Isolation and Characterization of a Thermophilic Oil-Degrading Bacterial Consortium

Gu Guizhou; Li Zheng; Zhao Dongfeng; Zhao Chaocheng

(1. College of Chemical Engineering, China University of Petroleum, Qingdao 266555; 2. College of Environmental Science and Engineering, Liaoning Shihua University, Fushun 113001)

Isolation and Characterization of a Thermophilic Oil-Degrading Bacterial Consortium

Gu Guizhou1,2; Li Zheng1,2; Zhao Dongfeng1; Zhao Chaocheng1

(1. College of Chemical Engineering, China University of Petroleum, Qingdao 266555; 2. College of Environmental Science and Engineering, Liaoning Shihua University, Fushun 113001)

In this study, a thermophilic oil-degrading bacterial consortium KO8-2 growing within the temperature range of 45—65 ℃ (with 55 ℃ being the optimum temperature) was isolated from oil-contaminated soil of Karamay in Xinjiang, China. Denaturing gradient gel electrophoresis (DGGE) showed that there were nine strains included in KO8-2, which originated from the genera ofBacillus,GeobacillusandClostridium. They all belonged to thermophilic bacteria, and had been previously proved as degraders of at least one petroleum fraction. The crude oil degraded by KO8-2 was analyzed by infrared spectrophotometry, hydrocarbon group type analysis and gas chromatography. The results indicated that the bacterial consortium KO8-2 was able to utilize 64.33% of saturates, 27.06% of aromatics, 13.24% of resins and the oil removal efficiency reached up to 58.73% at 55 ℃ when the oil concentration was 10 g/L. Detailed analysis showed that KO8-2 was able to utilize the hydrocarbon components before C19, and the n-alkanes ranging from C20—C33were signif icantly degraded. The ratios ofnC17/Pr andnC18/Ph were 3.12 and 3.87, respectively, before degradation, whereas after degradation the ratios reduced to 0.21 and 0.38, respectively. Compared with the control sample, the oil removal efficiency in KO8-2 composting reactor reached 50.12% after a degradation duration of 60 days.

thermophilic bacteria; bacterial consortium; oil-contaminated soil; biodegradation; DGGE

1 Introduction

Petroleum is used as the principle source of energy. With the increase of oil production, serious soil pollution caused by mismanagement or accidents in the process of oil exploitation, refining, transport, utilization and disposal has become a noticeable social and environmental issue[1-2]. Bioremediation technology is considered to be the most promising and environment-friendly method for treating contaminated soil sites because of its low cost, insignificant environmental impact, and absence of secondary pollution[3-4]. Up to now, many mesophilic genera have been found to be capable of utilizing crude oil or some components of oil as the sole source of carbon, such asRhodococcussp.[5],Pseudomonasp.[6],Acinetobactrsp., etc.[7]However, in the oil-contaminated torrid regions during summer time, the air temperature frequently exceeds 40 ℃ and the surface soil temperature becomes even higher, so only thermophilic oil-degrading microorganisms can propagate. Since then, five thermophilic genera have been found, which areThermoleophilumsp.[8],Ther-momicrobiumsp.[9],Bacillussp.[10],Geobacillussp.[10-11]; andThermussp.[12]. Most studies have focused on using these thermophilic bacteria to degrade one petroleum fraction or light crude oil[13-14]. However, isolating crudeoil-degrading bacteria in order to degrade high-concentration crude oil and bioremediation of oil-polluted soil have been rarely reported up to now.

