Se (IV) triggers faster Te (IV) reduction by soil isolates of heterotrophic aerobic bacteria: formation of extracellular SeTe nanospheres
© Bajaj and Winter; licensee BioMed Central. 2014
Received: 1 August 2014
Accepted: 17 November 2014
Published: 26 November 2014
Selenium and Tellurium have many common chemical properties as both belong to group 16 of the periodic table. High toxicities of Se and Te oxyanions cause environmental problems in contaminated soils and waters. Three strains (C4, C6 and C7) of selenite reducing and nanoparticle forming aerobic bacteria which were isolated from agricultural soils of India containing high concentrations of Se were investigated after 3.5 months of freeze-storage for their resistance against the toxic oxyanion tellurite and its reduction to non toxic elemental form Te0 as well as nanoparticles formation.
Strains C4, C6 and C7 reduced tellurite at maximum reduction rates of 2.3, 1.5 and 2.1 mg Te (IV)/L/d, respectively and produced extracellular Te0 nanospheres as revealed from SEM-EDX analysis. Production of extracellular Te nanospheres has been described seldom. Further, concurrent reduction of both selenite and tellurite by bacteria was examined as these toxic oxyanions are often present together in natural environments, mine tailings or wastewater from copper refining. Interestingly, bioreduction of 100 mg/L selenite in shake flasks was not much affected by the presence of 10 mg/L tellurite but tellurite reduction rate increased 13 fold with selenite in the medium. The concurrent reduction of these oxyanions resulted in rarely described bioformation of extracellular nanoparticles composed of both Se and Te, reported first time for aerobically growing heterotrophic non-halophilic bacterial cultures. Duganella violacienigra, the closely related strain to C4 was also found to be resistant to oxyanions of Se and Te.
Selenite reducing heterotrophic non-halophilic aerobic bacteria revived from 3.5 months freeze storage could successfully reduce toxic tellurite to non toxic elemental form and produced extracellular nanospheres during detoxification. Presence of relatively less toxic selenite in the medium triggers bioreduction of more toxic tellurite leading to formation of extracellular SeTe nanospheres which are sought by solar and optical recording media industry because of their excellent photovoltaic and optical properties. The bacterial cultures investigated in this study could be exploited commercially to remediate not only selenite and tellurite-contaminated soil and water but also for green synthesis of extracellular Se, Te and Se + Te nanospheres.
Selenium and tellurium share many common characteristics e.g. both metalloids are found with copper- and sulfur-bearing ores and exist in four oxidation states i.e. +VI, +IV, 0, and –II in nature. Oxyanions (IV and VI) of both elements are soluble and mobile thus bioavailable and toxic. Elemental (zero valent) Te and Se are non soluble. Both Te and Se are found in relatively low abundances in earth’s crust, yet high toxicities of these elements cause environmental problems in contaminated soils and waters e.g. in agricultural lands or wastewater discharges from industrial activities or mine tailings ,. Due to an increased inclination towards biological treatments, microorganisms capable of reducing toxic oxyanions have been investigated by many researchers -. Relative to arsenic, not many studies discussing Se geo-chemical-microbial cycling in the environment have been reported and the studies focusing on Te are even less than Se. Till now, the biological function of Te and its microbial oxidation is not known. Since concentrations as low as 1 μg/L of tellurite are toxic to most bacteria ,, it is especially used in selection media for Te resistant microorganisms which are often pathogens, e.g. Shiga toxin-producing E.coli ,. Owing to their resemblance to sulfur, Te and Se disrupt the thiol chemistry within cells leading to toxicity in bacteria not resistant to these compounds . In the last decade, many anaerobic bacteria capable of both selenium and tellurium reduction to non toxic forms have been isolated from extreme environments like ocean hydrothermal vents , or the highly alkaline water of Monolake, California ,, but competent aerobic non-halophilic heterotrophic bacteria reducing both Se and Te are not yet known. Concurrent Te and Se reduction has also not been investigated for chalcogen reducing heterotrophic aerobic bacterial cultures except for a few studies describing synergistic reduction of Te + Se by halophilic bacteria ,,.
As described previously , four strains of aerobic bacteria from Se enriched agricultural soils in India were isolated that were able to produce extracellular Se nanospheres during selenite reduction to Se0. The strains C1 and C4 (GenBank NCBI accession numbers JQ745646 and JQ745647) were found to be closely related to Duganella violacienigra species and strains C6 and C7 (GenBank NCBI accession numbers JQ745648 and JQ745649) were related to Agrobacterium tumefaciens species . In the present study strains C4, C6 and C7 were revived after 3.5 months of freeze-storage and were investigated for their resistance against selenite as well as against tellurite. Tellurium is the next element to Se in chalcogen group 16 of the periodic table and shares many properties with each other. Scanning- and transmission electron microscopy (SEM and TEM) with energy dispersive X-ray (EDX) analysis were carried out to observe nanoparticle formation of elemental Se and Te produced as a result of detoxification. The effect of the presence of Se (IV) on Te (IV) reduction was further examined to elucidate bioremediation when multiple detrimental ions are present as it often occurs in natural environments or copper refining. Since isolate C4 was closely related to Duganella violacienigra  which is one of the few known species of this genus therefore, the type strain D. violacienigra DSM 15887 was also examined for its resistance against Te (IV) and Se (IV) as it has not been investigated before.
