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Hexavalent Chromium Reduction by Anoxybacillus Rupiensis Isolated from Hot Water Spring of Dhapoli, Maharashtra, India

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Hexavalent Chromium Reduction by Anoxybacillus Rupiensis Isolated from Hot Water Spring of Dhapoli, Maharashtra, India Environ & m m en u ta le l o B r t i o e t P e f c h Gursahani and Gupta, J Pet Environ Biotechnol 2015, 6:4 o Journal of Petroleum & n l a o l n o r DOI: 10.4172/2157-7463.1000232 g u y o J ISSN: 2157-7463 Environmental Biotechnology Research Article Open Access Hexavalent Chromium Reduction by Anoxybacillus rupiensis isolated From Hot Water Spring of Dhapoli, Maharashtra, India Gursahani YH1* and Gupta SG2 1Department of Biotechnology, Government Institute of Science, Nipatniranjan Nagar, Caves Road, Aurangabad (M.S), India 2Government Institute of Forensic Science, Nipatniranjan Nagar, Caves Road, Aurangabad (M.S), India Abstract A novel thermophile, aerobic, Gram positive, spore forming bacterium isolated from hot water springs of Dhapoli, Maharashtra; India. The isolate was able to ferment a wide spectrum of sugars, polyols, and polysaccharides like xylan, glycogen and starch. Optimal growth was observed at 60°C, and pH at 6.5. Phylogenetic analysis of the whole 16S rRNA gene sequence clustered the strain T4 with the representatives of the genus Anoxybacillus and with Geobacillus tepidamans. The G+C content of the genomic DNA was 41.7%. Fatty acid profile (major fatty acids iso-C15:0 and iso-C17:0) confirmed the affiliation of the strain to the genus Anoxybacillus. Anoxybacillus rupiensis showed 75% of Cr (VI) reduction after 24 hours of incubation. Our results have confirmed that Anoxybacillus rupiensis is one of the most prominent thermophiles that could be exploited for the treatment of chromium bearing effluents. Keywords: Thermophiles; Hot water springs; Anoxybacillus rupiensis; at Dhapoli hot water springs but their ability was not assessed for 16sRNA gene analysis; Cr (VI) reduction. biodegradation purposes. In this study the phylogenetic, physiological and biochemical properties of a novel isolate Anoxybacillus rupiensis Introduction and the assessment of its ability to reduce Cr (VI) on lab scale, are The first representative of the genus Anoxybacillus,A. pushchinoensis described. was described by Pikuta [1] as strictly anaerobic and an emended Material and Methods description of the species was published later on Pikuta [2] according to which this species should be considered as aerobic anaerobe and Sample collection and enrichment the genus Anoxybacillus should be emended to aerobic anaerobes and The hot water was collected from the hot water spring source into facultative anaerobes. Although the name of the genus Anoxybacillus sterile steel thermos. Soil Samples were also collected from the same means ‘‘without oxygen Bacillus’’, according to Pikuta [1], most of the source at the depth of 1 m and taken in 100 ml sterile bottles. Both species described grow well aerobically and even for some species dilution plate method and enrichment method were used for isolation anaerobic growth was registered only under certain conditions [3]. The of aerobic thermophilic bacteria [6]. Bacillacea family members are good source of bacteria for bioprocessing and biotransformation involving whole cells or enzymes. In contrast to From the enrichment method, 10 g or 10 ml of samples were Bacillus and Geobacillus, Anoxybacillus is relatively new genus that was transferred into 100 ml of Thermus broth media. Incubation was proposed in the year 2000. Because these bacteria are alkali tolerant performed in stationary shaker at 60°C until turbidity was observed. thermophiles, they are suitable for many industrial applications. Then 500 µl of the broth was plated on the Thermus media plates; More than a decade after the first report of Anoxybacillus, where in Gelarite was used as a solidifying agent. For the dilution plate knowledge accumulated from fundamental and applied studies suggest method, 10 g soil or 10 ml of water samples were transferred in 90 ml that the genus can serve as a good alternative in many applications of 0.85% saline water. The mud samples only were pasteurized for 10 related to environmental waste water treatment, dye degradation [4] minutes at 80°C in a waterbath in order to kill most of the vegetative enzyme technology, and possibly bioenergy production [5]. There are cells and thus to eliminate non-spore forming bacteria. Following the many reports regarding the reduction of Cr (VI) which is soluble and heat treatment, 1ml aliquots from each sample was transferred in 9 ml toxic form into Cr (III) insoluble non- toxic form by mesophilic aerobic of 0.85% saline water and 6 fold dilutions were prepared. One ml of the bacteria. A great deal of work has been carried out on aerobic reduction dilutions was plated on Thermus solid media and incubated for 24h at of Cr (VI) by Pseudomonas sp. Literature