Journal of Petroleum Science and Engineering 43 (2004) 247 258 www.elsevier.

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Effect on crude oil by thermophilic bacterium

Ruixia Hao *, Anhuai Lu, Yishan Zeng
Department of Geology, Peking University, Beijing, 100871 PR China Received 26 June 2003; accepted 13 February 2004

Abstract The objective of this study is to demonstrate the basic characteristics of thermophilic strain TH-2 and evaluate the effect of strain TH-2 on different crude oils. Strain TH-2 is a non-motile, Gram-negative, oil-degrading thermophile that was isolated from a reservoir of the Shengli oil field in East China. The cells of strain TH-2 were grown at high temperatures up to 85 jC in the neutral to alkaline pH range. Depending on the culture conditions, the organisms occurred as single rods, or as filamentous aggregates. Strain TH-2 was grown chemoorganotrophically and produced volatile fatty acids and such surfactants as 1,2benzenedicarboxylic acid-bis ester, dibutyl phthalate, and di-n-octyl phthalate. It could use different organic substrates (acetate, D-glucose, fructose, glycerol, maltose, pyruvate, starch, sucrose, and xylose). Laboratory studies have demonstrated that the strain affected different crude oils, converted and degraded various components and changed the physical and chemical properties of crude oils. The strain TH-2 degraded crude oil and growth of the bacterium on crude oil resulted in loss of aromatic hydrocarbons, resins, and asphaltenes. The bioconversion of crude oils leads to an enrichment in lighter hydrocarbons and an overall redistribution of these hydrocarbons. The interactions of microorganisms with crude oils are variable and depend on the microbial species and the chemical compositions of crude oils. Different interactions may influence the efficiency of processes in which single or mixed microbial species are used for the oil treatment and may also suggest possible combinations of biological and chemical technologies. D 2004 Elsevier B.V. All rights reserved.
Keywords: Thermophilic bacterium; Metabolite; Crude oil; Conversion; Degradation

1. Introduction Thermophilic bacteria can be isolated from hot springs, hot pipes or factories and other habitats, which are only heated transiently, and from permanently cold places. Many researchers have been engaged in studying thermophiles. It is reported that 140

* Corresponding author. Tel.: +86-10-82309539(H), +86-1062754156(O); fax: +86-10-62754156. E-mail address: rxhao@pku.edu.cn (R. Hao). 0920-4105/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.petrol.2004.02.017

species of 70 genera of thermophiles have been discovered in high temperature environments, and they have extensive prospects of application (He et al., 2000). In the Shengli oil field of East China, most of reservoirs under EOR (Enhanced Oil Recovery) processes have extreme physical conditions with temperature of 60 90 jC and depths of 1000 2000 m. This harsh environment seems to be unsuitable for microbial growth. However, it was discovered that extreme thermophilic bacteria existed in deep-sea, hydrothermal vent with high temperatures and pressures (Brock,

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1978; He et al., 2000; Kristiansson and Stetter, 1992). Similarly, many thermophilic bacteria with optimum growth temperature from 45 to 85 jC have been isolated from oil fields (Beeder et al., 1995; Cayol et al., 1995; Cochrane et al., 1988; Fardeau et al., 1996, 1997; Jeanthon et al., 1995; LHaridon et al., 2001, 2002; Miroshnichenko et al., 2001; Nilsen et al., 1996a,b; Orphan et al., 2000; Ravot et al., 1995; Ress et al., 1995; Sanchez et al., 1993; Takahata et al., 2001). Interactions between microorganisms and crude oils under laboratory and reservoir conditions have been studied over the past 40 years (Premuzic and Lin, 1999). A wide range of studies has dealt with biodegradation, the role of sulfate-reducing bacteria in reservoir fouling, corrosion and microbial enhanced oil recovery. Results from these studies have been extensively reviewed as provided by Tissot and Welte (1978), Yen (1990) and Premuzic and Woodhead (1993). Microbial enhanced oil recovery (MEOR) involves the use of microorganisms and their metabolic products to increase oil recovery. However, it is necessary that the microorganisms can grow and metabolize in the hot conditions in the reservoir. The use of microbes in MEOR involves knowledge of the many biochemical changes they impart to oil. This study examines the bioconversion and biodegradation in various crude oils. A microbial cultures ability to metabolize a chosen substrate can be determined by monitoring (a) growth on the substrate, (b) the loss of the substrate from the culture medium, or (c) the production of a specific metabolic end product (Fedorak et al., 1983). In studying the conversion and degradation of different components in crude oils, all three approaches have been used. Thermophilic bacteria have been reported in a vast array of literature, but the thermophilic bacteria isolated from oilfields and effects on crude oil have only been detailed in unpublished reports. The thermophilic bacterium, strain TH-2, which converted and degraded crude oils, was isolated from Shengli oil field in China. The primary purpose of this study is to demonstrate the basic characteristics of thermophilic strain TH-2 and evaluate the effect of strain TH-2 on various crude oils.

