Elsevier

Journal of Hazardous Materials

Volume 344, 15 February 2018, Pages 749-757
Journal of Hazardous Materials

Research Paper
Biotransformation and detoxification of selenite by microbial biogenesis of selenium-sulfur nanoparticles

https://doi.org/10.1016/j.jhazmat.2017.10.034Get rights and content

Highlights

  • A. brasilense is able to efficiently reduce toxic selenite to Se0S0-nanoparticles.

  • Reduction was also possible in environmental waters supplemented with selenite.

  • Biogenic nanoparticles are Se8-nSn structured spheres, most likely Se6S2.

  • Se0S0- nanoparticles occur extracellularly with an average size of 400 nm.

  • Se0S0-nanoparticles form a (destabilized) colloidal suspension (ζ-potential −18 mV).

Abstract

This study combines the interaction between the toxic oxyanions selenite and selenate and the plant growth promoting bacterium Azospirillum brasilense with a comprehensive characterization of the formed selenium particles. As selenium is an essential trace element, but also toxic in high concentrations, its state of occurrence in nature is of major concern. Growth of the bacterium was affected by selenite (1–5 mM) only, observable as a prolonged growth lag-phase of 3 days. Subsequently, selenite reduction occurred under aerobic conditions resulting in extracellularly formed insoluble Se0 particles. Complementary studies by microscopic and spectroscopic techniques revealed the particles to be homogeneous and stable Se8-nSn structured spheres with an average size of 400 nm and highly negative surface charge of −18 mV in the neutral pH range. As this is the first study showing Azospirillum brasilense being able to biotransform selenite to selenium particles containing a certain amount of sulfur, even if environmental waters supplemented with selenite were used, they may significantly contribute to the biogeochemical cycling of both elements in soil as well as to their soil-plant transfer. Therefore, microbial biotransformation of selenite under certain circumstances may be used for various bio-remediation and bio-technological applications.

Introduction

In the present time, water contamination by substances such as heavy metals, toxic organics, radionuclides or nanoparticles poses an increasing problem of global concern, especially in drinking water treatment and in the purification of process water from many industrial applications [1]. For the improvement of water quality in the contaminated streams, detailed knowledge on chemical, biological and physical interaction of these noxious chemicals are mandatory. With respect to synthetic submicron- or nanoparticles in water matrices, respective knowledge is still limited. In this context, selenium with its ambivalent biologic characteristics is an especially challenging case. For living organisms, selenium is a key trace element, but the healthy level between selenium deficiency (<40 μg/day) and acute selenium poisoning (>400 μg/day) is rather narrow [2].

Selenium can exist in different oxidation states. The oxyanions selenate [SeO42−] and selenite [SeO32−] represent soluble species in aqueous media, whereas the reduced species Se0 and Se2− form mainly colloidal particles or hardly soluble precipitates [3]. Chemical equilibrium speciation information on selenium oxidation state are challenging, discussed and summarized in “Chemical Thermodynamics of Selenium.” by OECD Nuclear Energy Agency [4]. The standard electrode potential of the redox couples are given in the supplementary information. Selenium is ubiquitous in natural environments (e.g. associated with various sulfide ores of copper, silver, lead, mercury and uranium) and has also anthropogenic origins, e.g. coal burning for power generation, agricultural irrigation of seleniferous soils [1], [5], [6], [7], [8]. Besides the potential chemotoxicity, the isotope 79Se as a fission product with a long half-life (∼3.27 × 105 years [9]) is present in spent nuclear fuel. Several national reports for the long-term safety assessment of high-level nuclear waste disposals show that 79Se is one of the radionuclides that dominate the long-term dose rate [10], [11].

These days, selenium becomes more and more important for technological applications, in consequence of its photoelectric and semiconducting properties [5]. Respective releases from industrial process waters and wastes have to be considered, too. Eventually, to understand the cycling of selenium in the environment is of great importance for the well-being of humans as well as for saving resources.

One possible way to address these hazards is to transform toxic soluble selenium species into insoluble selenium species like nanoparticles promoting their technological separation by sedimentation, coagulation and filtration.

Reactions between selenium and microorganisms can significantly influence the selenium oxidation state and therefore the transport through geological environment. Recently, many investigations have shown that bacteria are able to form Se0 particles under anaerobic as well as aerobic conditions (e.g. Geobacter sulfurreducens, Veillonella atypical, Bacillus subtilis, Bacillus cereus, Shewanella putrefaciens, Agrobacterium sp., Pseudomonas aeruginosa, Stenotrophomonas maltophilia) [12], [13], [14], [15], [16], [17], [18], [19].