Results and discussions
Selenite reduction after revival of freezed cultures
Incubation with Tellurite
Bacterial reduction of tellurite and its deposition occur almost exclusively intracellular ,,. Bioformation of extracellular Te nanoparticles is not common and almost all reports describe extracellular deposits as nanorods or needle like nanoprecipitates ,,. The formation of extracellular Te nanospheres was only described by the Te (IV) respiring anaerobic Sulfurospirillum barnesii . This strain also reduced Se (IV) and other metalloids as terminal electron acceptors ,. Production of extracellular spherical Te nanoparticles by heterotrophic aerobic bacteria has not yet been reported. Only a few studies described extracellular Te deposition by aerobic bacteria as Te nano rods  or nanoparticles of up to 300 nm with an undefined shape . Recently, Borghese et al.  described reduction of Te (IV) and Se (IV) oxyanions into extracellular needle like Te0 and globular Se0 nano precipitates during anaerobic growth of the facultative photosynthetic bacterium Rhodobacter capsulatus. The formation of extracellular nano precipitates was dependent on the carbon source that was supplied for bacterial growth. The rate of chalcogen reduction was constant at different concentrations of lawsone that was added as a redox mediator to facilitate the formation of external Te nanoparticles. In the present study, no external redox mediator was required but glucose as a carbon source was essential and effective for both Se (IV) and Te (IV) reduction leading to spherical nanoparticles of Se (0) and Te (0) [; Figure 2d and e) which is reported first time for aerobically growing heterotrophic non-halophilic bacterial cultures. Extracellular nanoparticles could be separated from bulk media and might be used for possible applications where a high surface to volume ratio of spherical shape is advantageous.
Incubation of cultures in the presence of both Selenite and Tellurite
Se and Te oxyanions in the group of chalcogens can be reduced by both, anaerobic and aerobic bacteria. The majority of these reduction processes form chalcogen-containing organics e.g. volatile organometalloids and may produce elemental precipitates or nanoparticles through similar mechanisms via (1) enzymatic reduction, (2) methylation, (3) dissimilatory reduction concomitant with sulfate reduction, (4) Painter-type reactions with the thiols of proteins as well as glutathione and (5) a chemical reaction with the siderophore thiocarboxylic acid and the products of its hydrolysis ,. Turner et al.  proposed a mechanism of tellurite toxicity in which tellurite oxyanions enter a cell via uptake by a phosphate transport system. Subsequently they were reduced to elemental tellurium by glutathione or other reduced thiols. Reactive oxygen species (O2 ‐) produced during reduction of tellurite may be detoxified by superoxide dismutase of aerobic bacteria. A similar mechanism was proposed by Kessi and Hanselmann  for selenite reduction in the glutathione synthesizing cyanobacteria and in α, β, and γ groups of proteobacteria (Gram-negative bacteria), thus being incompatible with strict anaerobes that lack oxidative stress enzymes. In the present study, all test strains were Gram negative proteobacteria. Strain C4 was phylogenetically closely related to β Proteobacteria, Duganella violacienigra spec. whereas, strains C6 and C7 were closely related to α Proteobacteria, Agrobacterium tumefaciens spec. . The presence of Se oxyanions might have triggered glutathione reductase and other enzymes required to counteract Te (IV) and reactive oxygen species. A positive effect of Se oxyanions on tellurite reduction has previously only been reported by Amoozegar et al.  who observed a 25% increase in the removal of 0.5 mM tellurite from a 10% NaCl containing nutrient broth in the presence of 6 mM sodium selenite by a halophilic bacterium, Salinicoccus strain QW6, which was isolated from salty environments of Iran. In addition, in their recent study , improved reduction of 0.1 mM and 0.2 mM Te (IV), respectively by halophilic Halomonas elongata DSM2581 and halotolerant Enterococcus faecalis PTCC1237 was reported in the presence of Se (IV). Increased Te (IV) reduction efficiency in presence of Se (IV) particularly by halophilic or halotolerant bacteria was assumed to be due to the presence of sodium and potassium cations in tellurite and selenite solutions, which were necessary not only for growth and survival of the bacteria but also for enzymatic activities. Alternatively, it might be due to tellurite reductase induced by selenite ions ,,. However, in the present study of non-halophilic/halotolerent aerobic bacteria, Se (IV)-triggered Te (IV) reduction might be due to an increased reductase activity rather than due to an effect on Na+/K+ -ATPase influx.