2. Materials and methods 2.1. Experimental design First, collecting the samples (crude oil) from the oil well, and selecting culture media to enrich, culture, and isolate the thermophilic bacterium (strain TH-2). Second, in order to understand more the basic features of strain TH-2, doing various physiological and biochemical tests, such as determining growth curve and rate, optimum growth temperature and pH, the capability of strain TH-2 to use different organic substrates, oxidase test, methyl red and indole test, catalase test, and so on. Third, analyzing the metabolic products of strain TH-2. Fourth, assaying the surface tension of the cell-free extract. Fifth, effecting of strain TH-2 on crude oils with various viscosities, and analyzing the physical and chemical features of all the crude oil samples before and after biotreatment. 2.2. Collection of samples The samples were taken from several wells in the Dongxin reservoir; the original temperatures were 60 80 jC, and the original pH was 7.1. The samples were collected at the wellhead of the producing wells in sterile glass bottles, which were then tightly sealed with rubber stoppers. The samples were transported to the laboratory and stored during transport and afterward at ambient temperatures. 2.3. Culture media For the samples tested, strain TH-2 was first enriched in an enrichment medium (EM1). It contained per liter: (NH4)2 SO4, 1.3 g; yeast extract, 1.0 g; pancreatic digest of casein, 1.0 g; KH2PO4, 0.28 g; MgSO 4 7H 2 O, 0.247 g; CaCl 2 2H 2 O, 0.074 g; FeCl36H2O, 0.019 g; hexadecane, 25 ml; and salt solution 1.0 ml. Salt solution contained per liter: Na2B4O710H2O, 4.4 g; MnCl2.4H2O, 1.8 g; ZnSO4 7H2O, 0.22 g; CuCl2H2O, 0.05 g; Na2MoO42H2O, 0.03 g; VOSO42H2O, 0.33 g (Pooks, 1997). The isolation medium (IM) for isolation, it contained per liter: tryptone, 2.5 g; yeast extract, 2.5 g; Na2HPO412H2O, 0.43 g, MgCl26H2O, 0.2 g, KH2PO4, 54.0 mg, nitrilotriacetic acid, 0.1 g; CaSO42H2O, 40.0 mg; ferrous citrate solution, 0.5

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ml; and trace element solution, 1.0 ml (Pooks, 1997). The experimental medium (EM2) for culture, stock maintenance and experimental studies contained per liter: NaNO3, 0.5 g; MgSO4.7H2O, 0.01 g; NaCl, 5.0 g; K2HPO4, 2.7 g; NH4Cl, 0.1 g; peptone, 3.0 g; yeast extract, 0.5 g; glucose, 3.0 g; and beef extract, 1.0 g. The medium Y to evaluate the capacity of the strain to use organic substrates, it contained per liter: NaNO3, 0.5 g; MgSO47H2O, 0.01 g; NaCl, 5.0 g; K2HPO4, 2.7 g; NH4Cl, 0.1 g; and yeast extract, 0.2 g. The medium YG for metabolites assay was consisted of the following components, in g/l: 0.5 yeast extract, 5.0 glucose, 0.5 NaNO3, 0.01 MgSO4.7 H2O, 5.0 NaCl, 2.7 K2HPO4, and 0.1 NH4Cl. 2.4. Enrichment culture and isolation procedure In order to isolate strain TH-2 from the reservoir samples, the samples were amended with carbon and nitrogen sources to stimulate the growth of indigenous microorganisms. About 10 ml of EM1 medium was inoculated with approximately 1 ml of original crude oil samples, which was performed immediately after the samples arrived. The enrichment was set up at 70 jC and atmospheric pressure by shaking in a rotary shaker at 150 rev min 1 for 3 5 days. After 3 days of incubation, the EM1 medium contained about 106 rod-shaped cells per ml. The enrichment culture could be successfully transferred to the same medium. Dominant species in the enrichment culture were isolated by serially diluting the cultures and plating them on the isolation medium (IM) with agar. Plates were incubated in oven at 70 jC. Successive transfers on solid media obtained pure cultures. In order to improve the growth yield of this culture, an experimental medium (EM2 medium) was designed based on the results of a chemical analysis of the reservoir samples. EM2 medium was supplemented with different nutrition (NaCl, NaNO3 , NH4Cl, K2HPO4, glucose, beef extract and peptone). The concentration of the organism was about 107 cells per ml under this medium. 2.5. Growth characteristics Study of the temperature and pH range, as well as substrates used, were determined in a water rotatory