Petroleum is a kind of complicated hydrocarbons mixture and is generally classified into four groups: saturates, aromatics, polars or resins, and asphaltenes[15]. Therefore, biodegradation of crude oil must be completed by different microbes with special functions[16]. Most related studies used defined mixed culture composed of purified single oil-degrading bacterial strains as the research object[17-19]. However, small change in dosage of each bacterial strain and inhibition that may exist between the strains would have negative effects[19]. There are a seriesof microbial communities optimized through natural selection in petroleum-contaminated soil[20-22], and the functions of different strains have stabilized after longterm evolution. Some strains cannot degrade the target pollutants directly; however, they can grow and propagate in intermediate metabolites of other strains serving as the carbon source. Some strains have a competitive relationship with other strains, but the presence of another strain can offset the negative effect[23]. And from a commercial perspective, mixed bacterial group does not need the use of sterilized fermenter and culture media, which would require high capital investment and operating cost[24]. Therefore, such natural microbial communities having inter-species cooperativity are more efficient microorganism for degrading oil.

The Karamay oilfield is located on the edge of desert in western China, and most areas are covered with the Gobi desert. The surface-soil temperature is as high as 60 ℃ in summer. Temperature plays an important role in controlling the nature and efficiency of microbial hydrocarbon degradation[25], which is a major limiting factor for insitu bioremediation. This study was conducted to isolate a high-efficiency thermophilic bacterial consortium that was capable of utilizing crude oil as the sole sources of carbon and energy to study the degrading characteristics and use DGGE to evaluate the predominance of microbial population, which will provide important reference for insitu bioremediation of oil-polluted extreme environment.

2 Materials and Analytical Methods

2.1 Collection of samples

Oil contaminated soil and crude oil used in the study were sampled from the Karamay oilfield in Xinjiang, China. 64 soil samples were taken from the surface layer (0—10 cm deep) at ten different locations of Karamay oilfield, stored in pre-sterilised glass bottles and transported to the laboratory for analyses. All the soil samples were collected in triplicate. Physicochemical properties of soil samples are shown in Table 1.

The oil sample was obtained from Baikouquan oil well of Karamay oilfield and stored in pre-sterilized glass bottles. Physicochemical properties of soil samples are shown in Table 2.

Table 1 Physicochemical properties of soil samples

Table 2 Physicochemical properties of oil sample

2.2 Enrichment culture

5 g of soil sample and 1.0 g of oil sample were added into 100 mL of mineral salts medium (MSM which was composed of 0.2 g of KH2PO4, 0.6 g of Na2HPO4, 4.0 g of NaNO3, 0.3 g of MgSO4, 10 mL of trace metals solution, and 1 000 mL of distilled water to form a pH value of 7.2—7.5. The trace metals solution contained 200 mg of CaSO4, 200 mg of FeSO4·7H2O, 20 mg of MnSO4·H2O, 10 mg of NaMo·2H2O, 20 mg of CuSO4, 10 mg of CoSO4·7H2O, 5 mg of H3BO3, and 1 000 mL of distilled water, with the solution being shaken on a rotary shaker at a rotary speed of 150 rpm and a temperature of 55 ℃. After 7 days of cultivation, 5 mL of culture solution and 1 g of crude oil were inoculated to 100 mL of fresh MSM prior to further cultivation under the same condition. These steps were repeated for five times, and then the crude oil-degrading bacterial consortium was obtained, which was able to utilize crude oil as the sole sources of carbon and energy. The final enrichment suspension contained approximately 1×1010cells/mL, measured by the Petroff-Hausser counting chamber (Hausser Scientific, Horsham Pa. USA).

2.3 DNA extraction and PCR

The DNA of the oil-degrading bacterial consortium was extracted, using a Bacteria DNA Mini Kit (Tiangen Biotech (Beijing) Co., Ltd., China) following the manufacturer’s instructions.

The extracted DNA was amplified by PCR. For the amplification of 16S rDNA genes, the universal bacterial primers 341F (5'-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3') and 534R (5'-ATTACCGCGGCTGCTGG-3') were used. The amplification was conducted in the 9700PCR meter (Bio-Rad Laboratories, Hercules, USA), based on the procedure described by Ke (2008)[26].

2.4 DGGE and sequence analysis

16S rDNA-DGGE was performed using the DCode System (Universal Mutation Detection System, BIO-RAD) with a denaturing gradient ranging from 30% to 60%. Denaturation of 100% corresponds to 7 M urea and 40% (v/v) deionized formamide. Electrophoresis was run for 30 min at 20 V and for 8 h at 150 V and 60 ℃. The obtained gels were silver-stained and preserved according to literature description[27].

