Degradation Characteristics and Community Structure of a Hydrocarbon Degrading Bacterial Consortium

Li Zheng; Gu Guizhou; Zhao Chaocheng; Zhao Dongfeng

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

Degradation Characteristics and Community Structure of a Hydrocarbon Degrading Bacterial Consortium

Li Zheng1,2; Gu Guizhou1,2; Zhao Chaocheng2; Zhao Dongfeng2

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

A hydrocarbon degrading bacterial consortium KO5-2 was isolated from oil-contaminated soil of Karamay in Xinjiang, China, which could remove 56.9% of 10 g/L total petroleum hydrocarbons (TPH) at 30 ℃ after 7 days of incubation, and could also remove 100% of fluorene, 98.93% of phenanthrene and 65.73% of pyrene within 3, 7 and 9 days, respectively. Twelve strains from six different genera were isolated from KO5-2 and only eight ones were able to utilize the TPH. The denaturing gradient gel electrophoresis (DGGE) was used to investigate the microbial community shifts in five different carbon sources (including TPH, saturated hydrocarbons, fluorene, phenanthrene and pyrene). The test results indicated that the community compositions of KO5-2 in carbon sources of TPH and saturated hydrocarbons, respectively, were roughly the same, while they were distinctive in the three different carbon sources of PAHs. Rhodococcus sp. and Pseudomonas sp. could survive in the five kinds of carbon sources. Bacillus sp., Sphingomonas sp. and Ochrobactrum sp. likely played key roles in the degradation of saturated hydrocarbons, PAHs and phenanthrene, respectively. This study showed that specific bacterial phylotypes were associated with different contaminants and complex interactions between bacterial species, and the medium conditions influenced the biodegradation capacity of the microbial communities involved in bioremediation processes.

bacterial consortium; community structure; degradation characteristics; total petroleum hydrocarbons (TPH); denaturing gradient gel electrophoresis (DGGE)

1 Introduction

The discharge of environmentally harmful total petroleum hydrocarbons (TPH) during exploitation, production, refining, transportation and storage of crude oil and its products is causing the contamination of lands and waters around the world[1-2]. Contamination by TPH poses risks to plant, animal, and human health. In China, petroleum enterprises produce about 7000000 tons of seeping crude oil annually and the area of petroleum contaminated land reaches about 5000000 hectares[3]. Among a variety of remediation methods, bioremediation technology is identified as the most promising and environmentally friendly method for treating contaminated soil sites because of its low cost, insignificant environmental impact, and absence of secondary pollution[4-6]. Although the ability to monitor diversity structuring, stability, and long-term resilience during process management is the key requirements for monitoring and predicting bioremediation efficiency, the lack of accurate description of microbial diversity and functionality tends to limit the efficient bioremediation[7]. These deficiencies can affect the understanding of microbial community dynamics in remediation systems[7]. Fortunately, molecular technologies including cloning, denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), and internal transcribed spacer (ITS) oligonucleotide array[8-9]now can successfully provide profiles of bacterial community dynamics, which have been applied to investigate the microbial community shifts[10-11]. DGGE technology has many advantages in the study of community dynamics and diversity, but it also has some limitations. For example, DGGE could only analyze the primary populations greater than 1% in microbial community[12], while the size of detected DNA fragment is limited in 100—500 bpand cannot provide the information on microbial activity. Therefore, we combine the conventional spread plate method with molecular technology to further explore the microbial community structure and community dynamics. The objective of this paper was to investigate the effects of bacterial community on TPH and the efficiency for degradation of different polycyclic aromatic hydrocarbons (PAHs). Conventional spread plate method and molecular biotechnologies of the denaturing gradient gel electrophoresis (DGGE) were applied to profile the bacterial community in five different carbon sources, explore the functional microorganisms of the bacterial consortium, and analyze the collaborative symbiotic relationship in the microbial community.

2 Materials and Methods

2.1 Collection of samples

Oil-contaminated soil and crude oil used in the study were collected from the Karamay oilfield. The soil sample was taken from the surface layer (0—10 cm deep) of Karamay oilfield in pre-sterilized glass bottle and transported to the laboratory for analysis. Physicochemical properties of the soil sample are shown in Table 1.