shaker at 150 rpm. The temperature growth range was determined in the medium EM2, by incubation between 30 and 90 jC. The range of pH for growth was determined at 70 jC, in EM2 at pH values of 6, 7 and 8. For the following pHs, different buffers were used to replace the phosphate in the EM2 medium. For pH 9, 84 mM boric acid-NaOH; and for pH values of 5 and 4, 50 mM citric acidNa2HPO4. The pH of each medium was adjusted at 70 jC after autoclaving. To evaluate the capacity of strain TH-2 to use organic substrates, the low concentration complex medium Y was supplemented with different substrates (acetate, citrate, D-glucose, fructose, glycerol, lactose, maltose, mannitol, pyruvate, starch, sucrose, xylose) each at a concentration of 2 g/l. Growth curve was derived from the strain TH-2. Cells were inoculated into EM2 and incubated at 70 jC for 3 days. Samples for optical density were obtained every 3 h. Optical density was monitored at 550 nm using 1-cm cuvettes in an UV Visible spectrophotometer (Cary 50Bio) and the growth rate was calculated. 2.6. Analysis of metabolic products 2.6.1. Volatile fatty acids assays Strain TH-2 was incubated in 100 ml of the medium YG (250 ml conical flask) at 70 jC for 72 h. To 1 ml culture broth one drop 6N H2SO4 was added, the pH of broth decreased to 3.5. The supernatant was obtained by centrifugation of broth at 6000 g during 20 min at 4 jC. The final pH of supernatant maintained 2. Volatile fatty acids were measured by a gas chromatograph (Shimadzu GC14B) using a GDX103 column (0.25 Am 0.53 mm 30 m) with N2 as the carrier gas, at a flow rate of 65 ml/min (injection temperature, 240 jC; column temperature, 210 jC). 2.6.2. Esters and other metabolites measurements After 72 h of growth in the medium YG at 70 jC, cells were removed by centrifugation (8000 g, 60 min, 4 jC). The supernatant was extracted by the mixed chloroform-methanol (2:1) and then distilled. The final supernatant was collected with sterile bottles and analyzed by a gas chromatograph-mass spectrometer (Shimadzu QP5050A). The samples

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were chromatographed with a gradient temperature program. The initial temperature of 80 jC was held for 2 min, and then the temperature was increased to 250 jC at 10 jC/min and held at 250 jC for 10 min. The injection temperature was 280 jC, and the transport line was maintained at 260 jC. The helium flow was 0.5 ml/min, and injection volumes were 2 Al. 2.7. Surface tensions assays The strain was incubated in EM2 broth at 70 jC for 3 days, sampled every 12 h and centrifuged the broth and then measured the surface tension of the supernatant. The surface tension of the supernatant was determined using an interfacial tension meter (CSC-NOS.70545). Surface tensions (air-liquid) were measured by the Dunouy method. The pure water was used to calibrate the system and measurements were taken in replicates of 7. The reported values are expressed as the means and standard deviations (the standard deviation ranges from 0.1 to 0.2). All surface tension measurements were made at room temperature. 2.8. Bacterial influence on crude oil The tests are to determine effecting of strain TH2 on different crude oils. The crude oils used in the study were taken from the Dongxin oil reservoir. The selected oils were divided into four types according to viscosity: crude oil A has a density of 0.9803 g/cm3 and a viscosity of 6.031 Pa s, crude oil B has a density of 0.9538 g/cm3 and a viscosity of 1.283 Pa s, crude oil C has a density of 0.9668 g/cm3 and a viscosity of 1.657 Pas, and crude oil D has a density of 0.9417 g/cm3 and a viscosity of 0.2335 Pas. The viscosities of various crude oils were measured with DV III Brook Viscometer at 50 rpm and 50 jC. The inoculums were pregrown in 100 ml medium EM2 with 5% (vol/vol) different four crude oils individually at 70 jC for 24 h and used for the experiment. The previous cultures and 65% (vol/vol) four different sterilized oils were added respectively into 500 ml conical flasks with 200 ml medium EM2. The 300 ml medium EM2 and 70% (vol/vol) four various sterilized oils were also added respectively into 500

ml conical flasks as control for comparison. The flasks sealed with foam stoppers were incubated at 70 jC in a rotary shaker at 150 rpm for 5 days. After that time, the crude oils were sampled and analyzed. The group components (saturated hydrocarbons, aromatic hydrocarbons, resins, and asphaltenes) were extracted by chloroform, fractionated by the solvent and silica-aluminum column, and measured by an electronic balance, and the analytical error is 1%. The components of saturated hydrocarbons and aromatic hydrocarbons were analyzed by a gas chromatograph-mass spectrometer (HP5890II GC/5970B MSD), and the analytical error is 0.1%. The samples were chromatographed with a gradient temperature program. The initial temperature of 110 jC was held for 1 min, and then the temperature was increased to 320 jC at 4 jC/min and held at 320 jC for 22 min (saturated hydrocarbons), and to 335 jC at 3 jC/min and held at 335 jC for 11.68 min (aromatic hydrocarbons). The columns used were DB5-MS (60 m 0.25 mm; 0.25 Am film) for saturated hydrocarbons and BPX5 (30 m 0.22 mm; 0.25 Am film) for aromatic hydrocarbons. The injection temperature was 320 jC, and the transport line was maintained at 320 jC. The helium flow was 25 cm/s, and injection volumes were 2 Al. The contents of carbon, hydrogen, nitrogen and sulfur were analyzed by an Elementar vario EL. The analytical error of carbon and hydrogen is 1% and that of nitrogen and sulfur is 0.1%. All experiments were conducted in duplicate with duplicate heat-inactivated (autoclaved) controls. All data reported are averages for duplicate culture.

