Microbial-Based Bioremediation of Selenium and Tellurium Compounds

The chalcogens selenium (Se) and tellurium (Te) are rare earth elements, which are mainly present in the environment as toxic oxyanions, due to the anthropogenic activities. Thus, the increased presence of these chalcogen-species in the environment and the contamination of wastewaters nearby processing facilities led to the necessity in developing remediation strategies aimed to detoxify waters, soils and sediments. Among the different decontamination approaches, those based on the ability of microorganisms to bioaccumulate, biomethylate or bioconvert Se- and/or Te-oxyanions are considered the leading strategy for achieving a safe and eco-friendly bioremediation of polluted sites. Recently, several technologies based on the use of bacterial pure cultures, bacterial biofilms or microbial consortia grown in reactors with different configurations have been explored for Se- and Te-decontamination purposes. Further, the majority of microorganisms able to process chalcogen-oxyanions have been described to generate valuable Se- and/or Te-nanomaterials as end-products of their bioconversion, whose potential applications in biomedicine, optoelectronics and environmental engineering are still under investigation. Here, the occurrence, the use and the toxicity of Se- and Te-compounds will be briefly overviewed, while the microbial mechanisms of chalcogen-oxyanions bioprocessing, as well as the microbial-based strategies used for bioremediation approaches will be extensively described

Biogenic selenium and tellurium nanoparticles synthesized by environmental microbial isolates efficaciously inhibit bacterial planktonic cultures and biofilms

The present study deals with Se(0)- and Te(0)-based nanoparticles bio-synthesized by two selenite- and tellurite-reducing bacterial strains, namely Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. MPV1, isolated from polluted sites. We evidenced that, by regulating culture conditions and exposure time to the selenite and tellurite oxyanions, differently sized zero-valent Se and Te nanoparticles were produced. The results revealed that these Se(0) and Te(0) nanoparticles possess antimicrobial and biofilm eradication activity against Escherichia coli JM109, Pseudomonas aeruginosa PAO1, and Staphylococcus aureus ATCC 25923. In particular, Se(0) nanoparticles exhibited antimicrobial activity at quite low concentrations, below that of selenite. Toxic effects of both Se(0) and Te(0) nanoparticles can be related to the production of reactive oxygen species upon exposure of the bacterial cultures. Evidence so far achieved suggests that the antimicrobial activity seems to be strictly linked to the dimensions of the nanoparticles: indeed, the highest activity was shown by nanoparticles of smaller sizes. In particular, it is worth noting how the bacteria tested in biofilm mode responded to the treatment by Se(0) and Te(0) nanoparticles with a susceptibility similar to that observed in planktonic cultures. This suggests a possible exploitation of both Se(0) and Te(0) nanoparticles as efficacious antimicrobial agents with a remarkable biofilm eradication capacity

Antimicrobial activity of biogenically produced spherical Se-nanomaterials embedded in organic material against Pseudomonas aeruginosa and\ua0Staphylococcus aureus strains on hydroxyapatite-coated surfaces

In an effort to prevent the formation of pathogenic biofilms on hydroxyapatite (HA)-based clinical devices and surfaces, we present a study evaluating the antimicrobial efficacy of Spherical biogenic Se-Nanostructures Embedded in Organic material (Bio Se-NEMO-S) produced by Bacillus mycoides SelTE01 in comparison with two different chemical selenium nanoparticle (SeNP) classes. These nanomaterials have been studied as potential antimicrobials for eradication of established HA-grown biofilms, for preventing biofilm formation on HA-coated surfaces and for inhibition of planktonic cell growth of Pseudomonas aeruginosa NCTC 12934 and Staphylococcus aureus ATCC 25923. Bio Se-NEMO resulted more efficacious than those chemically produced in all tested scenarios. Bio Se-NEMO produced by B.\ua0mycoides SelTE01 after 6 or 24\ua0h of Na2 SeO3 exposure show the same effective antibiofilm activity towards both P.\ua0aeruginosa and S.\ua0aureus strains at 0.078\ua0mg\ua0ml(-1) (Bio Se-NEMO6 ) and 0.3125\ua0mg\ua0ml(-1) (Bio Se-NEMO24 ). Meanwhile, chemically synthesized SeNPs at the highest tested concentration (2.5\ua0mg\ua0ml(-1) ) have moderate antimicrobial activity. The confocal laser scanning micrographs demonstrate that the majority of the P.\ua0aeruginosa and S.\ua0aureus cells exposed to biogenic SeNPs within the biofilm are killed or eradicated. Bio Se-NEMO therefore displayed good antimicrobial activity towards HA-grown biofilms and planktonic cells, becoming possible candidates as new antimicrobials