Table 1 Physicochemical properties of the soil sample

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

2.2 Enrichment culture

5.0 g of soil sample and 1.0 g of oil sample were added in a 250 mL glass flask containing 100 mL of mineral salts medium (MSM) having the following composition (g/L): Na2HPO4—0.6, KH2PO4—0.2, NaNO3—4.0, MgSO4—0.3,CaCl2—0.01, FeSO4—0.01, and yeast extract—0.5. The mixture with a pH value of 7.2 was subjected to shaking on a rotary shaker at a rate of 160 r/min and 30 ℃. After 7 days of cultivation, 5 mL of culture solution and one g of crude oil was inoculated to 100 mL of fresh MSM for further cultivation under the same condition. The steps were repeated for five times, and then the crude oildegrading bacterial consortium was obtained, which was able to utilize crude oil as the sole source of carbon and energy. The final enrichment suspension contained approximately 1×1010cells/mL, as measured by the Petroff-Hausser counting chamber (Hausser Scientific, Horsham Pa. USA).

Table 2 Physicochemical properties of oil sample

2.3 Isolation and identification of single strains from the bacterial consortium

Isolation and purification procedures were carried out on LB (viz., the Luria-Bertani medium, containing 10 g of NaCl, 10 g of peptone, and 5 g of yeast extract, which were mixed with 20 g of agar in 1 000 mL of distilled water, with the solution pH value being adjusted to 7.0.) agar plates by conventional spread plate techniques. The size and color of the isolated colonies were recorded every day. Pure bacterial strains obtained thereby were kept on LB slant culture at 4 ℃.

Genomic DNA was extracted by means of a bacteria DNA mini kit (Tiangen Biotech (Beijing) Co., Ltd., China), and 16S rDNA was amplified in PCR using the genomic DNA as template, and the bacterial universal primers were as follows: F27(5’-CAGCGGTACCAGAGTTTGATCCTGGCTCAG-3’); R1492(5’-CTCTCTGCAGTACGGCTACCTTGTTACGACTT-3’). Amplification was conducted as follows: The initial denaturation was carried out at 94 ℃ for 5 min followed by 30 cycles of denaturationat 94 ℃ for 30 s, and annealing at 55 ℃ for 1 min with a gradient of ± 5 ℃, and then elongation at 72 ℃ for 1 min, and final elongation at 72 ℃ for 10 min. The PCR products were then purified with a QIAquick PCR purification kit (Qiagen Ltd., Maryland, USA).

Strains identification was carried out by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China.) Database search with the determined 16SrDNA sequence was conducted by using the BLASTN program provided by the National Center of Biotechnology Information (NCBI).

2.4 Analysis of the microbial community structure by DGGE

The DNA of the oil-degrading bacterial consortium in MSM 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 F341(5’-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3’) and R534(5’-ATTACCGCGGCTGCTGG-3’) were used. The amplification was conducted in a 9700PCR meter (Bio-Rad Laboratories, Hercules, USA), based on the procedure described by Ke, et al[13].

16S rDNA-DGGE was performed using the DCode system (Universal Mutation Detection System, BIO-RAD) with a denaturing gradient ranging from 30% to 60%. A denaturation of 100% corresponded to 7 M urea and 40% (v/v) deionized formamide. Electrophoresis was run for 30 min at 20 V and then 8 h at 150 V and 60 ℃. The obtained gels were silver-stained and preserved according to Watanabe, et al.[14]Prominent bands were excised from the gels, resuspended in Milli-q water overnight, reamplified, and purified for sequencing using a Highly 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 Degradation of TPH

Detailed analysis of chemical composition of residual TPH was performed by gas chromatography (GC) (SY/T 5779—1995). The analysis was carried out using a GC-6890(+) gas chromatograph (Agilent, USA) equipped with a flame ionization detector (GC-FID). A fused-silica capillary column (35 m×0.22 μm) was used with high purity N2(with a purity of over 99.99%) serving as the carrier gas. The oven temperature was programmed from 40 ℃to 320 ℃ at a temperature increase rate of 5 ℃/min. The combustion gas was H2introduced at a rate of 30 mL/min, and the combustion supporting gas was air fed at a rate of 300 mL/min. The sample injection volume was 1 μL and the split ratio was 1:50. Data acquisition and handling were computer assisted.

