This investigation was proposed to monitor the ability of isolated Bacillus spp. to transform toxic forms of selenium (selenium oxyanions) to non toxic selenium. These strains reduced up to 89% selenite on average at 37ºC after time of incubation. At higher initial concentrations (100, 200, 400, 600 and 800 µg ml-1), reduction value dropped to 31%. In the presence of other metals stresses (Co, Hg and Cr at a concentration of), the average selenite reduction percentage was 48%. This reduction value shifts from 87% to 94% with the increase in incubation time (from hrs to hrs). Reduction potential of these strains decreased 81% to 27% at various initial selenium concentrations in N-broth and acetate minimal media, respectively. With the increase in the sodium concentration of the media, the measured selenite reduction was above 95%. After exposure to the UV treatment B. pichinoty lost its ability to reduce selenite while B. endophyticus and B. foraminis reduced up to 96% and 71%, of selenite, respectively. Keywords: Selenite, bacteria, Bacillus, Bioremediation, heavy metal

Faisal, Muhammad

Siddiqa, Ayesha

Sultan, Shaiq

English

International Institute for Science, Technology and Education (IISTE): E-Journals

Journal of Environment and Earth Science                                                                                     www.iiste.org ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online) Vol.4, No.22  2014  196 Selenite detoxification by Bacillus spp isolated from indigenous polluted sites Ayesha Siddiqa, Shaiq Sultan and Muhammad Faisal* Department of Microbiology & Molecular Genetics, University of the Punjab, Lahore, Pakistan. Tel: +924235952811  E-mail: mohdfaysal@yahoo.com Abstract This investigation was proposed to monitor the ability of isolated Bacillus spp. to transform toxic forms of selenium (selenium oxyanions) to non toxic selenium. These strains reduced up to 89% selenite on average at 37ºC after time of incubation. At higher initial concentrations (100, 200, 400, 600 and 800 µg ml-1), reduction value dropped to 31%. In the presence of other metals stresses (Co, Hg and Cr at a concentration of), the average selenite reduction percentage was 48%. This reduction value shifts from 87% to 94% with the increase in incubation time (from hrs to hrs). Reduction potential of these strains decreased 81% to 27% at various initial selenium concentrations in N-broth and acetate minimal media, respectively. With the increase in the sodium concentration of the media, the measured selenite reduction was above 95%. After exposure to the UV treatment B. pichinoty lost its ability to reduce selenite while B. endophyticus and B. foraminis reduced up to 96% and 71%, of selenite, respectively. Keywords: Selenite, bacteria, Bacillus, Bioremediation, heavy metals  1. Introduction The main threats to human beings from heavy metals in their toxic forms are associated with exposure to their toxic insoluble oxyanions (Lampis et al., 2014). For proper growth and development these ions required in little amount as they are the part of vital enzymes, proteins and are involved in various physiological processes (Wang et al., 2014). Some elements are although indispensable for living system at low concentration but at higher concentration they become toxic and deleterious for growth and life (Yao et al., 2003). Bacteria living under subsurface are able to detoxify and degrade metals and other pollutants by the process of situ bioremediation (Chapelle, 2001). The bacterial removal of toxic metals in these days is very important and considered a vital process for better environment (Jonathan, 2003). Micro-organisms also have resistance mechanisms which introduce convert metal oxidation states (Tetteh et al., 2014), organometals, and radionuclide contaminants (Bajaj et al., 2014). Bacterial reduced the mobility of contaminats by reducing it in less harm state (Adeniji, 2004). Many microorganisms like bacteria, protozoan’s and algae are utilized for metal removal (Pena-Castro, 2004; Munoz et al., 2006; Munoz, 2006). Selenium is