Biogenic selenium nanoparticles: characterization, antimicrobial activity and effects on human dendritic cells and fibroblasts

Tailored nanoparticles offer a novel approach to fight antibiotic-resistant microorganisms. We analysed biogenic selenium nanoparticles (SeNPs) of bacterial origin to determine their antimicrobial activity against selected pathogens in their planktonic and biofilm states. SeNPs synthesized by Gram-negative Stenotrophomonas maltophilia [Sm-SeNPs()] and Gram-positive Bacillus mycoides [Bm-SeNPs(+)] were active at low minimum inhibitory concentrations against a number of clinical isolates of Pseudomonas aeruginosa but did not inhibit clinical isolates of the yeast species Candida albicans and C. parapsilosis. However, the SeNPs were able to inhibit biofilm formation and also to disaggregate the mature glycocalyx in both P. aeruginosa and Candida spp. The Sm-SeNPs() and Bm-SeNPs(+) both achieved much stronger antimicrobial effects than synthetic selenium nanoparticles (Ch-SeNPs). Dendritic cells and fibroblasts exposed to Sm-SeNPs(), Bm-SeNPs(+) and Ch-SeNPs did not show any loss of cell viability, any increase in the release of reactive oxygen species or any significant increase in the secretion of pro-inflammatory and immunostimulatory cytokines. Biogenic SeNPs therefore appear to be reliable candidates for safe medical applications, alone or in association with traditional antibiotics, to inhibit the growth of clinical isolates of P. aeruginosa or to facilitate the penetration of P. aeruginosa and Candida spp. biofilms by antimicrobial agents

Bacteria facing chalchogens: biogenic formation of Se and Te nanoparticles and evaluation of their antimicrobial potential

