Formation of Se (0) Nanoparticles by Duganella sp. and Agrobacterium sp. isolated from Se-laden soil of North-East Punjab, India
© Bajaj et al.; licensee BioMed Central Ltd. 2012
Received: 24 January 2012
Accepted: 20 May 2012
Published: 9 July 2012
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.
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.
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.
Selenium (Se) is an essential trace element and a constituent of selenoproteins which act as antioxidants, playing an important role in the protection of cellular damages from oxygen radicals and preventing the development of chronic ailments like cancer and heart diseases . Inspite of health benefits of Se at low concentrations, Se is highly toxic if its recommended daily dietary intake by adults exceeds the limit of 400 μg/d, causing selenosis . Carbon shales, phosphotic rocks and coal are rich natural sources of Se in the environment . Se concentrations ranging from 0.01 to 1200 mg/kg can be found in soils due to a number of factors such as the Se content of the parent rock, deposition of mining residues, magma and ashes from volcanic eruptions or seleniferous erosion materials or of residues from fossil fuel combustion. Also poor drainage, irrigation with Se-containing water and fertilization with Se-containing phosphate as well as topographic and climate conditions may be responsible for the elevated Se concentrations in soils . Besides the inorganic compounds of Se in mineral fertilizers, some organic selenium compounds also find their use in agriculture due to their bactericidal, fungicidal and herbicidal properties . Within the four inorganic Se oxidation states (−II, 0, +IV, +VI) found in nature, the oxyanions selenate and selenite are most mobile and detrimental as they are bioavailable and can easily be taken up by the plants from Se-rich soil or Se-containing irrigation water and thus enter the plant-animal/human food chain, posing a health risk for animals and humans [4, 6]. Diseases, such as skin lesions and hair fall as a result of consuming water or plants grown in north-east Punjab, India have been associated with the high Se concentration in soil and irrigation water of that area [4, 7]. The sources of Se in soil of that region are still unknown, but the high Se concentrations might, at least in part, stem from the use of Se-containing groundwater for irrigation and the practice of rice-wheat crop rotation. It was found in our previous study  that microbes in the top soils of Jainpur village in Punjab, which has the highest total Se concentration (up to 11.6 mg/kg) among all the collected sediment samples, have gained resistance to high soluble Se concentrations and could reduce selenate or selenite to Se (0). Due to the toxicity and changing bioavailability of selenium, most of the previous investigations were focused on the permanent removal or immobilization of selenium oxyanions by physical, chemical or biological processes. A permanent removal could be obtained by stimulating microbes for methylation of Se to generate volatile Se-compounds in soil or through direct or gravity filtration of insoluble elemental selenium (Se0) in water after microbial reduction of Se compounds . Soil bacteria play an important role in Se transformations from soluble toxic forms [Se (IV), Se (VI)] to insoluble non-toxic Se (0) as part of a detoxification mechanism which could well be exploited for bioremediation. The full redox cycle of Se in nature is dependent on geochemical as well as on microbial transformations by soil bacteria [3, 4]. These microbes could be exploited for periodic or permanent avoidance of Se toxicity, since the rate of Se (IV) or Se (VI) reduction is higher than that of Se (0) oxidation . A number of anaerobic and anoxic bacteria e.g. Geobacter sulfurreducens, Shewanella oneidensis, S. sp. HN-41, Veillonella atypica, Rhodospirillum rubrum, Sulfurospirillum barnesii, Bacillus selenitireducens and Selenihalanerobacter shriftii[6, 10–13] have been identified to form Se nanoparticles by reducing Se oxyanions to elemental Se (0) during the biogeochemical cycling of Se. To date the main research focus was laid on anaerobic Se reduction. Due to this, the variety of Se nanoparticle-forming aerobic bacteria is less known compared to anaerobic/anoxic bacteria and is confined mainly to species of Pseudomonas and Bacillus such as P. fluorescens, P. aeruginosa B. subtilis or B. megaterium[14–16]. Se nanoparticles are regarded as promising material for a number of applications in particular for the photovoltaic and semiconductor industry due to their high particle dispersion and unique electrical and optical properties [13, 17]. Because of their high activity in biological tissues and low toxicity, Se nanoparticles are getting attention for medical applications. Nano-Se has exhibited novel in vitro and in vivo antioxidant activities through the activation of seleno enzymes, and as chemo-preventive and -therapeutic agents . Se nanoparticles inhibit growth of Staphylococcus aureus and it has been suggested to use them as human medicine for preventing and treating S. aureus infections . Besides this, Se nanoparticles could be used to remove metallic pollutants like copper from aqueous solutions .