survey has confirmed only 60°C. The plates were covered with cooking bags to prevent drying of few reports on reduction of Cr (VI) by aerobic thermophilic bacteria. This generated interest to check the potential of the isolated aerobic thermophilic bacteria for Cr (VI) reduction. Generally, aerobic bacteria *Corresponding author: Gursahani YH, Department of Biotechnology, when grown under anerobic conditions, Cr (VI) serves as a terminal Government Institute of Science, Nipatniranjan Nagar, Caves Road, Aurangabad electron acceptor. During metabolism, electrons released as a result of (M.S), India, Tel: +919890497439; E- mail: [email protected] oxidation of energy sources, NAD and FAD are reduced to NADH2 and Received April 20, 2015; Accepted June 30, 2015; Published July 10, 2015 FADH 2. This in turn donates the electrons to the different redox carriers Citation: Gursahani YH, Gupta SG (2015) Hexavalent Chromium Reduction of electron transport chain of aerobic bacteria. Finally, the electrons are by Anoxybacillus rupiensis isolated From Hot Water Spring of Dhapoli, accepted by Cr (VI) and reduced into Cr (III). Maharashtra, India. J Pet Environ Biotechnol 6: 232. doi:10.4172/2157- 7463.1000232 Dhapoli, a small town in Ratnagiri district of Maharashtra State of India is rich in geothermal water sources, recording the highest Copyright: © 2015 Gursahani YH, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits temperature of 71°C and has pH 6.5. Although, thermophilic producers unrestricted use, distribution, and reproduction in any medium, provided the of biotechnologically valuable enzymes were isolated from Unhavre original author and source are credited. J Pet Environ Biotechnol ISSN: 2157-7463 JPEB, an open access journal Volume 6 • Issue 4 • 1000232 Citation: Gursahani YH, Gupta SG (2015) Hexavalent Chromium Reduction by Anoxybacillus rupiensis isolated From Hot Water Spring of Dhapoli, Maharashtra, India. J Pet Environ Biotechnol 6: 232. doi:10.4172/2157-7463.1000232 Page 2 of 5 Gelarite at this temperature. Single colonies were picked and they were out by Disha life Sciences, Ahmedabad, Gujarat, India. purified using streak plate method [6]. G+C content Culture medium and growth conditions G+C content of isolates were determined at the identification service The samples were enriched in Thermus medium, containing (g/l): of Genei; India. DNA was purified on hydroxyapatite according to the peptone (Difco)-3, NaCl (Difco)-1, yeast extract (Difco)-2, Beef extract procedure of Cashion [7]. G+C content was determined according to (Difco)-0.2, pH of the medium was adjusted before autoclaving to 7.0 Mesbah [8] by using HPLC. GC content was calculated from the ratio using 1 M NaOH. Cultures were incubated overnight at 60°C. The same of deoxyguanosine (dG) and thymidine (dT). medium supplied with 3% Gelarite (a substitute for agar) was used for obtain pure cultures. Cellular fatty acid analysis Phenotypic characterization Fatty acid and polar lipid content was determined as described by Nicolaus [9]. The fatty acid analysis was got done from Genei, Isolates were grown on sporulation media for 2 days. The cells were Bangalore, India. suspended in 3–5 μl of sterile distilled water on a glass slide and covered with another slide. Endospores were observed under the phase contrast Chromium reduction microscope. Chromium stock K2Cr2O7 (100 mM) was prepared of which 0.4 ml The effect of pH on growth was determined in the area 5.0–9.0 with was added to the serum bottles so as the final concentration of hexavalent 0.5 pH steps using acetate (for pH 5–5.5), phosphate (for pH 5.5–8.0) chromium in Yeast Starch Soluable Broth medium – (g/l) Soluble starch 15; Yeast extract 0.4; K HPO 0.23; KH PO 0.2; MgSO .7H O 0.05; or glycine-NaOH (for pH 8.0–9.0) buffers at concentration 0.05 M. pH 2 4 2 4 4 2 values were adjusted at room temperature. The influence of temperature Citric acid 0.052, 1000 ml distilled water; pH 5.5 is 2 mM. The culture on growth was determined at pH 6.5 at different temperature with medium was inoculated with 1 ml of the isolated culture. The bottle 5°C steps. Methods described by Smibert and Krieg were used for was sealed with rubber cap and aluminium foil seal to avoid medium physiological characterization of the strains. Catalase activity was evaporation and to maintain partial anaerobic conditions. A bottle was assayed by mixing a pellet of freshly centrifuged 6 h culture with a refrigerated (0 hr for initial chromium). The rest of the bottles were drop of 3% hydrogen peroxide. Their ability to utilize different sugars incubated at 60°C for estimation of residual Cr (VI). A pair of bottles was examined in a minimal salt medium (MSM) consisting of (g/l): was removed after every 2 hrs and was refrigerated. At the end of 24 hrs, all the refrigerated bottles were removed and the broth was subjected to (NH4)2HPO4, 1.0; KCl, 0.2; MgSO4, 0.1; thiamine, 1.10–6; bromthymol blue, 1.10–4.
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