Identification and Bio-Corrosion Behavior of Thermoanaerobacter CF1, a Thiosulfate Reducing Bacterium Isolated from Dagang Oil Field
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African Journal of Microbiology Research Vol. 6(12), pp. 3065-3071, 30 March, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR12.239 ISSN 1996-0808 ©2012 Academic Journals Full Length Research Paper Identification and bio-corrosion behavior of Thermoanaerobacter CF1, a thiosulfate reducing bacterium isolated from Dagang oil field Guihong Lan1, Huarong Hong2*, Dayong Qing1, Yan Wu1 and Haobo Tan3 1State Key Laboratory of Oil Gas Reservoir Geology and Exploitation, Southwest Petroleum University, Chengdu 610500, China. 2College of Basic Medicine, Chengdu Medical College, Chengdu 610083, China. 3China National Offshore Oil Corporation (CNOOC) Oilfield Technology Services Co. Ltd., Tanggu 300451, China. Accepted 8 March, 2012 A slightly curved rod strictly anaerobic, thermophilic bacterium was isolated from Dagang oil field, China. Its fundamental physiology and bio-corrosion characteristic were characterized. The growth of strain CF1 occurred at 45 to 78°C (optimum 60°C), pH 5.0 to 9.5 (optimum 7.1). The doubling time under optimal conditions was 45 min. Substrates that could be used included D-cellobiose, fructose, glucose, maltose, mannose, trehalose, melezitose, raffinose, ribose, sucrose, xylose and starch. 16S rRNA gene sequence analysis indicated that the isolate was most closely related to Thermoanaerobacter uzonensis (99.2%). The G+C content of the genomic DNA of strain CF1 was 33.5 mol%. The major cellular fatty acid contents were 14 : 0 (2.5%), 15 : 0 (65.8%), 16 : 0 (8.6%), 15-methyl 17:0 (8.1%), 14- methyl 17:0 (7.4%), 18 : 1v9c (1.7%) and 18 : 0 (4.8%). The strain reduced up to 1 M thiosulfate to elemental sulfur without sulfide formation. In the presence of thiosulfate, electrochemical method, weight loss and surface analysis showed the corrosion behavior of the isolate. A slight change of corrosion current was detected through electrochemical method. Elemental sulfur was found on the surface of the carbon steel, with less weight loss in the cultivation system. Key words: Thiosulfate reduction, element sulfur, bio-corrosion, carbon steel. INTRODUCTION Microbial thiosulfate reduction is an important process in thiosulfate to either elemental sulfur or hydrogen sulfide, the geochemical cycling of sulfur species under or both. Many of them belong to the genus anaerobic environment (Ravot et al., 1995). Thiosulfate is Thermoanaerobacter or Thermoanaerobacterium, a highly significant sulfur intermediate, that can be either stratum of oil fields. Numbers of themophilic microbes reduced to produce inorganic sulfur compounds or can reduce thiosulfate to either elemental sulfur or disproportionate (Jørgensen, 1990), in the anaerobic hydrogen sulfide. Most species of the genus conditions. The reduction of thiosulfate increases with Thermoanaerobcter reduce thiosulfate to hydrogen stratum depth, anaerobic organisms participate promi- sulfide, except 3 species. Thermoanaerobacter italicus nently in dissimilatory thiosulfate reduction (Lee et al., and Thermoanaerobacter uzonensis produce both sulfide 2007), indicating that thermophilic anaerobic microbes and elemental sulfur (Kozianowski et al., 1997; Wagner may be responsible for the function, in deep stratum of oil et al., 2008), and Thermoanaerobacter sulfurigignens fields. Numbers of thermophilic microbes can reduce produces elemental sulfur (Lee et al., 2007). It has been proven that the presence of thiosulfate in oil fields may increase the risk of biocorrosion of oil pipelines (Faudon et al., 1995; Duncan et al., 2009). A "thiosulfate shunt" *Corresponding author. E-mail: [email protected]. seems to exist in anoxic environment, and may be very 3066 Afr. J. Microbiol. Res. important to control the corrsion. So, its further control is chromatography (Sasser, 2001). therefore a key reaction in oil fields. In this paper, we report on one Thermoanaerobacter Polarization study isolate from Dagang oil field and which is typically for the genus Thermoanaerobacter, which reduces thiosulfate to Q235 carbon steel (Cangzhou City Rui Pipe Co., Ltd. China) was elemental sulfur only without the expected formation of used as a working electrode in this study, which composed of the sulfide. We describe its physiological characteristics and following elements with a mass ratio of 0.22% C, 0.05% Si, 0.48% biological corrosion with metabolism of thiosulfate. Mn, 0.012% P, 0.022% S. It was plugged in araldite with an exposed area of 1.0 cm2. The specimens were immersed at 60°C in a sterile medium, as well as the isolation inoculated in basal medium with 250 mM sodium thiosulfate. A platinum plate and a MATERIALS AND METHODS saturated calomel electrode (SCE) were used as a counter electrode and a reference electrode respectively (Kuang et al., Sample collection and bacterial isolation 2007). The Tafel polarization curves were obtained according to Anandkumar et al. (2009). Strain