2.6 Degradation of PAHs

PAHs degradation in liquid culture was performed by using washed cell suspensions pre-grown with corresponding PAH. The liquid culture (100 mL of MSM containing 10 mg of fluorene, phenanthrene, and pyrene, respectively) was inoculated by transferring 3 mL of pre-culture (MSM medium, OD600nm= 0.25) of the bacterial consortium (5%, v/v) and the initial pH value was adjusted to 7.0. The flasks were incubated in darkness at a rotation speed of 160 r/min and 30 ℃. The PAHs degradation efficiencies were detected by gas chromatography at 3 d, 5 d, 7 d, and 9 d, respectively.

The rest PAHs were extracted from the liquid culture with hexane three times. Then the organic phase was dehydrated with anhydrous sodium sulfate and then was concentrated through vaporization in a rotary evaporator to 1—2 mL. One μL of the organic phase was analyzed by the GC-6890(+) gas chromatograph (Agilent, USA) equipped with a flame ionization detector (GC-FID). A fused silica capillary column (35 m×0.22 μm) was used. The oven temperature was initially set at 80 ℃ for 1 min, and then was programmed at a temperature increase rate of 15 ℃/min to 255 ℃, at which the reaction was maintained for 1 min; and then was programmed further to 265 ℃ at a temperature increase rate of 1 ℃/min. The injector and detector temperature was 250 ℃ and 300 ℃, respectively. The carrier gas, helium, was adjusted to a linear velocity of 1 mL/min. The PAHs were identified by comparison of the retention times with those of PAH standards. All biodegradation experiments were performed intriplicates and the sterilized culture without inoculation was used as the negative control.

3 Results and Discussion

3.1 Degradation characteristics of the bacterial consortium

3.1.1 Degradation of saturated hydrocarbons by KO5-2

Figure 1 The GC-FID diagram of degradation of saturated hydrocarbons in crude oil

A bacterial consortium (named KO5-2) capable of degrading TPH was isolated from the soil sample, which had the activity to emulsify TPH at an initial concentration of 10 g/L at 30 ℃ in MSM after three weeks of selective enrichment by repeated subcultures. The components of saturated hydrocarbons before and after degradation by KO5-2 were examined by GC-FID (Figure 1). Isoprene compounds of pristane (Pr) and phytane (Ph) in crude oil were relatively stable[15], and microbial degradation could not affect their contents. Therefore, they served as biomarker compounds in crude oil. The ratios of nC17/Pr and nC18/Ph were important indicators to evaluate the hydrocarbon degradation[16]which were used to determine the degradation degree of crude oil. For this purpose, Pr and Ph were used as internal standards, and the decrease of nC17/Pr and nC18/Ph indicated the degradation degree of saturated hydrocarbons by KO5-2. According to Figure 1, after 7 days of degradation, the components boiling before C19almost disappeared, while the n-alkanes ranging from C20—C33were significantly degraded. Unresolved complex mixture (UCM) increased after degradation that indicated to the abundant insoluble substances produced during hydrocarbons degradation[17]. Before degradation, the ratios of nC17/Pr and nC18/Ph were 3.07 and 4.16, respectively, whereas the ratios reduced to 0.25 and 0.43 after degradation. Therefore, the TPH removal efficiency reached 56.9% on the seventh day (Table 4).

3.1.2 Degradation of PAHs by KO5-2

The degradation test of fluorene, phenanthrene and pyrene was carried out at a temperature of 30 ℃, a pH value of 7.0 and an initial sample concentration of 100 ppm, respectively. During the experiments, no other carbon sources or surfactants were added to MSM. Figure 2 shows the degradation results. The bacterial consortium KO5-2 could remove 100% of fluorene in three days of incubation and utilize 98.93% of three-ring phenanthrene in seventh days. Four-ring pyrene was the most difficult to degrade among the three samples, and attained a degradation efficiency of 65.73% after nine days. Therefore, with an increase in the number of benzene rings, the degradation efficiency of PAHs by KO5-2 decreased gradually. This outcome wasmainly attributed to the high molecular weight of PAHs with the following specific features: complex molecular structure, high stability, strong hydrophobicity, low water solubility, low bioavailability, and high toxicity. So, PAHs with high molecular weight could hardly be biodegraded completely.

Figure 2 Degradation of fluorene, phenanthrene and pyrene by KO5-2 at different incubation time

3.2 Community structure of the bacterial consortium 3.2.1 Isolation and identification of single strains from KO5-2

As shown in Figure 3, there were many rod-shaped bacteria in the enrichment culture of KO5-2 after 7 days of incubation, and OD600nmof the enrichment culture was 0.28. The black residue in micrograph was the un-degraded petroleum hydrocarbons.