Prominent bands were excised from the gels, resuspended in Milli-Q water overnight, reamplified, and purified for sequencing using a High Pure PCR Product Purification Kit (Tiangen Biotech (Beijing) Co., Ltd., China). Strains identification was carried out by the Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China).

2.5 Evaluation of oil-degrading potential in crude oil MSM

2.5.1 Infrared spectrophotometric analysis

Total crude oil concentration was measured by the infrared spectrophotometry (IR) method (HJ 637-2012, China) using an infrared photometer oil content analyzer (Model 510, Being ChinaInvent Instruments Co., Ltd.).

2.5.2 SARA analysis

Crude oil contains a complicated mixture of compounds which can be simply categorized into four components: saturates, aromatics, resins and asphaltenes[28]. In order to analyze the changes in oil fractions before and after degradation, SARA analysis was carried out (according to SY/T 5119—2008, China). Volatile oil fractions were eliminated by heating (for 48 h at 100 ℃) for experimental precision and asphaltenes were extracted byn-pentane precipitation (according to ASTM D2007—1991). The remaining oil was total hydrocarbons (TH). Total hydrocarbons were further fractionated by column chromatography to determine the saturates, aromatics and resins[29]. Extracted fractions were evaporated to constant weight for gravimetric quantification.

2.5.3 Gas chromatographic analysis

Detailed analysis of chemical composition of residual crude oil was made by gas chromatography (GC) (according to SY/T 5779—1995). The analysis was carried out using a GC-6890 (+) gas chromatograph (Agilent, USA) equipped with a flame ionization detector (GCFID). A fused-silica capillary column (35 m×0.22 μm) was used with high purity N2(with purity exceeding 99.99%) serving as the carrier gas. The oven temperature was programmed from 40 ℃to 320 ℃ at a heating rate of 5 ℃/min. The combustion gas was H2fed at a rate of 30 mL/min, and the assistant combustion gas was air introduced at a rate of 300 mL/min. The injection volume was 1 μL and the split ratio was 1:50. Data acquisition and handling was computer assisted.

2.6 Evaluation of oil-degrading ability in contaminated soil

In order to test the oil-degrading ability of the selected best-performing microbial group, composting was performed in the contaminated soil of Karamay oilfield. The composting reactor was a round pot without lid which contained 2 kg of air-dried contaminated soil (over 2 mm sieve). Besides, 5 m% of dry straws (5—10 mm in length) and 5% (v/w) of bacterial inocula were added and mixed. The composting reactor was placed in a biochemical incubator thermostat maintained at a constant temperature of 55 ℃. An uninoculated composting reactor was used to account for non-biologically mediated losses tested under the same conditions. The composting reactor was weighted every day and the lost water was replenished to maintain moisture content of 15%—20%. To keep aerobic conditions, the soil was turned over every two days. Soil was sampled at the 0, 10th, 20th, 30th,40th, 50th, and 60th day, respectively, from the composting reactor to measure residual oil content (according to the US EPA8440 method). Experimental sets and controls were run in triplicates.

3 Results and Discussion

3.1 Isolation and cultivation of bacterial consortia

Six bacterial consortia were isolated from 64 soil samples, which had the activities to emulsify the crude oil at 55 ℃ in MSM medium and were named after Karamy Oilfield (KO) on the serial number of soil samples. The oil degradation phenomena and oil removal efficiency are shown in Table 3. Selection of bacterial consortia was based on their crude oil biodegrading rates and cell density. In this study, KO8-2 with a highest oil removal efficiency and cell density was selected for further research.

Table 3 The phenomena and crude oil removal efficiency via degradation by thermophilic bacterial consortia

3.2 The dominant isolates of KO8-2

DGGE was used in evaluating the predominance of microbial population. Figure 1 shows the DGGE measured microbial community profile of KO8-2. Nine dominant bands were cut out for DNA sequencing. These sequences were compared with the GenBank databases, where high similarity (＞97%) was verified for some bacterial isolates (Table 4).