3. Results 3.1. Morphology and culture characters Cells of strain TH-2 stained Gram-negative and rods were shaped with dimensions 2 5 0.5 0.6 Am without spore. The strain was incubated in liquid medium EM2 at 70 jC for 24 h, the cells were distributed singly or filamentously. The strain TH-2 was put onto Petri dishes within the EM2 medium containing 15 g/l agar. After 3

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days of incubation at 70 jC, round colonies with diameters of about 1 mm had developed. The colonies were smooth, transparent, colorless or white, and could not produce pigment. 3.2. Growth characteristics Temperature growth range for strain TH-2 was 40 85 jC with the maximal growth rate at 70 jC. The strain was able to grow at pH values comprised between pH6.5 and 9.0, and thrives optimally around pH 7.5 7.8. Strain TH-2 required a low concentration of yeast extract for growth. Good growth was obtained on acetate, D-glucose, fructose, glycerol, maltose, pyruvate, starch, sucrose, and xylose. No growth occurred on citrate, lactose, and mannitol. The strain was grown chemoorganotrophically under aerobic condition with oxygen as the electron acceptor. The oxidase test was positive, while the methyl red and indole tests were negative. Packed cells showed gas production after a 3% (vol/vol) H2O2 solution was added, indicating the presence of catalase activity. 3.3. Metabolic products The strain TH-2 produced from YG medium volatile fatty acids, such as, acetic acid (286.6 ppm) and propionic acid (24.6 ppm). The gas chromatograph-mass spectrometer (GC-MS) analyses of the culture samples showed multiple peaks; we identified some metabolites peaks with matching of mass spectra to the known compounds in the GC-MS library. The main metabolites are as follows: 1,2-benzenedicarboxylic acid-bis ester (C16H22O4), dibutyl phthalate (C16H22O4), and di-n-octyl phthalate (C24H38O4). These metabolic products have various effects on crude oils. The ester is the better organic solvent, and is also low-molecular-weight surfactant with lower surface tension. It is by far the most abundant metabolites, accounting for about 92% of the total products. 3.4. Surface characters The close attention to oil-degrading bacterium is whether it can produce surfactants. It is established that

the strain TH-2 can produce surfactants, and the surface tension of the cell-free extract changed regularly with strain growth (Fig. 1), which was lowest (47.1 mN/m) at the peak of growth. Fig. 1 shows the absorbance (growth) and surface tension (surfactant production) plotted versus time for strain TH-2. Data for EM2 medium indicate a typical growth curve having lag, log, and stationary growth phases. Surfactant production was directly proportional to cell growth; as the cell density increased the surface tension decreased. The greatest decrease in surface tension was obtained during log phase (within 24 h). This curve shows that a 72-h incubation is sufficient for surfactant production in shake flask experiments. Surface tension dropped from 58.2 mN/m to 47.1 mN/m within the first 24 h and then stabilized over the next 48 h to the value between 47.4 and 48.9 mN/m. 3.5. Effect of strain TH-2 on crude oils Bioconversion often brings about some changes in contents of saturated hydrocarbons, aromatic hydrocarbons, resins and asphaltenes, and a reduction in contents of sulfur and nitrogen. Biodegradation is the complete transformation of organic molecules into inorganic substances. The effects of induced bioconversion of various crude oils on four major fractions are shown in Table 1. While compared to the sterile controls (abbr. SC) the relative percentages of saturated hydrocarbon, aromat-

Fig. 1. Surface tension (o) and strain TH-2 cell growth as optical density (E) over time. Culture reactors were maintained at 70 jC.

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Table 1 Distribution of saturated hydrocarbons, aromatic hydrocarbons, resins and asphaltenes in various crude oils before and after biotreatment Crude Saturated oil hydrocarbon (% w/w) SC A B C D 38.44 38.70 38.87 47.12 BT 37.32 37.74 38.09 53.27 Aromatic hydrocarbon (% w/w) SC 25.08 24.86 24.99 20.19 BT 22.44 24.76 22.38 15.63 Resin (% w/w) SC 28.66 27.40 28.08 29.33 BT 24.88 25.72 27.20 25.88 Asphaltene (% w/w) SC 6.19 5.93 6.26 2.88 BT 5.61 5.77 5.42 0.00

SC: sterile control. BT: biotreatment.

ic hydrocarbon, resin and asphaltene vary; there is a decrease in all cases except for the saturated hydrocarbon in oil D. It should be noted that the asphaltene of oil D was completely degraded by the strain (Table 1). The contents of carbon, hydrogen and nitrogen in various oils, compared to the sterile controls, were decreased after biotreatment (Table 2). The sulfur contents of oils A and D were not changed, but those of crude oils B and C were decreased (Table 2). Thermophile TH-2 could convert and/or degrade various components of different crude oils. Consistent with above-mentioned analyses, bioconversion of these oils leads to enrichment in lighter hydrocarbons and an overall redistribution of these hydrocarbons, as shown by the peak clusters as retention times of 20 30, 30 40 and 40 50 min (Figs. 2 5). The analytical results of the gas chromatograph of saturated hydrocarbons in various crude oils state that the strain converted C22 C25 heavy compounds of oil A, the C19 C30 heavy fractions of oil B, and the C22
Fig. 2. Gas chromatograms of the saturated hydrocarbon fraction of oil A, (a) sterile control, (b) treated with strain TH-2.