Tellurite reduction at various selenite concentrations and effect of tellurite re-feeding
Tellurite and Selenite reduction by Duganella violacienigra
Materials and methods
Isolation and identification of bacteria C4, C6 and C7 and their main physicochemical properties have been described in detail previously . All bacteria were isolated from mixed microbial aerobic cultures enriched from a high selenium containing soil obtained from agricultural fields in India. After log-phase growth of the pure cultures in medium without Se (IV) (according to Ghosh et al. ), the cell suspension was mixed with 86% w/v glycerol in the ratio of 3:1 and stored at -20°C in a deep freezer. After 3.5 months of storage the cultures were thawed and 2 ml of respective cultures were suspended in 20 ml medium containing 1 g/L glucose monohydrate and no Se (IV) in 100 ml shake flasks with cotton plugs. Flasks were incubated at 27°C on a rotary shaker at 110 rpm. These bacteria were adapted to high Se (IV) concentrations in successive incubations in fresh medium (40 ml) containing 1 g/L glucose monohydrate and increasing concentrations of Se (IV) with 30-40 mg/L increments during 1 to 1.5 months.
Chalcogens resistance assays
Growth medium  containing 0.03 g NH4Cl, 0.01 g NaCl, 0.01 g MgSO4, 0.15 g yeast extract and 0.5 g peptone per L of deionized water, pH 7.5 supplemented with 1 g/L glucose monohydrate and the indicated concentrations of Se (IV) as Na2SeO3·5H2O or Te (IV) as K2TeO3 (Sigma-Aldrich, Taufkirchen or Serva, Heidelberg, Germany) in 100 ml Erlenmeyer flasks was inoculated with 5% of pure cultures of the bacterial strains C4, C6 and C7, respectively. Concentrated solutions of each C6H12O6·H2O, Na2SeO3·5H2O and K2TeO3, respectively were sterilized separately and added to growth medium under sterile conditions as required. Controls containing inoculum and no chalcogens as well as sterile controls containing Se (IV), Te (IV) or both Se (IV) and Te (IV), respectively were carried out for all assays. Flasks were closed with cotton plugs and incubated at 27 ± 2°C on a rotary shaker at 110 rpm. Working volumes of the assays were 40 to 60 ml and the bacterial concentration was about 1.8 to 2.2 ×107 cells/ml in the flasks at the start of incubation.
Growth of Duganella violacienigrawith Te and Se
A culture of D. violacienigra (DSM 15887) also known as Pseudoduganella violaceinigra  was purchased from Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. A loop full of preserved bacteria was inoculated in 100 ml Erlenmeyer flasks containing 50 ml medium  with 0.5 g/L glucose monohydrate and incubated at 30°C on a shaker at 110 rpm. After appearance of the violet pigmentation, 10% of inoculum from this culture was transferred into fresh medium containing 20 mg/L of Se (IV) or Te (IV), respectively and 1 g/L glucose monohydrate. Conditions for incubation were same as described above. Control assays with only bacteria but without Te or Se and two sterile assays containing Se (IV) and Te (IV), respectively, in the medium were run in parallel.
Selenate and selenite were determined by ion-exchange-chromatography (ICS 90, Dionex) as described earlier . Tellurite in the samples was measured according to Turner et al.  by binding of Diethyldithiocarbamate (DDTC) with Te (IV) in the presence of Tris-HCl buffer and analyzing absorbance at 340 nm with a spectrophotometer. Glucose was determined with dinitrosalicylic acid-reagent according to Miller  by measuring the absorbance with a spectrophotometer at 550 nm. Nanoparticle formation was observed and characterized by electron microscopy equipped with energy dispersive spectroscopy as described previously . All chemicals used for analyses and medium were purchased from Carl-Roth, Karlsruhe, Germany except where stated otherwise.
After 3.5 months of freeze-storage, bacterial cultures C4, C6 and C7 isolated from soil, were revived and could retain their selenite reduction activities for soil detoxification. Beside selenite they also reduced tellurite and formed extracellular Te (0) nanospheres, which is a rare phenomenon during aerobic growth. Not much is known about detoxification of selenite and tellurite in aerobic conditions, while more information exists on the anaerobic respiration of these oxyanions. It was also possible to reduce both oxyanions concurrently and the presence of selenite induced much faster tellurite reduction rates. Owing to their similar structure and chemical properties, selenite might have triggered the uptake and reduction of more toxic tellurite during detoxification mechanism by bacteria. The optimum ratio of selenite : tellurite for faster tellurite reduction was 4:1. The three soil isolates could also produce rarely described extracellular nanospheres composed of both, Se + Te. Duganella violacienigra that is phylogenetically very close to strain C4, could also reduce both oxyanions. The capability of reducing selenite and tellurite particularly in aerobic conditions makes these strain worthy of possible exploitation in bioremediation as well as for environment friendly production of useful nanoparticles.
MB designed and carried out the research work and drafted the manuscript. JW was involved in the discussions of the research results and contributed to finalize the manuscript. Both authors have read and approved the final manuscript.
Mr. Volker Zibat, Mr. Mohammed Fotouhi Ardakni and Mr. Peter Pfundstein from Laboratory of Electron Microscopy (LEM), KIT are kindly acknowledged for their assistance in electron microscopy.
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