Figure 1 DGGE microbial community profile of KO8-2

Six bacteria were grouped in the genus ofBacillus, and two strains were related to the genus ofGeobacillus; one strain was related to the genus ofClostridium. These genera have the ability to form endospores, which allow the bacteria to survive in semiarid torrid climates characteristic of high temperature and non-stable or poor nutrient supply. Additionally, some bacteria in these genera have been reported to exist in oil polluted soil and have the ability to degrade petroleum components[30-32].

3.3 Temperature tolerance experiments

Temperature tolerance was tested through incubation of 5v% of KO8-2 (1.0×1010cells/mL) in 100 mL of MSM supplemented with 1 % (w/v) of crude oil in the 250-mL triangular flasks for 7 days on a rotary shaker (under a rotary speed of 150 r/min) at a specified temperature (Figure 2). An additional set of uninoculated bottles serving as controls was checked under similar incubation conditions in triplicates.

Table 4 Sequence analysis of partial bacterial 16S rDNA genes from dominant DGGE bands

The growth of oil-degrading bacteria was significantly affected by temperature. According to literature[33], below a certain temperature level the rate of metabolism of hydrocarbons increases with a rising temperature. With temperature continuing to rise, the toxicity of hydrocarbon film increases, leading to a decreasing microbial activity. However, at low temperature, oil viscosity increased, volatility of toxic short-chain alkanes decreased, and microbial proliferation postponed. Furthermore, microbial enzymatic activity could play a role within a certain temperature range. Therefore different microorganisms require a specific temperature range to realize their optimum growth.

Figure 2 indicates that the oil-degrading bacterial consortium KO8-2 had a relatively noticeable oil-degrading ability in the temperature range between 45 ℃ and 65 ℃(with oil removal efficiency exceeding 45%), and the optimum degradation rate was obtained at 55 ℃ (with an oil removal efficiency of 58.73%). When the temperature reached 70 ℃, the oil removal efficiency decreased significantly. The results showed that KO8-2 could not tolerate the high temperature of 70 ℃ and the optimum growth temperature was in the range between 45—65 ℃.

Figure 2 Crude oil removal efficiency of KO8-2 at different temperatures

3.4 Evaluation of oil-degrading ability of MSM

The SARA analysis of crude oil was used to examine the changes of oil fractions before and after degradation. Saturates and aromatics are composed of carbon and hydrogen atoms. However, besides carbon and hydrogen atoms, resins and asphaltenes also have other compounds that also contain sulfur, oxygen, nitrogen, and metal atoms. This method can determine the relative content of each fraction in the oil sample, and then the degradation rates shown in Table 5 can be calculated. It can be seen from Table 5 that 64.33% of saturates and 27.06% of aromatics were biodegraded by KO8-2, while the degradation rate of total petroleum hydrocarbons (TPH) was 51.66%. Saturates and aromatics were degraded quite efficiently, because the two fractions contained relatively low molecular weight hydrocarbons which tended to be most readily degradable. Although little information was reported about the bacteria that can degrade resins[16], the bacterial group KO8-2 was able to utilize 13.24% of resins. The degradation of asphaltenes was only 1.86%, which indicated that KO8-2 could not use this fraction. The resins and asphaltenes consisted of polar molecules[29], which could hardly be degraded by microbes. Especially, asphaltenes are large molecules that are colloidally dispersed in oil[28], tending to be more resistant to biodegradation.