By means of the conventional spread plate method, twelve strains with different colony morphology were isolated from KO5-2. Database search with the determined 16S rDNA sequence was conducted by using the BLASTN program emanated from the National Center of Biotechnology Information (NCBI). The results are shown in Table 3.

Figure 3 Micrograph of KO5-2 in oil MSM (magnification by 10×100)

Twelve strains from six different genera were isolated from KO5-2, among which, Pseudomonas sp.[18], Acinetobacter sp.[19], rthrobacter sp.[20]and Bacillus sp.[21]were oil-degrading bacteria reported more frequently. Pseudomonas aeruginosa belonging to Pseudomonas sp. was reported to produce surfactant[22]. Certain strains related to the genus of Bacillus were proved to be thermophilic oil-degrading bacteria[23]. Ochrobactrum sp. was able to degrade benzene series[24], which suggested that this species had the ability to cleave the aromatic ring.

3.2.2 TPH degradation efficiency of the twelve isolates

The TPH degradation efficiency of KO5-2 and the twelve isolates were investigated. Whether the TPH could be utilized or not by the isolates was studied through the observations indicating if the medium was turbid or if total petroleum hydrocarbons (TPH) were emulsified. Then the degradation ones were chosen to be sampled and measured and the results are shown in Table 4.

Eight isolates among twelve ones were able to utilize theTPH. Additionally, the ability to degrade TPH displayed by the eight isolates showed great difference. Among them, KO5-2-5 and KO5-2-11 which were grouped in the genus of Rhodococcus were able to degrade about 40% of the TPH in the first three days, and besides, the enrichment culture was a light-brown solution without oil slick, and had much of tiny flocculates at this time. The TPH degradation efficiency exceeded 50% on the seventh days. Among all the genera, the degradation rate of Rhodococcus sp. was the fastest, and its degradation efficiency was the best. The degradation rate of KO5-2-3, which belonged to Acinetobacter sp., was high during the initial incubation time. On the third day, the medium became turbid with a great deal of oil slick. As the incubation time prolonged, the surface slick of the MSM was reduced, but the color of medium was darker. The degradation rate was 39.2% by the seventh day, and then the increase in degradation rate was not obvious. Four islates (KO5-2-6, KO5-2-7, KO5-2-8 and KO5-2-12) corresponding to the genus of Bacillus sp. showed TPH degradation phenomenon after 7 days of incubation. Afterwards, the color of the medium became brighter gradually with the formation of more flocculates. On the 20thday, the degradation efficiency of the four isolates reached more than 45%. KO5-2-10 was grouped in the genus of Ochrobactrum, which could degrade 22.7% of TPH on the 10thday, and the degradation efficiency improved relatively insignificantly when the culture time further went on. Pseudomonas sp. had been reported to exist in oil polluted soil and had the ability to degrade petroleum components[18]. But KO5-2-1, KO5-2-2 and KO5-2-9 originated from KO5-2 could not utilize the TPH. KO5-2-9, which belonged to Pseudomonas aeruginosa, could obviously emulsify the TPH. The enrichment culture was transparent with a large number of dispersed oil particles. A great deal of foams were produced after shaking. Pseudomonas aeruginosa had been reported to produce surfactant[25]. It was suggested that KO5-2-9 was a bio-surfactant producing bacteria. KO5-2-4 was related to the genus of Arthrobacter which did not show the ability to degrade the TPH in the process of 20 days.

Table 3 Comparison of 16S rDNA fragment sequence of single colonies

Table 4 TPH degradation efficiency of isolates and KO5-2

Compared with the twelve isolates, the bacterial consortium KO5-2 had the fastest degradation rate and the best degradation efficiency[26]. The enrichment culture of KO5-2 was turbid in the first 24 h. Most tiny flocculates were formed in the next day. Until the third day, the TPH removal efficiency reached 45.7%. As the incubation time went on, the degradation efficiency of KO5-2 graduallyincreased. It reached 56.9% on the seventh day. Then the degradation efficiency increased slowly. This might occur because the medium was short of nutrients when the bacteria propagation continued. Besides, saturated hydrocarbons which were readily utilized by bacteria were less, and the remaining components could hardly be degraded since they realy had toxicity[27].