essential in a way that it is very crucial for organisms but in very low quantity and if this disturb then it is toxic (Zheng et al., 2014, Kim et al., 2014). Selenium is required for both animals and plants for their proper growth  (Li et al., 2014) while it may be toxic when present in high amount (Tinggi, 2003, Zheng et al., 2014). As part of 21st amino acid - selenocysteine - that is present in many enzymes is essential to all living organisms (Gouget et al., 2005). Selenium comes in environment through fertilizers, fossils fuel, metal processing and many others activities (Broadley et al., 2006). Bacteria isolated from selenite rich environment have developed a variety of resistance mechanisms, for instance; the oxidation, reduction or methylation of inorganic and organic selenium species (Ayano et al., 2014) some bacteria can use the selenate as electron acceptor (Gouget et al., 2005). Various environmental factors like time duration, selenium concentration, pH, temperature and moisture may impact on these reactions (Lenz, 2008). The gene fnr is involved for the reduction of selenite to elemental selenium (Yee et al., 2007). This study aims to determine the bio-transformation ability of Bacillus spp. at different physico-chemical factors so that they may be used practically for bioremediation purposes. 2. Material and Methods  Three isolated and identified strains (Bacillus foraminis-YAK-1 Accession No. JX203248, Bacillus endophyticus-YAK-7 Accession No. JX203252, Bacillus pichinotyi-YAM-2 Accession No. JX203257) were taken and routinely in N-Agar amended with 500 µg ml-1 of sodium selenite (Na2SeO3). Journal of Environment and Earth Science                                                                                     www.iiste.org ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online) Vol.4, No.22  2014  197 2.1 Selenite reduction assay Reduction experiments were undertaken in aerobic conditions. Ten ml of N-broth amended with Na2SeO3 in 20ml test tubes stopped with a cotton plug were prepared and autoclaved. Medium was inoculated with 24 hours old culture and incubated at 37ºC for 48 hours at two initial selenite (200 and 400 µg ml-1) concentration. After incubation selenite content was estimated using a modified method of Keka et al. (2011). 2.2 Selenite content determination With little modification of Keka et al. (2011) method was used for the estimation of selenium content. After the glass test tubes containing 10 ml of N-broth were autoclaved, aliquots of sterile 1M Na2SeO3 solution was taken and after inoculation were incubated for 48 hours at 37ºC. Red colored culture broth was stirred others centrifuge tubes. Solution was centrifuged (5000 rpm) for ten minutes; pellet and supernatant were separated. In order to remove non-metabolized selenite, ten ml of 1 M NaCl was added to wash off the pellets. Then ten ml of distilled water poured into the centrifuge tubes and centrifuged (5000 rpm) for ten minutes. Then pellet was suspended by vortex and its absorbance was determined by spectrophotometer at 500 nm. 2.3 Effect of UV radiation on bacterial selenite reduction Strains were exposed to UV radiation for 15, 30 and 60 minutes time duration. Colonies were selected, cultured and their ability to grow and reduce the selenite was determined. 3. Results 3.1 Bacterial strains Three identified strains (Bacillus foraminis-YAK-1 Accession No. JX203248, Bacillus endophyticus-YAK-7 Accession No. JX203252, Bacillus pichinotyi-YAM-2 Accession No.JX203257) were maintained on N-agar plate supplemented with sodium selenite. 3.2 Strain characterization These bacterial strains are gram positive and forms spores. All these strains are aerobic rods. These strains showed resistance in the presence of selenite. a.                                          b.             