CF1 was isolated from a reservoir water sample from an off shore oil-producing well (Dagang oil field) in Tianjin. Production fluids of Dagang oil field used for microbial analysis were collected Method for weight loss detection directly from production wellheads into sterile carboys. Size of 1 to 5 cm of Q235 carbon steel coupons were polished, degreased with aceton and air dried. The carbon steels were Medium, enrichment and isolation immersed in 250 ml of isolation medium incubated at 60°C. Duplicate systems were maintained, one inoculated with 5 ml of The modification of Hungate technique (Miller and Wolin, 1974) was strain CF1 and the other was blank control. Finally, the carbon used in the overall process. Enrichments were performed at 60°C steels were removed in pickling solutions, washed with water and by directly inoculating a 3 ml sample from the reservoir water into a dried and the average loss weight of the coupons in each culture basal medium containing (per litre of distilled water) 1 g KH2PO4, and uninoculated control were calculated. 0.5 g K2HPO4, 3 g NaCl, 0.5 g NaNO3, 1 g NH4Cl, 0.05 g CaCl2.2H2O, 0.2 g MgCl2.6H2O, 0.5 g Cysteine-HCl, 2 g yeast extract (Oxoid), 1 g Bio-trypticase (Oxoid), 5 g glucose , 10 ml of X-ray diffraction (XRD) analysis the trace element solution (Balch et al., 1979) and 0.1 mg resazurin. The carbon steel specimens were taken out and dried in cool air. The corrosion products were observed by XRD (X’ Pert MPD PRO) analysis. Physiological studies Physiological characteristics of strain CF1 were tested with different carbons, and a range of temperature and pH. The growth was RESULTS determined by counting and by measuring the increase in optical density at 600 nm. The substrates were tested in the presence or Petroleum reservoir characteristics absence of sodium thiosulfate (20 mM). For electron acceptors tests, 20 mM sodium thiosulfate, 20 mM sodium sulfite, 20 mM The oil field is exploited using water-flooding. The water sodium sulfate and 2% (w/v) elemental sulfur were added to the medium (Ravot et al., 1999). To test the tolerance of thiosulfate, a for injection is separated from the oil produced fluid and ranging concentration of sodium thiosulfate was added to the recycled. The formations are situated 1965 to 1976 m medium. The end products from glucose fermentation were below the sea floor and have a temperature of 75°C. The determined by gas chromatography (Chrisotomos et al., 1996). formation water of sodium hydrocarbonate type has a low salinity (4780 mg/l) and a pH of 8.3 with low concentration sulfate (8 mg/l). Phylogenetic analysis The 16S rRNA gene was selectively amplified from genomic DNA by PCR using 27F and 1492R as the primers. The productions Physiological studies were recovered by a DNA Recovery Kit, and ligated to pMD-19T cloning vector and transformed into Escherichia coli JM109. Three A strictly anaerobic, thermophilic bacterium, designated positive clones were selected randomly for sequencing analysis. Sequence analysis and phylogenetic tree constructions were done strain CF1, was isolated. The cells were Gram-positive using CLUSTAL_X 1.83 and MEGA 4.1 software, and the GenBank rod-shaped, straight to slightly curved approximately 0.5 accession number was HM228409. μm in width and 2 to 6 μm in length (Figure 1). Occasionally, cells up to 25 μm in length were observed. Shorter or longer cells frequently contained intermittent The DNA G+C content and cellular fatty acid contents coccoid cells (Figure 1), spores were oval and located at the terminal of the mother cell which have the typical Genomic DNA was extracted using a large-scale phenol/ chloroform-based procedure essentially as described by Wilson characteristics of Thermoanaerobacter (Wiegel and (1997). The DNA G+C content was measured by using HPLC Ljungdahl, 1981). Physiological analysis showed that (Mesbah et al., 1989). The celluar fatty acids were analyzed by gas yeast extract was necessary for growth on non- Lan et al. 3067 Figure 1. Electron micrograph of BF1. Figure 2. Morphology of cells and intracellular sulfur deposits were observed in strain CF1 (The lights pots in cell are sulfur deposits). proteinaceous substrates. Using glucose as carbon arabinose, sodium carboxymethylcellulose, xylan. When sources, growth occurred at 45 to 78°C (optimum 60°C), grown with 2 g glucose l-1 and 1 g yeast extract l-1, the at pH 5.0 to 8.5 (optimum 7.1), at NaCl 0 to 4.5% fermentation end products were detected with 0.29 g -1 -1 (optimum 0.5%). ethanol l , 0.07 g acetic acid l , some CO2 and H2, after At the optimum, the shortest observed doubling-time cultivation for 3 days. The presence of thiosulfate did not was 45 min. Some sugars were used as energy sources, affect the substrate utilization pattern. The effect of the substrates were the following: D-cellobiose, fructose, electron acceptors analysis showed that the strain CF1 glucose, maltose, mannose, trehalose, melezitose, used thiosulfate but not sulfur or sulfate. It could be raffinose, ribose, sucrose, xylose and starch.