Table 2 Variations in the concentrations of carbon, hydrogen, nitrogen and sulfur in various crude oils before and after biotreatment Crude Carbon oil (% w/w) SC A B C D 85.7 85.8 86.1 85.0 BT 85.2 85.5 86.0 84.3 Hydrogen (% w/w) SC 13.0 12.8 12.5 13.7 BT 11.2 11.6 11.2 13.3 Nitrogen Sulfur H/C (% w/w) (% w/w) (atom/atom) SC BT SC BT SC 0.6 0.6 0.7 0.5 0.5 0.5 0.5 0.3 0.2 0.2 0.2 0.3 0.2 0.1 0.1 0.3 1.82 1.79 1.74 1.93 BT 1.58 1.63 1.56 1.89

SC: sterile control. BT: biotreatment.

C31 heavy components of oil C; the C9 C16 and C29 C39 fractions of oil D were decreased (Fig. 6). It should be pointed out that the C24 and C25 fractions of oil A, the C25 C30 fractions of oil B, and the C12, C26 C31 compounds of oil C were completely degraded by strain. The physical and chemical properties of crude oils were changed with biotreatment. Data in Table 3 show that the viscosity and paraffin contents of crude oils,

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have direct and indirect effects on the properties of crude oils include (1) change of the physical and chemical features, (2) bioconversion of crudes and (3) biodegradation of oils. These functions are interpreted as consequence of microbial growth, bacterial enzymes, and metabolites. The thermophilic bacterium, strain TH-2, is able to grow at high-temperature environments, and the optimal growth temperature is 70 jC, and can grow

Fig. 3. Gas chromatograms of the saturated hydrocarbon fraction of oil B, (a) sterile control, (b) treated with strain TH-2.

with compared to the sterile controls, were decreased respectively by 10.1 55.4% and 16.2 37.7%.

4. Discussion The interaction between bacteria and crude oils is a complex biochemical process. In general, bacteria can
Fig. 4. Gas chromatograms of the saturated hydrocarbon fraction of oil C, (a) sterile control, (b) treated with strain TH-2.

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Fig. 5. Gas chromatograms of the saturated hydrocarbon fraction of oil D, (a) sterile control, (b) treated with strain TH-2.

up to 85 jC. The nutritional characteristics and the 16S rDNA analyses of strain TH-2 isolated from oil reservoir showed that it was closely related to the genus Thermus. Comparing strain TH-2 with other thermophilic bacteria isolated from oil fields, the strain TH-2 can metabolize a wide variety of lowmolecular-weight organic substrates, and convert and degrade various crude oils. Strain TH-2 could produce many metabolic products, which act as bridging

between the bacteria and crude oils and made them contact each other. Some reports (Boochan et al., 2000; Jain et al., 1992; Kirscner et al., 1980; Rosenberg et al., 1983) indicated that bacterial adherence to hydrocarbons are of benefit to grow and reproduce of microbes in crude oils and biodegradation of petroleum. Thermophilic bacteria isolated from a Venezuelan oil field could produce alcohols, short chain fatty acids and gases (CO2, H2), and ferment carbohydrates (Sanchez et al., 1993). However, effects on crude oils by them have not been reported. The unidentified hyperthermophiles (Stetter et al., 1993) and the thermophilic sulfate reducers (Rueter et al., 1994) have been reported to grow on crude oil and the sulfatereducing bacteria can oxidize hydrocarbons in crude oil. Archaeoglobus spp. and many thermophilic sulfate reducers utilize organic acids in oil reservoirs (Beeder et al., 1994, 1995; Nilsen et al., 1996b; Ress et al., 1995). The hyperthermophiles in the Kubiki oil reservoir produced acetate, lactate and gases (CO2, H2), but did not utilize acetate, lactate, or crude oil as a sole carbon and energy source (Takahata et al., 2000). The oils under discussion are complex mixtures representing different types of oils; however, all falling into definable categories. Chemically and biochemically caused changes in these categories involve multiple reactions within a complex mixture which follow distinct trends that can be followed by chemical markers. Thus, gas chromatographic analyses of four biodegraded crude oils, when treated with the same strain TH-2, show a similar response, but different in detail. Bioconversion of four oils leads to an enrichment in lighter hydrocarbons (shorter retention time). However, the relative distribution of hydrocarbons differs. The strain TH-2 and its metabolites changed the physical and chemical properties of crude oils. The heavy compounds of oil samples were decreased, and the light fractions increased. Also, the contents of aromatic hydrocarbons, resins and asphaltenes in various oils were reduced. As a result, the viscosity and paraffin contents of crude oils were decreased, for example, the viscosities of oils A, B and D were decreased respectively by 17.8%, 10.1% and 55.4%, and the paraffin contents of oils A, B and C were reduced respectively by 16.2%, 37.7% and 30.1%.

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Fig. 6. Distribution of different alkane groups in various crude oils before and after biotreatment (1) C7 C10; (2) C10 C15; (3) C15 C20; (4) C20 C25; (5) C25 C30; (6) C30 C35; (7) C35 C40.