In addition to the better separation of particulate selenium from water, nanoparticles attract special interest since their properties usually differ significantly from those of the bulk material. Especially Se0 nanoparticles have various attractive features, like higher biological activity [20], lower toxicity [21], [22] and larger surface area [23]. They have novel in vitro and in vivo antioxidant activities and provide new pathways for medical application like cancer treatment as well as anti-bacterial coating material [24], [25], [26], [27], [28]. In the photovoltaic and semiconductor industry Se0 nanoparticles are used because of their high particle dispersion and unique electrical and optical properties. Other practical applications in the field of nanotechnology are under development [23], [27], [29] Finally, recent studies have shown Se0 nanoparticles being good adsorbents for heavy metals such as Zn, Hg or Cu [29], [30], [31].

In the present study, the interaction of SeO32− and SeO42− with the plant growth promoting rhizobacterium Azospirillum brasilense was investigated, which were reported to have the ability to form Se0 nanoparticles [32], [33] earlier. As this bacterium might be used for biological fertilization also in regions with heavily selenite-loaded soils, its influence on the transfer of the toxic selenium oxyanion as well as the reduction potential needs to be further elucidated. As it was reported that Azospirillum forms the Se0 particles mainly inside the cells [32], [33], this bacteria might be helpful to prevent migration of (radio)toxic selenium through soil and water, as the selenium remains entrapped inside the biomass [34], [35]. Kamnev et al. [36] used A. brasilense to obtain extracellular Se nanoparticles, which were than further characterized by infrared spectroscopy and electron microscopy. In this study, after comparable growth experiments to Tugarova et al. [32], [33] special focus was set on the physico-chemical and structural characterization of the formed Se0 particles, confirming results already reported, but revealing also some more profound and interesting new structural aspects on the Se particles. The formation of hardly soluble Se(0) particles during reduction of selenium oxyanions might be of interest for an industrial application. Moreover, if the Se0 particles will be released from the biomass (e.g. cell death), the mobility of the selenium particles in the environment will be governed by their physico-chemical properties.

So in this study, the elemental selenium particles for further investigation were produced by Azospirillum brasilense. The process of microbial selenium reduction was tracked by inductively coupled plasma mass spectrometry (ICP-MS), hydride generation atomic absorption spectrometry (HG-AAS) and light microscopy. Scanning and transmission electron microscopy (SEM and TEM) with energy dispersive X-ray (EDX) microanalysis, Raman spectroscopy, X-ray absorption spectroscopy (XAS) and UV/Vis spectroscopy (UV/Vis) as well as zeta potential measurements and photon correlation spectroscopy (PCS) were used to characterize the formed selenium particles.

Section snippets

Medium and growth conditions

Growth medium for Azospirillum brasilense (DSMZ 1843) was a malate-containing Azo-medium (DSMZ 2007). Medium components (yeast extract 0.05 g, K2HPO4 0.25 g, FeSO4·7 H2O 0.01 g, Na2MoO4·2 H2O 1.00 mg, MnSO4·H2O 2.00 mg, MgSO4·7 H2O 0.20 g NaCl 0.10 g, CaCl2·2 H2O 0.02 g, (NH4)2SO4 1.00 g, Biotin 0.10 mg) were solved in 950 mL distilled water and pH was adjusted to 7.1 before autoclaving. After sterilization 25 mL each of filter-sterilized 20% glucose and 20% Na-malate were added.

Cells were grown under aerobic

Evaluation of reducing ability and growth of Azospirillum brasilense on selenite and selenate

To determine the reducing ability of A. brasilense for selenate and selenite, the bacterium was grown in the presence of 1 mM sodium selenate and sodium selenite, respectively. As shown in Fig. 1A, selenate was not reduced by A. brasilense. The slight increase of selenate concentration over time can be attributed to evaporation of medium during incubation. The growth behavior of the cells in presence of toxic selenate was similar to control cell suspensions without selenium oxyanions in the

Conclusions

Azospirillum brasilense belongs to the group of bacteria able to reduce selenite under aerobic conditions to extracellular spherical Se8-nSn structured particles in the submicron range. These results suggest that Azospirillum as a plant growth promoting rhizobacterium may help to prevent accumulation of selenium in crops cultivated on selenium-contaminated soils. Nevertheless, the cells are sensitive to the toxicity of selenite as indicated by the growth profiles showing a prolonged growth

Acknowledgements

The authors thank the beamline team at ROBL (ESRF Grenoble, France) for assistance during the XAS-measurements, the analytics group for elemental analysis and Stephan Weiß for assistance while nanoparticle characterization. Support by the Structural Characterization Facilities Rossendorf at IBC is gratefully acknowledged. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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