Figure 1. Effect of various temperature (28, 37 and 42ºC) and pH (5, 7 and 9) on selenite reduction. (a) at 200 µg 01020304050607080B.endophyticus B.pichinoty B.foraminis% Se (iv) reduction 5 7 90102030405060B.endophyticus B.pichinoty B.foraminis% Se (iv) reduction 5 7 9020406080100120B.endophyticus B.pichinoty B.foraminis% Se (iv) reduction 28°C 37°C 42°C01020304050B.endophyticus B.pichinoty B.foraminis% Se (iv) reduction 28°C 37°C 42°CJournal of Environment and Earth Science                                                                                     www.iiste.org ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online) Vol.4, No.22  2014  198 ml-1, (b) at 400 µg ml-1 of sodium selenite after 48 hours incubation period. 3.3 Selenite reduction 3.3.1 At different temperature At 37ºC all of the strains had maximum reduction of selenite at both selenite concentrations i,e; 200 and 400 µg ml-1. At 42ºC B. foraminis showed increased reduction potential than other strains, but marked decrease in reduction potential at higher concentration (Figure 1).  Figure 2. Effect of various initial selenite concentrations (100, 200,400, 600 and 800 µg ml-1) on selenite reduction potential of bacterial strains. 3.3.2 At different initial selenite concentration B. endophyticus and B. pichinoty showed the highest reduction at a concentration of 800 µg ml-1. B. foraminis had maximum reduction potential at 400 µg ml-1 concentration (Figure 2). 3.3.3 At different pH Strain B. endophyticus had maximum reduction potential at neutral pH when initially supplied with 200 µg ml-1. B. pichinoty had maximum reduction at pH 7 and concentration 400 µg ml-1. B. foraminis showed maximum reduction at pH 9 and concentration 400 µg ml-1 (Figure 1). a.                                  b.        Figure 3. Effect of various incubation time (48 and 96 hrs) and media (acetate minimal media, N-broth) on 020406080100120B.endophyticus B.pichinoty B.foraminis% Se (iv) reduction 100 200 400 600 800020406080100120B.endophyticus B.pichinoty B.foraminis% Se (iv) reduction 48 hours 96 hours020406080100120B.endophyticus B.pichinoty B.foraminis% Se (iv) reduction 48 hours 96 hours020406080100120B.endophyticus B.pichinoty B.foraminis% Se (iv) reduction Acetate Minimal Media N -broth020406080100120B.endophyticus B.pichinoty B.foraminis% Se (iv) reduction Acetate Minimal Media N -brothJournal of Environment and Earth Science                                                                                     www.iiste.org ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online) Vol.4, No.22  2014  199 selenite reduction potential of bacterial strains (a) at 200, (b) at 400 µg ml-1. 3.3.4 In presence of heavy metals B. endophyticus and B. foraminis reduces selenite optimally in the presence of mercury and cobalt stress respectively, but all other strains showed decreased reduction potential in increased concentration of other heavy metals. 3.3.5 At different incubation time A common trend was observed in all the strains that as the incubation time was increased reduction potential also increased. After 48 hours of incubation B. foraminis showed the highest reduction potential while B. pichinoty showed lowest reduction potential. After 96 hours incubation duration the same results were obtained with the slightly increase in reduction potential (Figure 3). 3.3.6 In presence of different media B. endophytics showed maximum reduction of selenite in acetate minimal media at concentration 200 µg ml-1 with slightly decrease in reduction potential at higher concentration i.e. 400 µg ml-1. B. pichinoty showed reduction less than B. endophytics at concentration 200 µg ml-1 but showed an increase in reduction at 400 µg ml-1. B. foraminis showed minimum reduction at both concentrations with slight increase in reduction at 400 µg ml-1 (Figure 3).   Figure 4. Effect of salinity (0%, 3%, 5% and 7%) on selenite reduction potential of bacterial strains at 400 µg ml-1 after 48 hours incubation period. 3.3.7 Effect of salinity on selenite reduction At 400 µg ml-1 concentration of sodium selenite, B. foraminis showed maximum reduction in 3% salinity, B. pichinoty showed maximum reduction in 5% salinity and B. endophyticus showed maximum reduction in 7% salinity. All of these strains showed the same trend of gradual decrease in reduction potential with the increase in salinity. When the concentration of selenite is increased from 200 to 400 µg ml-1, all of the strains had shown a decrease in reduction potential (Figure 4).  