Octa Journal of Environmental Research Jul.–Sept., 2017 International Peer-Reviewed Journal ISSN 2321 3655 Oct. Jour. Env. Res. Vol. 5(3): 206-213 Available online http://www.sciencebeingjournal.com Octa Journal of Environmental Research Research Article CULTIVATION, ISOLATION AND IDENTIFICATION OF SULFATE REDUCING BACTERIA EMPLOYING HUNGATE TECHNIQUE Vipul Kashyap and Chand Ram Grover* Synbiotic Functional Foods & Bioremediation Research Laboratory, Dairy Microbiology Division, ICAR-National Dairy Research Institute, Karnal-132001, Haryana, India Corresponding Author’s E-mail: dmcaft2011@gmail.com Received: 17 th Sept. 2017 Revised: 29 th Sept. 2017 Accepted: 30 th Sept. 2017 Abstract: Obligate anaerobes are the most complicated establishment in the nature, which survive only in absence of molecular oxygen. The major challenge to cultivate anaerobes is lack of well-equipped anaerobic facility in the laboratory. Hungate roll tube technique is generally used for anaerobic isolation, wherein roll tubes are employed for isolation of bacterial colonies. In the present study the roll tubes were replaced with serum bottles to cultivate and isolate sulfate reducing bacteria as these are more convenient and reliable. A total 5 isolates were recovered from open drains of Tung Dhab region of Amritsar district (Punjab), India based on colony morphology which were further screened on the basis of gram staining, and 16S rRNA analysis. Sulfate reducing bacteria (BN-15) was finally identified as Clostridium indolis BN-15. Substrate i.e. short chain volatile fatty acids and NaCl utilization patterns were also observed for finally screened isolate. Keywords: Anaerobic isolation; Clostridium species; Hungate roll tube; Sulfate reducing bacteria. Postal Address: Synbiotic Functional Foods & Bioremediation Research Laboratory, Dairy Microbiology Division, ICAR-National Dairy Research Institute, Karnal-132001, Haryana, India Phone: +91 184 2259182 INTRODUCTION Sulfur exists in the form of sulfate in sediments or pyrite/gypsum in rocks, where following the reduction/oxidation of sulfate/sulfide, nourish many micro-flora and fauna. Sulfur is a major constituent of the biogeochemical sulfur cycle as sulfur has a broad range of oxidation states, from -2 to +6. It can be transformed both chemically and biologically to various sulfur intermediates. Sulfate is consumed by sulfate reducing bacteria (SRB) and companions of archaea/bacteria, which uses it as a terminal electron acceptor for its reduction into sulfide when combined with enzymes and amino acids. Biochemical reactions representative of oxidation (chemolithotrophic sulfate reducers) and reduction process (dissimilatory sulfate reducers) generates metabolic energy. Sulfate reducing bacteria are widely distributed in nature, dominated mainly in extreme environments like marine sediments estuarine, saltmarsh sediments, environment of landfills, freshwater lakes, activated sludge systems, and acidic sediments representing its obligate anaerobic nature (Pimenov et al. 2014; Xia et al. 2014; Andrea et al. 2015; Brand et al. 2015; Colin et al. 2017; Cui et al. 2017; Yang et al.2017; Kharrat et al. 2017). The SRB are effectively adapted to almost all the ecosystem of the planet, where stringent anaerobic conditions prevail. In these ecosystems, they have to face severe physicochemical conditions. The SRB can be gathered into seven phylogenetic ancestries, two among the archaea and five among the bacteria which are constructed based on the comparative study of 16S rRNA sequences (Muller et al. 2014). Depending upon the substrate requirement sulfate reducers can be categorized into two groups: (a) Organotrophs- a microbial community which uses carbon complexes, such as lactate, acetate, and pyruvate as electron donors for e.g. bacterial community