As microbes apparently play an important role in the biogeochemical cycle of Se in natural environment, Se resistance of indigenous aerobic microbes in Se rich sediments of the Punjab region in India and biotransformation of selenite to non toxic elemental Se (0) were investigated in this study. From the mixed microbial culture that was enriched in the presence of Se, four strains of bacteria were isolated and identified. Both, the mixed culture as well as the two Duganella sp. and the two Agrobacterium sp. isolates were capable of producing exogenous Se (0) nanoparticles by reducing Se (IV) under aerobic conditions. Generation of Se (0) nanoparticles by strains of these two genera has not been reported previously. Duganella is a rarely described genus with the most well known species D. violacienigra, a violet-black pigmented bacterium isolated from forest soils in China .
In this study we report the isolation of pure cultures of two Duganella species and two Agrobacterium species from Se rich sediments of Punjab. Genus assignment of the isolates was done by 16 S rDNA alignment, Se (IV) reduction by ion chromatography and qualitative examination of biosynthesized nanoparticles with scanning and transmission electron microscopy (SEM and TEM) and by energy dispersive X-ray spectroscopy (EDX).
Results and Discussions
Enrichment of aerobic selenite reducing bacteria from soil of the Punjab area in India
Isolation of pure cultures, phylogenetic identification and selenite reduction
Phenotypic characteristics of four strains isolated after enrichment from Se-containing soils in north-east Punjab, India
Liquid medium after 7 d
Single suspended cells
very good: white-yellow, flat colonies, 2–4 mm Ø
very good: white-yellow, flat colonies with dark raised center, 2–4 mm Ø
very good: white-yellow, flat colonies up to 3 mm Ø
very weak: orange-yellow, round colonies upto 1.5 mm Ø
very good: orange-yellow, round, little slimy colonies with raised yellow center and colorless edge upto 4 mm Ø
very good: orange-yellow, round, little slimy colonies with raised yellow center and colorless edge upto 2.5 mm Ø
very good: white colonies with flattened center upto 2 mm Ø
very good: white colonies with flattened center upto 3 mm Ø
very good: yellowish-white, very slimy colonies with a dark center upto 4 mm Ø
Yeast-Mannitol agar with 0.025% Congo red
very good: slimy colonies, pink color > confirm Agrobacterium sp. as according to Bergey’s manual Rhizobium sp. forms white colonies on this medium
1/10 Tryptic-Soy broth + 100 μg/ml Cycloheximide (OD 578 nm)
Spore forming agar
very good: no spores observed under microscope
Growth on SP medium
The 16 S rDNA gene sequence of strain C 6 (1352 bases) showed 97% sequence similarity to that of Agrobacterium tumefaciens strain JDC-49 and 98% to that of Rhizobium sp. SYF-5, respectively, while the 16 S rDNA gene sequence of strain C 7 (1294 bases) also showed 97% sequence similarity to Agrobacterium tumefaciens strain JDC-49. The next highest sequence similarity with only 94% was found with Agrobacterium tumefaciens strain IAM 1204. The alignment of the 16 S rDNA gene sequences of strain C 6 with strain C 7 showed only 88.2% sequence similarity, confirming that C 6 and C 7 were genetically different. It is well known that Agrobacterium and Rhizobium are closely related. Phenotypic as well as 16 S rDNA sequence analyses had revealed difficulties in distinguishing these two genera as separate monophyletic clades, due to which an amalgamation of these two genera has been suggested . Nevertheless, the genome structure and some phenotypic characteristics clearly set Agrobacterium apart from other members of the family Rhizobiaceae. According to Bergey’s Manual of Systematic Bacteriology , one important test to distinguish between the genera Agrobacterium and Rhizobium is growth on 0.025% Congo red-containing yeast extract-mannitol agar (YEMA). When grown on this medium Agrobacterium forms large and stained colonies, whereas Rhizobium forms only small, white, translucent colonies. Both of our strains, C 6 and C 7, formed slimy pinkish red colonies on YEMA indicating that they do not belong to the genus Rhizobium but more likely to the genus Agrobacterium (Table 1). Agrobacterium sp. are Gram negative soil bacteria and are well known for their ability of horizontal gene transfer to plants. Hunter et al.  isolated a selenite reducing Rhizobium sp. from a laboratory bioreactor treating simulated groundwater and classified it as R. selenireducens sp. nov. This bacterium was related to but was genetically divergent from R. radiobacter (syn. Agrobacterium tumefaciens) or R. rubi (syn. A. rubi). No report is available describing selenite reduction by Agrobacterium species except for a brief note of selenate and selenite reduction by A. tumefaciens in a study investigating in situ Raman and X-ray spectroscopy to monitor microbial activities under high hydrostatic pressure . Interestingly the two genera to which our four Gram negative isolates (C 1, C 4, C 6, C 7) from Se-rich soil in India belong were not mentioned by Ghosh et al. , who isolated 8 strains of Se-tolerant bacteria from Se-contaminated sediments of three different regions in India. One of these regions was the present study area. All of the newly isolated strains were catalase and oxidase positive and were tested for other phenotypic characteristic as presented in Table 1. The 16 S rDNA gene sequences of all four strains have been submitted to the GenBank at NCBI. The accession numbers are JQ745646 for strain C 1 (Duganella sp.), JQ745647 for strain C 2 (Duganella sp.), JQ745648 for strain C 6 (Agrobacterium sp.) and JQ745649 for strain C 7 (Agrobacterium sp.).