Figure 5. Effect of UV radiations on selenite reduction potential of bacterial strains (a) at 200 µg ml-1, (b) at 400 µg ml-1 after 48 hours incubation period 3.3.8 Effect of UV treatment on selenite reduction of strains 020406080100120B.endophyticus B.pichinoty B.foraminis% Se (iv) reduction 0% 3% 5% 7%020406080100120B.endophyticus B.foraminis% Se (iv) reduction 15 min 30 min 60 minJournal of Environment and Earth Science                                                                                     www.iiste.org ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online) Vol.4, No.22  2014  200 Exposure of strains to the UV radiation for various time duration led to the loss of ability to tolerate the selenite in case of B. pichinoty whereas B. endophyticus showed optimal reduction by the culture exposed to UV radiation for 30 minutes while B. foraminis showed optimal reduction after 15 minutes exposure (Figure 5). 4. Discussion Biotransformation of selenite as well as selenate into elemental selenium is known as dissimilatory selenate/selenite reduction (Narasingarao and Häggblom, 2007). A number of remediating techniques including reduction techniques have been developed for the removal of toxic forms of selenium (Dungan and Frankenberger, 1999). In present study three Bacillus species transformed toxic selenite into nontoxic insoluble elemental selenium aerobically at different physico-chemical parameters since its reduction under these factors prove these strains are important for bioremediation process. All of the strains exhibited optimal biotransformation at 37ºC and showed different optimal transformation at different pH, B. endophyticus reduced slenite optimally at pH range 5-7, B. foraminis and B. pichinoty at 5-9 in the presence of different concentration. With the gradual increase in the selenite content in medium all strains showed decreased reduction. When the selenite concentration reached 800 µg ml-1, selenite reduction reduced to 30 to 34%. It was observed that high initial reduced reduction ability of strain as observed in Pseudomonas stutzeri, reduction potential decreased as selenite concentration increased above 19mM (Lortie et al., 1992). Since selenite reduction is not an independent process, a number of other pollutants are also present that can either inhibit or enhance the reduction process. These strains showed reduced transformation in the presence of other metals. When Shewanella oneidensis was grown aerobically it will reduced selenite mainly at end of stationary stage (Klonowska et al., 2005). These Bacillus spp. showed increased reduction potential over time. The amount of Se volatilized by the cultures increased with the growth of the cultures over time (Desouza et al., 2001). Greater the incubation time greater will be the reduction potential. The heterotrophic selenium respiring bacteria utilize carbon for selenium reduction (Oremland et al., 1999; Losi and Frankenberger, 1997; Cantafio et al., 1996). Strains exhibited improved reduction potential in acetate minimal media. All strains showed abundant growth and increased reduction in saline medium as compared to N-agar, but reduction potential was gradually decreased with increasing salinity. B. endophyticus and B. foraminis showed the ability to withstand the prolonged UV rays exposure while retaining their ability to bio-transform the selenite while B. pichinoty was sensitive to UV rays and showed no growth at all. These strains showed their remarkable ability to transform and detoxify toxic oxy-anion of selenium present in industrial effluents over the wide environmental parameter range. References Adeniji, A. (2004). Bioremediation of arsenic, chromium, lead and mercury. USEPA, Washington, D.C. Bajaj, M., & Winter, J. (2014). Se (IV) triggers faster Te (IV) reduction by soil isolates of heterotrophic aerobic bacteria: formation of extracellular Se Te