Moreover, the physical properties of oils including the flow ability were improved. The interactions between microorganisms and different crude oils occur through complex biochemical and chemical reactions. These reactions depend on multiple variables within and the interface of a multicomponent system consisting of organic aqueous, and inorganic components. The speed of the biochemical action on bacteria and crude oil was faster within 7 days; it would be gradually lowered after 7 days

(Aldrett et al., 1997). The experiments in this study lasted 5 days. If the nutrients were sufficient, especially nitrogen and phosphorus, the biochemical action could be sustained for 28 days; and the components of crude oils would be changed further; otherwise, it needed to complement nutrients and continuous culture. Some chemical markers can be used to diagnose the biochemical interactions between bacteria and crude oils. They include the distribution of saturated

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Table 3 Changes of viscosity and paraffin content of various crude oils before and after biotreatment Crude Viscosity (Pa s) oil SC BT Content of paraffin (%) Percent SC decreased (%) 2.71 6.16 6.34 17.37 BT Percent decreased (%) 16.24 37.66 30.13

A B C D

6.031 1.283 1.657 0.2335

4.955 17.84 1.153 10.13 ND 0.1041 55.42

2.27 3.84 4.43 31.89

Precision on viscosity is 1%. Precision on paraffin content is 1% (oil A, B, C) and 2% (oil D). SC: sterile control. BT: biotreatment. ND: not determined.

hydrocarbons, aromatic hydrocarbons, resins and asphaltenes, and the contents of sulfur and nitrogen. The use of chemical markers in the monitoring of the interaction between different bacteria and various crude oils allows us to determine the efficiency of the biochemical conversion and/or degradation of the petroleum. In the present study, all the organic compounds of oils A, B, C and D were changed (Table 1). As for the oil D with low viscosity, saturated hydrocarbons were increased, which resulted in the increasing of paraffin contents, although aromatic hydrocarbons, resins and asphaltenes were reduced. From the data in Fig. 6, it can be seen that the bioconversion of heavy hydrocarbons into light compounds. This implies that the hydrogen contents (and H/C ratio) of those oils increased with decreasing carbon number. However, H, and C contents, and H/C ratios of oils, were reduced (Table 2). This fact is due to the partial degradation of organic compounds into inorganic compounds.

The analytical results of GC of saturated hydrocarbons in various crude oils demonstrate that the values of parameters of crude oils were changed with biotreatment (Table 4). Except for crude oil D, the carbon numbers of main peak of GC profiles of saturated hydrocarbons in various crude oils were decreased, and the ratios of Pr/n-C17 of those oils were reduced. However, the ratios of Ph/n-C18 of other oils were increased except for oil B. The decrease of the ratios of Pr/n-C17 and Ph/n-C18 was determined by two reasons, one reason is that strain TH-2 maybe first effect on pristane and phytane than C17 and C18; the other reason is that most heavy fractions were converted to light fractions resulted in increasing of C17 and C18. The lighter fraction contents of saturated hydrocarbons in crude oils A, B and C, were increased, which showed also in Fig. 6. The experimental data of this study and several reports (Fedorak et al., 1983; Forrester et al., 1983; Premuzic et al., 1996; Rabus et al., 1999; Solanoserena et al., 1999) indicated the biotreatments of microorganisms on crude oils have resulted in following changes: (1) emulsification, (2) acidification, (3) qualitative and quantitative transformations in the light and heavy fractions of crudes, (4) decrease in organic sulfur content and chemical changes in the fractions containing sulfur compounds, and (5) reduction in organic nitrogen content with concurrent biochemical conversion of polar nitrogen containing compounds. These studies have also generated information supporting the view that the biochemical interactions between crude oils and microorganism follow distinct trends characterized by a group of chemical markers (Premuzic et al., 1993). The qualitative and changes in hydrocarbon composition depend on the

Table 4 Variation of parameters of different cured oils before and after biotreatment Crude oil A B C D AC21/AC22 SC 2.38 2.23 1.16 0.76 BT 13.42 4.95 6.12 0.74 Pr/n-C17 SC 2.88 3.80 1.41 0.52 BT 2.40 2.28 1.14 0.52 Ph/n-C18 SC 3.14 3.54 1.44 0.75 BT 3.31 2.81 2.09 0.76 Pr/Ph SC 0.69 0.87 0.73 0.56 BT 0.69 0.63 0.57 0.52 Carbon number of main peak SC 19 19 23 22 BT 16 18 16 22

SC: sterile control. BT: biotreatment.

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microbial species and the chemistry of the crude oils.

Acknowledgements This work was supported by China National Petroleum Corporation and the Ministry of Science and Technology of China, National Key Program for Basic Research, Grant No. 2001CCA02400. We thank Shengli Petroleum Administrative Bureau for contribution of samples and information about the oil reservoirs. We are grateful to M. Xu for analysis of metabolites, to Z. Liu for identify of bacteria, and to Y. Pan, X. L. Geng and X. Wang for laboratory assistance. Furthermore, we are indebted to two anonymous reviewers for their constructive comments and suggestions, which led to a major revision of the manuscript.