Table 5 Results of SARA analysis

The component of saturated hydrocarbons before and after degradation by KO8-2 was examined by GC-FID (Figure 3). Isoprene compounds of pristane (Pr) and phytane (Ph) in crude oil were relatively stable[34], and microbial degradation could not affect these compounds. Therefore, they were biomarker compounds in crude oil. The ratios ofnC17/Pr andnC18/Ph were important indicators for evaluating the hydrocarbon degradation[35]. They were used to determine the degradation degree of crude oil. For this purpose, Pr and Ph were used as internal standards, and the decrease ofnC17/Pr ratio andnC18/Ph ratio could indicate the degradation degree of crude oil by KO8-2. According to Figure 3, after degradation, the components before C19almost disappeared, while then-alkanes ranging from C20—C33were significantly degraded. Unresolved complex mixture (UCM) increased after degradation which indicated that abundant amount of insoluble substances was formed during hydrocarbons degradation[36]. Before degradation, the ratios ofnC17/Pr ratio andnC18/Ph were 3.12 and 3.87, respectively, whereas their ratios reduced to 0.21 and 0.38, respectively, after degradation. It can be identified that saturated hydrocarbons had been effectively degraded by KO8-2.

3.5 Evaluation of oil-degrading potential in the contaminated soil

The performance of bioaugmentation for composting of oil-contaminated soil using the bacterial consortium KO8-2 was tested. Oil removal ef ficiency between KO8-2 composting reactor and the control composting reactor was compared in Figure 4. It can be seen from the curve of oil removal efficiency in KO8-2 composting reactor that there was no significant degradation at the initial period (within 0—20 days), because the consortium was experiencing an adaptation phase and could not affect the oil degradation. When the experimental time increased further (from 20 days to 30 days), KO8-2 had adapted to the contaminated soil and the growth of bacteria was in a logarithmic phase, so the oil removal efficiency increased fast. At the end of this stage, the cells of the bacteria were activated, and their metabolizable energy was strongest with the bacteria propagating well. Furthermore, most alkanes which accounted for a large percentage of the oil were easily degradable, so the oil removal efficiency increased quickly. After 30 days, the oil removal efficiency continued to increase but the degradation rate began to level off. The reason was that the bacteria reached the end of the stationary phase and their metabolic rate decreased. On the other hand, the rest of the oil was more difficult to degrade. After a degradation duration of 60 days, the oil removal efficiency in KO8-2 composting reactor reached 50.12%, while the oil removal efficiency of the control sample without addition of bacterial con-sortium was merely 7.13%. Therefore, adding highefficiency oil bacterial consortium could improve the speed and effectiveness of bioremediation.

Figure 3 Degradation of saturated hydrocarbons in crude oil

Figure 4 Comparison of oil removal efficiency between KO8-2 and control composting reactors

4 Conclusions

(1) An oil bacterial consortium KO8-2 which could degrade the Karamay crude oil efficiently at a relatively high initial concentration (10 g/L) was found. The bacterial consortium was able to grow in the temperature range of 45—65 ℃, and the optimum temperature was 55 ℃with the oil degradation rate reaching 58.73%.

(2) Nine dominant strains were identified in KO8-2 and six of them were grouped in the genus ofBacillus, two strains were related to the genus ofGeobacillus,and one strain was related to the genus ofClostridium. They all belonged to thermophilic bacteria, and had been previously proved as degraders of at least one petroleum fraction. (3) KO8-2 was able to degrade 64.33% of saturates, 27.06% of aromatics and even 13.24% of resins. It could utilize the hydrocarbon components before C19, and the n-alkanes ranging from C20—C33were significantly degraded. The ratios ofnC17/Pr andnC18/Ph were 3.12 and 3.87, respectively, before degradation, whereas after degradation the ratios reduced to 0.21 and 0.38, respectively. The oil-degrading ability of KO8-2 in the oil-contaminated soil was tested by composting. Compared with the control sample, the oil removal efficiency in KO8-2 composting reactor reached 50.12% after a degradation duration of 60 days.

Acknowledgments:The authors gratefully acknowledge the support provided by the Research & Technology Development Project of China National Petroleum Corporation (No. 2008D-4704-2).

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Recieved date: 2013-01-21; Accepted date: 2013-03-28.

Professor Zhao Chaocheng, E-mail: zhao8021@sina.com; Telephone: +86-532-86981719.