nanospheres. Microbial Cell Factories, 13, 168 Ayano, H., Miyake, M., Terasawa, K., Kuroda, M., Soda, S., Sakaguchi, T. and Ike, M. (2014). Isolation of a selenite-reducing and cadmium-resistant bacterium Pseudomonas sp. strain RB for microbial synthesis of CdSe nanoparticles, Journal of Bioscience and Bioengineering, 117, 576-81 Broadley, M.R., White, P. J., Bryson, R.J., Meacham, M.C., Bowen, H.C., Johnson, S.E., Hawkesford, M.J., McGrath, S.P., Zhao, F-J., Breward, N., Harriman, M. and Tucker, M. (2006). Biofortification of UK food crops with selenium. The Proceedings of the Nutrition Society, 65, 169–181 Chapelle, F. H. (2001). Ground-Water Microbiology and Geochemistry. New York. Wiley Gouget, B. et al. (2005). Resistance, accumulation and transformation of selenium by the Cyanobacterium synechocystis sp. PCC6803 after exposure to inorganic Se(VI) or Se (IV). Molecular Microbiology, 5, 683-689 Journal of Environment and Earth Science                                                                                     www.iiste.org ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online) Vol.4, No.22  2014  201 Jonathan, R. L. (2003). Microbial reduction of metals and radionuclides. FEMS Microbiology Reviews, 27, 411- 425 Kim, Y.H., Lee, H.S., Kwon, H.J., Patnaik, B.B., Nam, K.W., Han, Y.S., Bang, I.S. and Han, M.D. (2014). Effects of different selenium levels on growth and regulation of laccase and versatile peroxidase in white-rot fungus, Pleurotus eryngii. World Journal of Microbiology and Biotechnology, 30, 2101-9 Li, B., Liu, N., Li, Y., Jing, W., Fan, J., Li, D., Zhang, L., Zhang, X., Zhang, Z. and Wang, L. (2014). Reduction of selenite to red elemental selenium by Rhodopseudomonas palustris strain N. PLoS One, 9, e95955. Lenz, M. (2008). Biological selenium removal from wastewaters. Ph.D. Thesis, Wageningen University, Wageningen, The Netherlands Lampis, S., Zonaro, E., Bertolini, C., Bernardi, P., Butler, C.S. and Vallini, G. (2014). Delayed formation of zero-valent selenium nanoparticles by Bacillus mycoides SeITE01 as a consequence of selenite reduction under aerobic conditions. Microbial Cell Factories, 13, 35. Munoz, R., Alvarez, M.T., Munoz, A., Terrazas, E., Guieysse, B. and Mattisasson, B. (2006). Sequential removal of heavy metals ions and organic pollutants using an algal-bacterial consortium. Chemosphere, 63, 903-991. Munoz, R., & Guieysse, B. (2006). Algal-bacterial processes for the treatment of hazardous contaminants: a review. Water Research, 40, 2799-2815. Pena-Castro, J.M., Mart, N.O.F., Esparza-Garca, F. and Canizares-Villanueva, R.O. (2004). Heavy metals removal by the microalga Scenedesmus incrassatulus in continuous cultures. Bioresourse Technology, 94, 219-222. Tetteh, A.Y., Sun, K.H., Hung, C.Y., Kittur, F.S., Ibeanu, G.C., Williams, D. and Xie, J. (2014). Transcriptional Response of Selenopolypeptide Genes and Selenocysteine Biosynthesis Machinery Genes in Escherichia coli during Selenite Reduction. International Journal of Microbiology, doi:  10.1155/2014/394835 Wang, Y., Fang, W., Huang, Y., Hu, F., Ying, Q., Yang, W. and Xiong, B. (2014). Reduction of selenium-binding protein 1 sensitizes cancer cells to selenite via elevating extracellular glutathione: A novel mechanism of cancer-specific cytotoxicity of selenite. Free Radical Biology and Medicine, 29, 186-196 Yao, H., Xu, J. and Huang, C. (2003). Substrate utilization pattern, biomass and activity of microbial communities in a sequence of heavy metal-polluted paddy soils. Geoderma, 115, 139-148 Zheng, S., Su, J., Wang, L., Yao, R., Wang, D., Deng, Y., Wang, R., Wang, G. and Rensing C. (2014). Selenite reduction by the obligate aerobic bacterium Comamonas testosteroni S44 isolated from a metal-contaminated soil. BMC Microbiology, 14, 204. doi:10.1186/s12866-014-0204-8  

Selenite detoxification by Bacillus spp isolated from indigenous polluted sites

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Selenite detoxification by Bacillus spp isolated from indigenous polluted sites

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