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Crude-Oil-Degrading Thermophilic Bacterium Isolated from an Oil Field

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Crude-Oil-Degrading Thermophilic Bacterium Isolated from an Oil Field 175 Crude-oil-degrading thermophilic bacterium isolated from an oil field Ruixia Hao, Anhuai Lu, and Guanyu Wang Abstract: Thermophilic bacterium strain C2, which has the ability to transform crude oils, was isolated from the reser- voir of the Shengli oil field in East China. The Gram-negative, rod-shaped, nonmotile cells were grown at a high tem- perature, up to 83 °C, in the neutral to alkaline pH range. Depending on the culture conditions, the organism occurred as single rods or as filamentous aggregates. Strain C2 was grown chemoorganotrophically and produced metabolites, such as volatile fatty acids, 1,2-benzenedicarboxylic acid, bis(2-ethylhexyl)ester, dibutyl phthalate, and di-n-octyl phthalate. It could metabolize different organic substrates (acetate, D-glucose, fructose, glycerol, maltose, pyruvate, starch, sucrose, xylose, hexadecane). The G+C content (68 mol%) and the 16S rRNA sequence of strain C2 indicated that the isolate belonged to the genus Thermus. The strain affected different crude oils and changed their physical and chemical prop- erties. The biochemical interactions between crude oils and strain C2 follow distinct trends characterized by a group of chemical markers (saturates, aromatics, resins, asphaltenes). Those trends show an increase in saturates and a decrease in aromatics, resins, and asphaltenes. The bioconversion of crude oils leads to an enrichment in lighter hydrocarbons and an overall redistribution of these hydrocarbons. Key words: thermophile, metabolite, crude oil, degradation, conversion. Résumé : La souche de bactéries thermophiles C2, qui a la capacité de transformer les pétroles bruts, a été isolée d’un réservoir du champs pétrolifère de Shengli dans la Chine orientale. Les cellules Gram-négatives en forme de bâtonnets et non motiles ont été cultivées à température élevée jusqu’à 83 °C sous un pH neutre à alcalin. Selon les condition de cultures, l’organisme s’est présenté sous forme de bâtonnets simples ou d’agrégats filamenteux. La souche C2 a été cultivée de manière chimio-organotrophe et a produit certains métabolites, tel que des acides gras, l’acide bis-ester 2-benzenedicarboxylique, le dibutyl phthalate et le di-n-octyl phthalate. Elle a pu métaboliser différents substrats organiques (acétate, D-glucose, fructose, glycérol, maltose, pyruvate, amidon, sucrose, xylose, hexadécane). Le contenue en G+C (68 mol%) et la séquence de l’ARNr 16S de la souche C2 indiquent que l’isolat appartient au genre Thermus. La souche a altéré divers pétroles bruts et a modifiée les propriétés physiques et chimiques des pétroles bruts. Les For personal use only. interactions biochimiques entre les pétroles bruts et la souche C2 empruntent des tendances définies par un ensemble de marqueurs chimiques (saturés, aromatiques, résines, asphaltènes). Ces tendances révèlent une augmentation des huiles saturées et une diminution des aromatiques, des résines et des asphaltènes. La bioconversion des pétroles bruts mène à l’enrichissement en hydrocarbures plus légers et en une redistribution généralisée de ces hydrocarbures. Mots clés : thermophile, métabolite, pétrole brut, dégradation, conversion. [Traduit par la Rédaction] Hao et al. 182 Introduction environments, and they have extensive prospects for applica- tion (He et al. 2000). Many thermophilic bacteria with opti- Temperature is one of the most important variables in our mum growth temperatures from 45 to 90 °C have been environment. Bacteria have traditionally been divided into isolated from oil fields (Jeanthon et al. 1995; L’Haridon et three main groups, psychrophiles, mesophiles, and thermo- al. 2001, 2002; Miroshnichenko et al. 2001; Orphan et al. philes. Thermophilic bacteria can be isolated from hot 2000; Ravot et al. 1995; Sanchez et al. 1993; Takahata et al. springs, hot composts, hot pipes, or factories and from other 2000, 2001). However, crude-oil-degrading thermophilic habitats that are only heated transiently (Kristiansson and bacteria isolated from oil fields have not been reported up to Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Canadian Science Publishing on 04/19/17 Stetter 1992). Many researchers have been engaged in study- present. The main interest in thermophiles in recent years ing thermophiles. It is reported that 140 species of 70 genera has been on two fronts. On the scientific front there has been of thermophiles have been discovered in high temperature the discovery of many new extremely thermophilic archaea, some of which can grow at 100 °C and higher (Baross and Received 27 June 2003. Revision received 3 December 2003. Deming 1983). On the biotechnological front there has been Accepted 8 December 2003. Published on the NRC Research the realization that thermophilic microorganisms can serve Press Web site at http://cjm.nrc.ca on 2 April 2004. as excellent sources for more thermostable biocatalysts than presently available (Kristiansson and Stetter 1992). 