The Agrobacterium isolates, strains C 6 and C 7, were also grown with glucose in the presence or absence of selenite. During growth they reduced 26% and 25% of 80 mg/l Se (IV) in 2 days, respectively and no further reduction occurred after prolonged incubation (Figure 4c). When grown in the absence of glucose no Se (IV) at all was reduced by both cultures (not shown). This suggested that in the absence of glucose, storage material such as glycogen or poly hydroxyl butyrate in strains C 1 and C 4 might have delivered a restricted amount of reducing equivalents for some Se (IV) reduction and that strains C 6 and C 7 lack storage products. An exponential decrease of selenite by all strains without a lag phase was observed when glucose was present to deliver reducing equivalents (Figure 4).
Electron microscopy and EDX analysis of pure cultures
Not many studies are available describing aerobic Se reduction and production of Se (0) nanoparticles in comparison to investigations on anaerobic/anoxic reduction. In this study, two strains of Duganella sp. and two strains of Agrobacterium sp. were isolated from Se-polluted agricultural soils of Punjab, India. The four strains were able to reduce selenite under aerobic conditions producing Se nanospheres on the cell surface as indicated by SEM/TEM-EDX analyses. The Duganella strains C1 and C4 could reduce selenite more rapidly than the Agrobacterium strains C 6 and C 7. To date no Duganella species were reported to have the capability to reduce selenite to Se (0) and similar studies with Agrobacterium have rarely been undertaken. The formation of uniformly round Se (0) nanoparticles by these bacteria under aerobic growth conditions may serve for detoxification and is of particular interest for nanoparticle production, as aerobic cultures could easily be handled. Further research is required to investigate the stability of these biosynthetic nanoparticles in different environments, their quantitative separation from biomass and EPS and their catalytic reactions. In ecosystems with a high geogenic background of Se compounds, the ability of indigenous soil bacteria to reduce soluble selenite to insoluble and thus non-toxic Se(0) could be a means to prevent Se uptake by plants for fodder and food production. If selenite-reducing bacteria in the rhizosphere could be activated at least during the cropping season to inactivate the selenite in the soil and from irrigation water at least a periodical “bioremediation” could be maintained in selenite containing soils of Punjab. For a permanent removal of selenite from drinking water biofiltration columns might be applied in small-scale or huge biofilters in large-scale to precipitate selenite from the raw water source as elemental Se. However, further investigations are necessary to find out possible interactions of selenite-reducing bacteria with other soil bacteria for ion removal.
Based upon our previous survey , Jainpur village (N31°08.082’ E076°11.776) located in north-eastern part of Punjab, India was selected for soil sampling in March 2011, as we found high Se concentrations in those soils. The soil samples were transferred to the laboratory in sealed plastic bags and kept at 4°C until further use. The top layer from 0–2 cm of soil profiles was selected for preparing soil slurries for isolation of bacteria under aerobic conditions.
Bacterial isolation and identification
Soil slurry was prepared by suspending 5 g soil from the top layer in 100 ml tap water. Twenty ml portions of slurry were incubated in 100 ml Erlenmeyer flasks under gentle shaking overnight at 28 ± 2°C. From this slurry a mixed bacterial culture capable of reducing selenite was enriched by multiple transfers of 2.5% of the initial soil suspension and later on of the cell suspension to enrichment medium (EM), described by Ghosh et al. . To this medium 133.2 mg/l of Na2SeO3.5H2O (= 40 mg/l Se (IV)) and 1 g/l glucose monohydrate as the main carbon source were added. The selenium concentration in EM was increased step wise up to concentrations of 160 mg/l of Se (IV) by adding the respective amount of Na2SeO3.5H2O (up to 533 mg/l). Before each Se increment, bacterial activity was monitored visually (red color formation of elemental Se in EM) and by measuring Se (IV) reduction with an ion chromatograph (IC). Four strains of bacteria were isolated from the Se (IV) reducing enrichment culture by picking single colonies from Petri dishes that contained 160 mg/l Se (IV) in EM-Medium and 1.5% agar. Selenite reduction was indicated by a red color of the colonies. The colonies were picked and re-grown in liquid EM containing 160 mg/l Se (IV). The reduction of Se (IV) was confirmed again by red coloration of the medium and by measuring selenite reduction with IC. The culture suspension was streaked once more onto agar plates and single colonies were again inoculated into liquid medium. This procedure was repeated a third time to ensure purity of the cultures.