References
Aldrett, S., Bonner, J.S., Mills, M.A., Autenrieth, R.L., Stephens, F.L., 1997. Microbial degradation of crude oil in marine environments tested in a flask experiment. Water Res. 31, 2840 2848. Beeder, J., Nilsen, R.K., Rosnes, J.T., Torsvik, T., Lien, T., 1994. Archaeoglobus fulgidus isolated from hot North Sea oil field waters. Appl. Environ. Microbiol. 60, 1227 1231. Beeder, J., Torsivik, T., Lien, T., 1995. Thermodesulforhabdus morvegicus gen. nov. and sp. nov., a novel thermophilic sulfatereducing bacterium from oil field water. Arch. Microbiol. 164, 331 336. Boochan, S., Britz, M.L., Stanlty, G.A., 2000. Degradation and mineralization of high-molecular-weight polycyclic aromatic hydrocarbons by defined fungal bacteria cocultures. Appl. Environ. Microbiol. 66, 1007 1019. Brock, T.D., 1978. Thermophilic Microorganisms and Life at High Temperatures. Springer-Verlag, New York. Cayol, J.L., Ollivier, B., Patel, B.K.C., Ravot, G., Magot, M., Ageron, E., Grimont, P.A.D., Garcia, J.L., 1995. Description of Thermoanaerobacter brockii subsp. lactiethylicus subsp. nov., isolated from a deep subsurface French oil well, a proposal to reclassify Thermoanaerobacter finnii as Thermoanaerobacter brockii subsp. finnii comb. nov., and an emended description of Thermoanaerobacter brockii. Int. J. Syst. Bacteriol. 45, 783 789. Cochrane, W.J., Johne, P.S., Sanders, P.F., Holt, D.M., Mosley, M.J., 1988. Studies on the thermophilic sulfate-reducing bacteria from a souring North Sea oil field. Soc. Pet. Eng., Prod. Eng. 18368, 301 316.

Fardeau, M.L., Cayol, J.L., Magot, M., Patel, B.K.C., Ollivier, B., 1996. Effect of thiosulfate as electron acceptor on glucose and xylose oxidation by Thermoanaerobacter finnii and a Thermoanaerobacter sp. isolated from oil field water. Res. Microbiol. 147, 159 165. Fardeau, M.L., Ollivier, B., Patel, B.K.C., Magot, M., Thomas, P., Rimbault, A., Rocchiccioli, F., Garcia, J.L., 1997. Thermotoga hypogea sp. nov., a xylanolytic thermophilic bacterium from an oil-producing well. Int. J. Syst. Bacterial. 47, 1013 1019. Fedorak, P.M., Foght, J.M., Westlake, D.W.S., 1983. Comparative studies on microbial degradation of aromatics and saturates in crude oil. In: Zajic, J.E., Cooper, D.G., Jack, T.R., Kosaric, N. (Eds.), Microbial Enhanced Oil Recovery. Pennwell Publishing, Tulsa, pp. 162 172. Forrester, P.I., Pearson, D., Roth, J., 1983. Microbial degradation of Athabasca bitumen. In: Zajic, J.E., Cooper, D.G., Jack, T.R., Koaric, N. (Eds.), Microbial Enhanced Oil Recovery. PennWell Publishing, Tulsa, pp. 156 161. He, Z., Peng, Q., Chen, J., 2000. Biology of Thermophiles. Scientific Press, Beijing, p. 234. In Chinese. Jain, D.K., Lee, H., Trevors, J.T., 1992. Effect of addition of Pseudomonas aeruginosa UG2 inocula or biosurfacants on biodegradation of selected hydrocarbons in soil. J. Ind. Microbiol. 10, 87 93. Jeanthon, C., Reysenbach, A.L., Haridon, S.L., Gambacorta, A., Pace, N.R., Glenat, P., Prieur, D., 1995. Thermotoga subterranean sp. nov., a new thermophilic bacterium isolated from a continental oil reservoir. Arch. Microbiol. 164, 91 97. Kirscner, Z., Rosenberg, I.Z., Gutnick, D., 1980. Incorporation of 32P and growth of Pseudomonas UP-2 on n-tetracosane. Appl. Environ. Microbiol. 40, 1086 1093. Kristiansson, J.K., Stetter, K.O., 1992. Thermophilic bacteria. In: Kristjansson, J.K. (Ed.), Thermophilic Bacteria. CRC Press, Boca Raton, FL, pp. 1 17. LHaridon, S., Miroshnichenko, M.L., Hippe, H., Fardeau, M.L., Bonch-Osmolovsksya, E.A., Stackebrandt, E., Jeanthon, C., 2001. Thermosipho geolei sp. nov., a thermophilic bacterium isolated from a continental petroleum reservoir in Western Siberia. Int. J. Syst. Evol. Microbiol. 51, 1327 1334. LHaridon, S., Miroshnichenko, M.L., Hippe, H., Fardeau, M.L., Bonch-Osmolovsksya, E.A., Stackebrandt, E., Jeanthon, C., 2002. Petrotoga olearia sp. nov and Petrotoga sibirica sp. nov., two thermophilic bacteria isolated from a continental petroleum reservoir in Western Siberia. Int. J. Syst. Evol. Microbiol. 52, 1715 1722. Miroshnichenko, M.L., Hippe, H., Stackebrandt, E., Kostrikina, N.A., Chernyh, N.A., Jeanthon, C., Nazina, T.N., Belyaev, S.S., Bonch-Osmolovsksya, E.A., 2001. Isolation and characterization of Thermococcus sibiricus sp. nov. from a Western Siberia high-temperature oil reservoir. Extremophiles 5, 85 91. Nilsen, R.K., Beeder, J., Torsvik, T., 1996a. Methanococcus thermolithotrophicus isolated from North Sea oil field reservoir water. Appl. Environ. Microbiol. 62, 728 7731. Nilsen, R.K., Torsvik, T., Lien, T., 1996b. Desulfotomaculum thermocisternum, sp. nov., a sulfate reducer isolated from a hot North Sea oil reservoir. Int. J. Syst. Bacteriol. 46, 397 402. Orphan, V.J., Ttaylor, L., Hafenbradl, D., Delong, E.F., 2000. Cul-