1 R. Hao, A. Lu, and G. Wang. Department of Geology, In the Shengli oil field of East China, most of the reser- Peking University, Beijing, 100871 China. voirs under enhanced oil recovery processes have extreme 1Corresponding author (e-mail: [email protected]). physical conditions. The oil wells range from 1000 to Can. J. Microbiol. 50: 175–182 (2004) doi: 10.1139/W03-116 © 2004 NRC Canada 176 Can. J. Microbiol. Vol. 50, 2004 2000 m deep, with in situ temperatures between 60 and Enrichment culture and isolation procedure 90 °C and the average in situ pressure 10 MPa. In deeper To isolate strain C2 from the reservoir samples, the samples geological layers, increases in temperature and pressure pro- were amended with carbon and nitrogen sources to stimulate vide suitable conditions to support thermophilic organisms. the growth of indigenous microorganisms. Ten millilitres of Petroleum reservoirs constitute ecological niches from EM1 was inoculated with approximately 1 mL of original which various physiological types of thermophilic and crude-oil samples; inoculation was started immediately after hyperthermophilic microorganisms have been isolated or de- the samples arrived. The enrichment culture was set up at tected. Physiological types isolated from these biotopes in- 70 °C and atmospheric pressure, with shaking in a rotary clude sulfate reducers (Beeder et al. 1994; Stetter et al. shaker at 150 r/min for 3–5 days. After 3 days of incubation, 1993; Tardy-Jacquenod et al. 1996), sulfur reducers (Stetter the EM1 contained about 106 rod-shaped cells/mL. The en- et al. 1993), methanogens (Ng et al. 1989), fermentative bac- richment culture could be successfully transferred to the teria (Davey et al. 1993; Grassia et al. 1996), manganese and same medium. iron reducers (Greene et al. 1997), and dibenzothiophene- Dominant species in the enrichment culture were isolated degrading bacteria (Bahrami et al. 2001). Similar types of by serially diluting the cultures and plating them on agar thermophiles have also been isolated from other high- containing IM. Plates were incubated in an oven at 70 °C. temperature environments, such as aquatic hot springs and Successive transfers on solid media isolated pure cultures. shallow water and deep-sea hydrothermal vents (He et al. To improve the growth yield of this culture, EM2 was de- 2000; Kristiansson and Setter 1992; Lowe et al. 1993). De- signed based on the results of a chemical analysis of the res- spite the description of an increasing number of new ervoir samples. EM2 was supplemented with different thermophilic species, relatively little information is available nutriments. The initial concentration of the organism was on the composition of microbial assemblages in these unique about 107 cells/mL in this medium. subsurface environments. This report describes a crude-oil- degrading thermophilic bacterium, strain C2, isolated from a Growth characteristics petroleum reservoir located in the Shengli oil field of East Studies of temperature and pH range, as well as the sub- China. strates used, were carried out in a water rotatory shaker at 150 r/min. The temperature growth range was determined in EM2 by incubating samples at 30–85 °C. The range of pH Materials and methods for growth in EM2 was determined at 70 °C and at pH val- ues of 6, 7, and 8. For the following pHs, different buffers were used to replace the phosphate in the EM2: for pH 9, Collection of samples 84 mmol/L boric acid – NaOH, and for pH values of 5 and 4, The samples were taken from several wells in the Dongxin 50 mmol/L citric acid – Na HPO . The pH of each medium oil reservoir. The in situ temperatures were between 60 and 2 4 was adjusted to 70 °C after autoclaving. 80 °C, and the in situ pH was 7.1. The samples were col- For personal use only. To evaluate the capacity of strain C2 to use organic substrates, lected at the wellhead of the producing wells in sterile bot- the low concentration complex medium Y was supplemented tles, which were then sealed with rubber stoppers. The with different substrates (acetate, citrate, D-glucose, fructose, samples were transported to the laboratory and were stored glycerol, lactose, maltose, mannitol, pyruvate, starch, sucrose, during transport and afterward at ambient temperatures. xylose, hexadecane), each at a concentration of 2 g/L. For growth curves, cells were inoculated into EM2 and in- Culture media cubated at 70 °C. Samples for optical density were obtained Strain C2 was enriched in enrichment medium (EM1) every 3 h. Optical density was monitored at 550 nm using 1-cm containing the following (per litre): (NH4)2SO4, 1.3 g; yeast cuvettes in an UV-visible spectrophotometer (Cary 50Bio), extract, 1.0 g; pancreatic digest of casein, 1.0 g; KH2PO4, and the growth rate was calculated. 0.28 g; MgSO4·7H2O, 0.247 g; CaCl2·2H2O, 0.074 g; FeCl3·6H2O, 0.019 g; hexadecane, 25 mL; and salt solution, Analysis of metabolic products 1.0 mL (Pooks 1997). The isolation medium (IM) contained the following (per litre): tryptone, 2.5 g; yeast extract, 2.5 g; Volatile fatty acids assays Na2HPO4·12H2O, 0.43 g; MgCl2·6H2O, 0.2 g; KH2PO4, Strain C2 was incubated in 100 mL of YG medium (250- 54.0 mg; nitrilotriacetic acid, 0.1 g; CaSO4·2H2O, 40.0 mg; mL conical flask) at 70 °C for 72 h.
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