chemical synthesis approaches [171].

pounds (e.g., trypan blue dye) [177], as well as an efficient bio-sensor for H<sup>2</sup>

properties, due to quantum confinement effects [188].

toxic and bioavailable elemental forms (i.e., Se0

**5. Summary**

TeO3 2− removal from wastewater using a UASB bioreactor was also recently investigated by Mal and coworkers, which inoculated a UASB reactor with anaerobic granular sludge fed with lactate as carbon source, with a hydraulic retention time of 12 h at 30°C [165]. In the UASB reactor, firstly a concentration of 10 mg L−1 of TeO3 2− was added, which was subsequently increased after 42 days to 20 mg L−1. Te-oxyanion removal started immediately after the initial TeO3 2− addition [165]. Particularly, after the first 3–4 weeks of sludge incubation in the reactor, a significant improvement of TeO<sup>3</sup> 2− removal efficiency was observed, suggesting an adaptation of the microbial consortium to the presence of this oxyanion [165]. Furthermore, TeO3 2− was almost completely bioconverted to its elemental state in the form of Te-nanostructures associated with the loosely bound EPS fraction surrounding the sludge, suggesting a pivotal role played by EPS and its functional groups in the biogenesis of Te-nanoprecipitates. In this regard, the possibility to easily recover Te-nanostructures associated with the EPS fraction opens new possibility to combine oxyanion removal with the recovery of Te0 [165].