I calcogenuri selenio e tellurio possono essere presenti in tracce nell'ambiente. I due elementi sono insolubili in acqua e privi di tossicit\ue0 nella loro forma elementale (Se0 e Te0), mentre gli ossianioni selenito (SeO32-) e tellurito (TeO32-) sono solubili in acqua e tossici per gli organismi. La tossicit\ue0 di questi ossianioni \ue8 dovuta alla loro attivit\ue0 ossidante che pu\uf2 interferire con le funzione cellulari fondamentali. I batteri hanno sviluppato diversi meccanismi per la trasformazione di forme metalliche ossianioniche a forme non tossiche. Infatti, diversi ceppi batterici hanno evidenziato la capacit\ue0 di ridurre selenito a selenio elementale in diverse condizioni, con la conseguente formazione di nanoparticelle con diverse morfologie, sia all'interno che all'esterno della cellula microbica. Similarmente, la riduzione di tellurito a tellurio elementale \ue8 stata osservata in differenti frazioni cellulari di diverse specie batteriche. Questi aspetti sono particolarmente interessante dal momento che il recente sviluppo delle nanotecnologie ha attirato un crescente interesse nello sfruttamento dell'abilit\ue0 naturale dei sistemi biologici di produrre nanoparticelle con propriet\ue0 definite sia nel citoplasma che nello spazio extracellulare. Questo processo pu\uf2 essere infatti considerato come un'alternativa economica ed ecosostenibile ai tradizionali metodi chimici e fisici di sintesi. Nel presente progetto di dottorato \ue8 stata valutata sia la possibilit\ue0 di utilizzare isolati batterici per produrre nanoparticelle di selenio e tellurio che la possibile applicazione di questi nanomateriali. Nella prima parte della tesi \ue8 stato analizzato il meccanismo coinvolto nella formazione di nanoparticelle biogeniche in diversi ceppi batterici. In particolare, la riduzione del selenito a selenio elementale \ue8 stata valutata nei ceppi Stenotrophomonas maltophilia SeITE02, Bacillus mycoides SeITE01, mentre la riduzione di selenito e tellurito \ue8 stata analizzata nel ceppo Ochrobactrum sp. MPV1. Sulla base dei dati discussi nel presente lavoro di tesi, \ue8 stato evidenziato sia il coinvolgimento di composti contenenti gruppi tiolici (glutatione per Stenotrophomonas maltophilia SeITE02 e Ochrobactrum sp. MPV1, bacillo-tioli per Bacillus mycoides SeITE01) che di enzimi intra o extracellulari. D'altro canto, la riduzione del tellurito da parte di Ochrobactrum sp. MPV1 pu\uf2 essere legata all'attivit\ue0 riduttiva di enzimi intracellulari NADH dipendenti, i quali evidenziano un meccanismo di riduzione diverso da quello responsabile per la riduzione del selenito. Nella seconda parte della tesi, le propriet\ue0 chimico-fisiche delle nanoparticelle biogeniche prodotte da questi ceppi batterici sono state studiate. SeNPs biogeniche hanno evidenziato dimensioni simili (dipendenti dal tempo di incubazione) ed elevata stabilit\ue0 (potenziale Z negativo). Anche le TeNPs estratte da Ochrobactrum sp. MPV1 hanno rivelato elevata stabilit\ue0 (potenziale Z positivo). E' stata inoltre studiata l'attivit\ue0 antibatterica di queste nanoparticelle biogeniche contro ceppi di riferimento. SeNPs hanno evidenziato attivit\ue0 antibatterica a concentrazioni basse. L'effetto tossico di queste nanoparticlle pu\uf2 essere correlato alla produzione di specie reattive dell'ossigeno dopo l'esposizione alla coltura batterica. I dati raccolti fino ad ora suggeriscono che l'attivit\ue0 antibatterica sembra essere strettamente correlata alle dimensioni delle nanoparticelle: \ue8 stato infatti osservato che la pi\uf9 alta attivit\ue0 \ue8 stata mostrata dalle nanoparticelle di dimensioni inferiori. E' importante notare, in particolare, che batteri cresciuti sottoforma di biofilm hanno evidenziato una tolleranza simile a quella evidenziata dalle colture planktoniche. Sono stati inoltre effettuati test comparativi mirati all'analisi dell'attivit\ue0 antibatterica di nanoparticelle di selenio estratte da due ceppi microbici differenti, Stenotrophomonas maltophilia SeITE02 e Bacillus mycoides SeITE01. Queste nanoparticelle biogeniche hanno evidenziato un potenziale antimicrobico pi\uf9 elevato di quello esibito da nanoparticelle di selenio sintetizzate chimicamente. Infine, \ue8 stata misurata su fibroblasti umani e cellule dendritiche sia la tossicit\ue0 che l'induzione della produzione di citochine da parte di questi nanomateriali: questi esperimenti hanno evidenziato l'assenza di tossicit\ue0 e induzione di produzione di citochine solo a concentrazioni molto elevate (250 e 500 mg/L). Tutti questi risultati aprono alla prospettiva di un possibile sfruttamento di