258

R. Hao et al. / Journal of Petroleum Science and Engineering 43 (2004) 247258 herence to hydrocarbons. In: Zajic, J.E., Cooper, D.G., Jack, T.R., Kosaric, N. (Eds.), Microbial Enhanced Oil Recovery. PennWell Publishing, Tulsa, pp. 114 123. Rueter, P., Rabus, R., Wilkes, H., Aeckersberg, F., Rainey, F.A., Jannasch, H.W., Widdel, F., 1994. Anaerobic oxidation of hydrocarbons in crude oil by new types of sulphate-reducing bacteria. Nature 372, 455 458. Sanchez, G., Marin, A., Vierma, U., 1993. Isolation of thermophilic bacteria from a Venezuelan oil field. In: Premuzic, E.T., Woodhead, A. (Eds.), Microbial Enhancement of Oil RecoveryRecent Advances. Elsevier, New York, pp. 383 389. Solano-serena, F., Marchal, R., Ropars, M., Lebeault, J.M., Vandecasteele, J.P., 1999. Biodegradation of gasoline: kinetics, mass balance and fate of individual hydrocarbons. J. Appl. Microbiol. 86, 1008 1016. Stetter, K.O., Huber, R., Blochl, E., Kurr, M., Eden, R.D., Fielder, M., Cash, H., Vance, I., 1993. Hyperthermophilic archaea are thriving in deep North Sea and Alaskan reservoirs. Nature 365, 743 745. Takahata, Y., Nishijima, M., Hoaki, T., Maruyama, T., 2000. Distribution and physiological characteristics of hyperthermophiles in the Kubiki oil reservoir in Niigata, Japan. Appl. Environ. Microbiol. 66, 73 79. Takahata, Y., Nishijima, M., Hoaki, T., Maruyama, T., 2001. Thermotoga petrophila sp. nov. and Thermotoga naphthophila sp. nov., two Hyperthermophilic bacteria from the Kubiki oil reservoir in Niigata, Japan. Int. J. Syst. Evol. Microbiol. 51, 1901 1909. Tissot, B.P., Welte, D.H., 1978. Petroleum Occurrence and Formation. Springer-Verlag, New York. Yen, T.F., 1990. Microbial Enhanced Oil Recovery: Principle and Practice. CRC Press, Boca Raton, FL.

ture-dependent and culture-independent characterization of microbial assemblages associated with high-temperature petroleum reservoirs. Appl. Environ. Microbiol. 66, 700 711. Pooks, L.C., 1997. Handbook of Microbiological Media, second edition. CRC Press, Boca Raton, FL, p. 1371. Premuzic, E.T., Lin, M.S., 1999. Induced biochemical conversion of heavy crude oils. J. Pet. Sci. Eng. 22, 171 180. Premuzic, E.T., Woodhead, A., 1993. Microbial Enhancement of Oil RecoveryRecent Advances. Elsevier, New York. Premuzic, E.T., Lin, M.S., Racaniello, L., 1993. Chemical markers of induced microbial transformations in crude oils. In: Premuzic, E.T., Woodhead, A. (Eds.), Microbial Enhancement of Oil RecoveryRecent Advances. Elsevier, New York, pp. 37 54. Premuzic, E.T., Lin, M.S., Lian, H., Zhou, W.M., Yablon, J., 1996. Microbial interactions in crude oils: possible impact on biochemical versatility on the choice of microbial candidates. Paper presented at the 1996 International Conference on Microbial Enhanced Oil Recovery, pp. 551 569. Rabus, R., Wilkes, H., Schramm, A., Harms, G., Behrends, A., Amann, R., Widdel, F., 1999. Anaerobic utilization of alkylbenzenes and n-alkanes from crude oil in an enrichment culture of denitrifying bacteria affiliating with the h-subclass of Proteobacteria. Environ. Microbiol. 2, 145 157. Ravot, G., Magot, M., Fardeau, M.L., Patel, B.K.C., Prensier, G., Egan, A., Garcia, J.L., Llivier, B., 1995. Thermotoga elfii sp. nov., a novel thermophilic bacterium from an African oil-producing well. Int. J. Syst. Bacterial. 45, 308 314. Ress, G.N., Grassia, G.S., Sheeny, A.J., Dwivedi, P.P., Patel, B.K.C., 1995. Desulfacinum infernum gen. nov., sp. nov., a thermophilic sulfate-reducing bacterium from a petroleum reservoir. Int. J. Syst. Bacterial. 45, 85 89. Rosenberg, M., Gutnick, D.L., Rosenberg, E., 1983. Bacterial ad-