Formation of Se (0) nanoparticles by Duganella sp. and Agrobacterium sp. isolated from Se-laden soil of North-East Punjab, India

Abstract

Background: Selenium (Se) is an essential trace element, but is toxic at high concentrations. Depending upon the geological background, the land use or on anthropogenic pollution, different amounts of Se may be present in soil. Its toxicity is related to the oxyanions selenate and selenite as they are water soluble and bioavailable. Microorganisms play an important role in Se transformations in soil and its cycling in the environment by transforming water-soluble oxyanions into water insoluble, non-toxic elemental Se (0). For this study, soil samples were collected from selenium-contaminated agricultural soils of Punjab/India to enrich and isolate microbes that interacted with the Se cycle.

Results: A mixed microbial culture enriched from the arable soil of Punjab could reduce 230 mg/l of water soluble selenite to spherical Se (0) nanoparticles during aerobic growth as confirmed by SEM-EDX. Four pure cultures (C 1, C 4, C 6, C 7) of Gram negative, oxidase and catalase positive, aerobic bacteria were isolated from this mixed microbial consortium and identified by 16 S rDNA gene sequence alignment as two strains of Duganella sp. (C 1, C 4) and two strains of Agrobacterium sp.(C 6, C 7). SEM/TEM-EDX analyses of the culture broth of the four strains revealed excretion of uniformly round sharply contoured Se (0) nanoparticles by all cultures. Their size ranged from 140-200 nm in cultures of strains C 1 and C 4, and from 185-190 nm in cultures of strains C 6 and C 7. Both Duganella sp. revealed better selenite reduction efficiencies than the two Agrobacterium sp.

Conclusions: This is the first study reporting the capability of newly isolated, aerobically growing Duganella sp. and Agrobacterium sp. from soils of Punjab/India to form spherical, regularly formed Se (0) nanoparticles from water soluble selenite. Among others, the four strains may significantly contribute to the biogeochemical cycling of Se in soil. Bioconversion of toxic selenite to non-toxic Se (0) nanoparticles under aerobic conditions in general may be useful for detoxification of agricultural soil, since elemental Se may not be taken up by the roots of plants and thus allow non-dangerous fodder and food production on Se-containing soil.

Figure 1

Selenite reduction by the mixed microbial culture isolated from agricultural soil. Selenite reduction at different Se (IV) concentrations (a) and development of red coloration in cultures after 5.5 h (b), 23 h (c) and 48 h (d) of incubation.

Figure 2

Scanning electron microscopy of mixed cultures. SEM of the mixed culture of bacteria (a) and at higher magnification along with bright round Se (0) nanoparticles (b). SEM-EDX spectrum of 3 targeted points of nanoparticle agglomerates, entrapped in EPS (c).

Figure 3

Colonies of four strains on agar plates showing selenite reduction to red-colored elemental selenium. The strains C 1, C 4, C 6 and C 7 are represented as 1, 4, 6 and 7, respectively, in the illustration.

Figure 4

Selenite reduction in the absence of glucose and selenate reduction by different strains. Se (IV) reduction by strains C 1 (a) and C 4 (b) in the presence and absence of glucose and by strains C 6 and C 7 in the presence of glucose (c). Figure 4dshows reduction of Se (VI) by strains C 1 and C 4.

Figure 5

Nanoparticles formation by strain C 1. SEM of strain C 1 (Duganella sp.) grown without selenite (a) and in the presence of selenite, forming extracellular Se (0) nanoparticles (b). Figure 5c shows SEM- EDX spectra of 5 targeted points in the insert, all confirming Se (0) nanoparticles in cultures of C1.

Figure 6

Nanoparticle formation by strain C 4. SEM of Duganella strain C 4 grown without selenite (a) and in the presence of selenite (b). Figure 6c shows agglomerates of Se nanoparticles. Figure 6d shows SEM- EDX spectra of the Se (0) nanoparticles from 4 targeted points in insert.

Figure 7

Transmission electron microscopy of nanoparticles formed by strain C 4. TEM of Duganella strain C 4 grown without selenite (a) and in the presence of selenite (b, c), showing Se (0) nanoparticles (some of them specified with diameter) along with bacteria and TEM-EDX spectrum of nanoparticles (d) confirming Se (0) nanoparticles.

Figure 8

Nanoparticle formation by strain C 6. SEM of strain C 6 (Agrobacterium sp.) grown without selenite (a) and in the presence of selenite (b), showing nanoparticles along with bacteria. SEM- EDX spectra of 4 targeted points (c) - all confirming selenium (0) nanoparticles.

Figure 9

Nanoparticle formation by strain C 7. SEM of C 7 (Agrobacterium sp.) grown without selenite (a) and in the presence of selenite (b), showing nanoparticles along with bacteria. Figure 9c shows SEM- EDX spectra of 4 targeted points confirming selenium (0) nanoparticles.