nanoparticelle di selenio e tellurio come efficati agenti antimicrobici con un'elevato potere di eradicazione di biofilm.The chalcogens selenium and tellurium can be found as trace elements in the environment. Selenium and tellurium in their elemental form (i.e. Se0 and Te0) are insoluble in water and almost non-toxic, while the oxyanions selenite (SeO32-) and tellurite (TeO32-) are highly soluble in water and toxic to biological systems. The toxicity of these oxyanions has been ascribed to their oxidizing activity, which can interfere with fundamental cellular functions. Bacteria have developed different mechanisms for the transformation of metal oxyanions to non-toxic forms. In fact, a number of bacterial strains has revealed the capacity to reduce selenite to elemental selenium in different conditions, with formation of nanoparticles with various morphological characteristics, either inside the cells or extracellularly. Similarly, tellurite reduction to elemental tellurium nanoparticles has been reported in different cellular compartments within a wide range of bacterial species. This appears particularly worth of note since the recent boost of nanotechnology has attracted growing interest in the exploitation of the natural ability of biological systems to generate nanomaterial with well-defined properties both in the cytoplasm and outside the bacterial cells. Indeed, this can be considered as a green and ecofriendly alternative to the chemical and physical methods conventionally used to synthesize nanomaterials. In the present PhD work, the utilization of bacterial strains to produce selenium and tellurium nanoparticles along with the possible applications of these biogenic nanomaterials have been evaluated. In the first part of this dissertation, the mechanisms involved in the biogenic formation of nanoparticles by different microbial isolates have been analyzed. In particular, the reduction of selenite to elemental selenium nanoparticles has been studied in Stenotrophomonas maltophilia SeITE02, Bacillus mycoides SeITE01, while the reduction of both selenite and tellurite has been evaluated in Ochrobactrum sp. MPV1. On the basis of the evidences discussed in this thesis, the involvement of both thiolic compounds (glutathione for Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. MPV1, bacillithiols for Bacillus mycoides SeITE01) and intracellular/extracellular enzymes can be singled out. On the other hand, tellurite reduction in Ochrobactrum sp. MPV1 may be attributed to the activity of an intracellular NADH-dependent enzyme, exhibiting a reducing mechanism different from that involved in selenite reduction. In the second part of the dissertation, biogenic nanomaterials produced by the microbial strains of interest are characterized in terms of physico-chemical parameters as well as for their biological activity. Biogenic SeNPs showed similar sizes (dependent on incubation time) and high stability (negative \u3b6-potential). In this respect, also biogenic TeNPs from Ochrobactrum sp. MPV1 revealed a high stability (positive \u3b6-potential). Moreover, the antimicrobial potential of these biogenic nanomaterials has been investigated against reference strains. In particular, Se0 nanoparticles exhibited antimicrobial activity at quite low concentrations. Toxic effects of both Se0 and Te0 nanoparticles can be related to the production of reactive oxygen species upon exposure of the bacterial cultures. Evidence so far achieved suggests that the antimicrobial activity seems to be strictly linked to the dimensions of the nanoparticles: indeed, the highest activity was shown by nanoparticles of smaller sizes. In particular, it is worth noting that bacteria tested in biofilm mode of growth responded to the treatment by Se0 and Te0 nanoparticles with a susceptibility similar to that observed in planktonic cultures. Comparative tests were also performed with biogenic SeNPs extracted from two different bacterial strains, namely Stenotrophomonas maltophilia SeITE02 and Bacillus mycoides SeITE01. These biogenic nanoparticles showed a higher antimicrobial potential than exerted by the chemically synthesized ones. Finally, toxicity and induction of cytokine production by these nanomaterials were tested on human fibroblasts and dendritic cells, evidencing on the one hand no toxic effects, while on the other induction of cytokine production only at high concentrations (250 and 500 mg/L). All these results open the perspective of a possible exploitation of both Se0 and Te0 nanoparticles as efficacious antimicrobial agents with a remarkable biofilm eradication capacity