DNA extraction from cells of the four selected strains was performed with chloroform-phenol. Universal eubacterial 16 S rDNA sequencing primers 27 F (5-AGAGTTT GATCCTGGCTCAG-3) and 1492R (5-GGTTACCTTGT TACGACTT-3)  were used for identification of the strains. The amplification of the 16 S rDNA gene by polymerase chain reaction (PCR) was carried out in a Biometra Thermocycler T Gradient. The amplified products were sent for DNA-sequencing to Seqlab Laboratories, Göttingen, Germany. The resulting nucleotide sequences were compared with known sequences of the database at National Center for Biotechnology Information (NCBI) by using Basic Local Alignment Search Tool (BLAST). Sequence alignment analyses of the four strains were conducted using MEGA4 . Details of the methods for DNA isolation and molecular identification of isolates have been described elsewhere . For morphological characterization of the four isolates a 1000 x magnification phase contrast microscope (Zeiss Axioskop, Göttingen, Germany) was used. Physiological and biochemical tests were performed as described in Bergey’s Manual . Production of acids from sugars was tested at 27°C after incubation of cultures for 1 day according to Hugh and Leifson . Growth of cultures on liquid and agar media was tested by incubation at 27°C for up to 7 days. Other biochemical assays (Table 1) were performed using microplates and the Micronaut-IDS/STREP 2 identification system (Merlin diagnostics GmbH, Bornheim-Hersel,Germany) following the manufacturer’s instructions. All biochemicals were of microbiological grade and were purchased from Carl Roth (Karlsruhe), Merck (Darmstadt) or Fluka (Steinheim), Germany.
Selenite reduction assay
All assays to investigate selenite reduction by mixed or pure microbial cultures were carried out in duplicate in 250 ml Erlenmeyer flasks containing 100 ml enrichment medium (EM). The flasks were incubated on a shaker at 110 rpm and 28 ± 2°C. The different concentrations of selenite or selenate in the assays were obtained by adding the required volume of stock solutions of 13.32 g/l Na2SeO3.5H2O (= 4 g/l Se IV) or 4.78 g/l Na2SeO4 (= 2 g/l Se VI). No inoculum was added to sterile controls. For other assays the percentage of the inocula was as indicated in the results.
Selenite concentrations in the samples were determined with an ion chromatograph (Dionex ICS-90) employing an AS9-HC 4 mm × 250 mm (IonPac®) analytical column. The eluent was 9 mM Na2CO3 and H2SO4 acid was used as a regenerate. The sample volume was 1.2-1.5 ml. Bacteria and other particles were pelleted by centrifugation at 6700 g for 7–8 min. The supernatant was once more centrifuged at 9660 g for 4–5 minutes in a Microfuge (Eppendorf, Hamburg) before analysis. All chemicals used for analyses were of analytical grade and were purchased from Merck/VWR (Darmstadt) or Carl Roth (Karlsruhe), Germany. The optical density of the cell suspension was measured at 578 nm using an UV LKB Biochrom Ultrospec II spectrophotometer (Cambridge, United Kingdom). The samples for scanning electron microscopy (SEM) + energy-dispersive X-ray spectroscopy (EDX) were prepared by mounting them on silicon wafers (Plano, Wetzler, Germany). For transmission electron microscopy (TEM) + EDX), formvar-coated copper grids 200 mesh (Plano, Wetzler, Germany) were used as a sample support. SEM was performed using a LEO 1530 Gemini microscope with a Schottky field emitter and TEM with a Philips CM 200 FEG/ST microscope. Electron microscopy was done by the Laboratory for Electron Microscopy (LEM) at Karlsruhe Institute of Technology, Germany.
We thank Prof. K.S. Dhillon, Prof. U.S. Sadana and their team at PAU, Ludhiana for helpful advices to select sampling sites and for assistance during sample collection. We also thank Prof. Dr. Th. Neumann and Dr. E. Eiche of IMG, KIT, who were part of the sampling team. Sampling was made possible by a travel grant provided by International Bureau of BMBF, Bonn to Th. Neumann, E Eiche and M Bajaj.
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