Biotechnological approach to selenite detoxification through the formation of Se0 nanoparticles by means of a Bacillus mycoides strain isolated from the rhizosphere of Astragalus bisulcatus

Selenium is a trace element commonly found in the earth\u2019s crust. It belongs to the Group 16 (chalcogens) of the Periodic Table and occurs in a variety of oxidation states in the environment. In particular, the predominant Se species in oxic conditions are the oxyanions selenite (SeO32 - ) and selenate (SeO42 - ), with the former exerting the highest toxicity. Interestingly, the ability to reduce SeO32 - into the non - toxic elemental form is widespread among microorganisms. The present work investigates on the re duction mechanisms of selenite to zero valent selenium nanoparticles by Bacillus mycoides SeITE01, a bacterial strain isolated from the rhizosphere of the Se - hyperaccumulator legume Astragalus bisulcatus. The strain SeITE01 exhibits resistance to SeO32 - up to 25 mM and is capable of complete reduction of 0.5 and 2.0 mM SeO32 - within 12 and 24 hours, respectively. SeITE01 also demonstrated to convert 91% of the selenite initially added to the growth medium into elemental selenium, with cultures developing a deep red color characteristic of crystalline monoclinic Se0. However, Se0 production was delayed respect to selenite depletion in the culture medium. Characterization of red Se0 precipitate by using transmission electron microscopy, scanning electron micro scopy and UV - Vis spectroscopy revealed the presence of extracellular spherical nanoparticles. In few cases, also intracellular nanoparticles were detected. Size of such selenium nanoparticles range from 50 to 400 nm in diameter, according to the different incubation times. SeITE01 protein fractions were assayed for selenite reduction activity, which can be associated to membrane proteins and spent culture medium after NADH addition. On the basis of the results gained so far, two different mechanisms for the synthesis of selenium nanoparticles have been proposed. The involvement of proteins/peptides able to extracellularly reduce selenite and a possible reduction of selenite by membrane reductase

Antimicrobial activity of Se0/Te0–based nanoparticles of bacterial origin

In the last few decades, the emergence of bacterial resistance to antibiotics has become a common phenomenon in both community and hospital setting. As a consequence, the effectiveness of antibiotic treatment of bacteriabased infection has progressively decreased. In particular, the treatment of biofilm-associated infection is problematic, since bacteria grown in biofilm mode are more tolerant to conventional antibiotics and biocides compared to free swimming cells. Therefore, it’s necessary to develop and test new antimicrobial compounds having both bactericidal potential and biofilm eradication activity against multidrug-resistant bacteria. In recent years, the employment of metallic nanoparticles has emerged as an alternative to the use of organic compounds as antimicrobial agents. Several studies has been focused particularly on the antimicrobial activity of silver nanoparticles: however other metal or metalloid nanoparticles have exhibited a promising bactericidal capability. However, one of the major drawback for the employment of nanoparticles is the cost associated with the traditional physical-chemical methods of synthesis and the production of toxic substances as byproduct. For these reasons, there is a considerable amount of interest in developing new and eco-friendly processes for the manufacturing of nanoparticles. In the present work, Se0 and Te0-based nanoparticles were bio-synthesized employing the selenite and telluritereducing capability of two bacterial strains isolated from polluted environments: Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. E. By regulating culture conditions and exposition time, we were able to produce nanoparticles of different dimensions, between 50 and 200nm. The nanoparticles were tested against planktonic and biofilms cultures of three common pathogenic strains: Escherichia coli JM109, Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC 25923. We evaluated both the inhibition activity against biofilm and planktonic growth and the eradication activity against biofilms established for 24 hours. To measure these parameters we determined both the minimum biocidal concentration (MBC) and the minimum biofilm eradication concentration (MBEC). In addition, we observed the effect of increasing concentrations of nanoparticles on biofilm structure using Confocal Laser Scanning Microscopy (CLSM). Our results indicate that both Se0 and Te0 nanoparticles possess antimicrobial and biofilm eradication activity. In particular Se0 nanoparticles exhibited antimicrobial activity at lower concentration. Preliminary data suggests that the activity seemed to be dependent on the dimension of the nanoparticles: indeed, the highest activity was shown by the nanoparticles smaller in size. The key observation is that bacteria growth in biofilm mode didn’t exhibit a higher level of resistance against the nanoparticles antimicrobial action. Results described in this study suggest a possible application of both Se0 and Te0 nanoparticles as an effective antimicrobial agent with a high biofilm eradication capacity

Elemental selenium nanoparticles efficiently bio-synthesized by Stenotrophomonas maltophilia SeITE02 possess promising antimicrobial activity

In the present study, Authors analyse dynamics of growth, selenite reduction rate and elemental selenium formation in cultures of Stenotrophomonas maltophilia, strain SeITE02, originally isolated from the rhizosphere of the Se hyperaccumulator legume Astragalus bisulcatus, grown in a seleniferous soil. Selenium nanoparticles synthesized by S. maltophilia SeITE02 were tested for their possible antibacterial activity against Staphylococcus aureus ATTC25923 and Pseudomonas aeruginosa PAO1 grown either as planktonic cells or in biofilm mode

Delayed formation of zero-valent selenium nanoparticles by Bacillus mycoides SeITE01 as a consequence of selenite reduction under aerobic conditions

Selenite (SeO3 2-) oxyanion shows severe toxicity to biota. Different bacterial strains exist that are capable of reducing SeO3 2- to non-toxic elemental selenium (Se0), with the formation of Se nanoparticles (SeNPs). These SeNPs might be exploited for technological applications due to their physico-chemical and biological characteristics. The present paper discusses the reduction of selenite to SeNPs by a strain of Bacillus sp., SeITE01, isolated from the rhizosphere of the Se-hyperaccumulator legume Astragalus bisulcatus. Results: Use of 16S rRNA and GyrB gene sequence analysis positioned SeITE01 phylogenetically close to B. mycoides. On agarized medium, this strain showed rhizoid growth whilst, in liquid cultures, it was capable of reducing 0.5 and 2.0 mM SeO3 2- within 12 and 24 hours, respectively. The resultant Se0 aggregated to form nanoparticles and the amount of Se0 measured was equivalent to the amount of selenium originally added as selenite to the growth medium. A delay of more than 24 hours was observed between the depletion of SeO3 2 and the detection of SeNPs. Nearly spherical-shaped SeNPs were mostly found in the extracellular environment whilst rarely in the cytoplasmic compartment. Size of SeNPs ranged from 50 to 400 nm in diameter, with dimensions greatly influenced by the incubation times. Different SeITE01 protein fractions were assayed for SeO3 2- reductase capability, revealing that enzymatic activity was mainly associated with the membrane fraction. Reduction of SeO3 2- was also detected in the supernatant of bacterial cultures upon NADH addition. Conclusions: The selenite reducing bacterial strain SeITE01 was attributed to the species Bacillus mycoides on the basis of phenotypic and molecular traits. Under aerobic conditions, the formation of SeNPs were observed both extracellularly or intracellullarly. Possible mechanisms of Se0 precipitation and SeNPs assembly are suggested. SeO3 2- is proposed to be enzimatically reduced to Se0 through redox reactions by proteins released from bacterial cells. Sulfhydryl groups on peptides excreted outside the cells may also react directly with selenite. Furthermore, membrane reductases and the intracellular synthesis of low molecular weight thiols such as bacillithiols may also play a role in SeO3 2- reduction. Formation of SeNPs seems to be the result of an Ostwald ripening mechanism

Selenium and tellurium nanomaterials

major advantages, drawbacks as weOver the last 40 years, the rapid and exponential growth of nanotechnology led to the development of vari- ous synthesis methodologies to generate nanomaterials different in size, shape and composition to be applied in various fields. In particular, nanostructures composed of Selenium (Se) or Tellurium (Te) have attracted in- creasing interest, due to their intermediate nature between metallic and non-metallic elements, being defined as metalloids. Indeed, this key shared feature of Se and Te allows us the use of their compounds in a variety of applications fields, such as for manufacturing photocells, photographic exposure meters, piezoelectric devices, and thermoelectric materials, to name a few. Considering also that the chemical-physical properties of ele- ments result to be much more emphasized when they are assembled at the nanoscale range, huge efforts have been made to develop highly effective synthesis methods to generate Se- or Te-nanomaterials. In this context, the present book chapter will explore the most used chemical and/or physical methods exploited to gener- ate different morphologies of metalloid-nanostructures, focusing also the attention on thell as the safety related to these synthetic procedures