3.2.3 Analysis of the microbial community shifts in different carbon sources

Figure 4 DGGE profile and its schematic diagram of KO5-2 in different carbon sources

DGGE was used to investigate the microbial community shifts in different carbon sources (TPH, saturated hydrocarbons, fluorene, phenanthrene and pyrene), explore the functional microorganisms of the bacterial consortium, analyze collaborative symbiotic relationship in the microbial community, and further reveal the degradation characteristics of KO5-2. The results are shown in Figure 4. The technology of PCR-DGGE could directly reflect the microbial community structure[28], detect the dominant strains, which were difficult to isolate and culture, and reveal the structure of the bacterial consortium. Different bands in DGGE fingerprints represented 16S rDNA V3 gene fragments of different bacteria. To a certain extent, the brightness of the bands in each lane could reflect how much the relative bacterial biomass accounted for. The number of the band could intuitively reflect the genetic diversity of bacterial communities in the sample[29]. It can be seen from Figure 4 that there were differences in the number of bands and brightness of each lane, which explained that KO5-2 showed different community structures in different carbon sources and the number of strains and the preponderant strains were also different. The number of bands in Lane A and Lane B was equal to seven, but there was a coarse and bright band (Band 3) in Lane B which was fine and light in the same position of Lane A. This explained that the community compositions of KO5-2 in carbon source of TPH and saturated hydrocarbons, respectively, were roughly the same, but there was preponderant strain (Band 3) in the culture of saturated hydrocarbons serving as the carbon source. This occurred probably because saturated hydrocarbons were the major parts of TPH, both of which were similar to the stress effect of KO5-2. But the saturated hydrocarbons were more easily to be utilized by KO5-2, resulting in the preponderant strain that was more prominent. Lane C, Lane D and Lane E had different numbers of bands, which were five, seven and four, respectively. It took place because the community structures of KO5-2 were distinct in the three different carbon sources of PAHs. The community composition of KO5-2 was the most abundant in the carbon source of phenanthrene (Lane D), which became simple in the carbon source of pyrene (Lane E). This occurred probably because the four-ring structure of pyrene was characteristic of high stability, low bioavailability, and greater toxicity, so that the stress was greater and the selectivity was higher as compared to KO5-2.

The brightness and number of DGGE fingerprints were analyzed to provide the macroscopic information on the microbial community structure. In order to determine the microbial species with which the bands were related, we excised the prominent DGGE bands of Figure 5 and sequenced the recycling DNA. The results are shown in Table 5.

Upon combining Figure 4 and Table 5, we can see that Band 7 and Band 10 corresponded to Rhodococcus sp. and Pseudomonas sp., respectively, which appeared in the five lanes. It was explained that the two genera of bacteria had strong adaptability which could survive inthe five kinds of carbon sources. Rhodococcus sp. had been reported as it could degrade petroleum components, meanwhile it had the capability for degradation of PAHs[30]. The similarities between the Band 10 related strain and Pseudomonas aeruginosa strain zgkd2 were 100%, so we speculated that it might be the bio-surfactant producing bacteria KO5-2-9. (This speculation needs to be further verified.) The Band 1 related Sphingomonas sp. just appeared in the carbon sources of PAHs (Lane C, Lane D and Lane E). Sphingomonas sp. was the key PAHs-degrading genus which was able to utilize naphthalene, phenanthrene and fluoranthene[31], as well as benzanthracene and benzo(a)pyrene[32]. Band 3, Band 4 and Band 5 were only found in the carbon sources of TPH and saturates (Lane A and Lane B), among which the Band 3 related Bacillus sp. was the preponderant strain in the culture of saturates serving as the carbon source, which was probably the key saturates degrading bacteria. Band 9 was coarse and bright which just appeared in the carbon sources of phenanthrene (Lane D), indicating that Ochrobactrum sp. was the key bacterium of the bacterial consortium and might be the functional bacterial strain capable of degrading phenanthrene. The rest of the band related strains were identified as uncultured bacterium clone, which was probably present in the environment that could not be isolated.

Table 5 Clone sequencing results of bands fromDGGE profile

4 Conclusions

A bacterial consortium KO5-2 was isolated from the oilcontaminated soil, which was able to degrade 56.9% of TPH with an initial concentration of 10 g/L at 30 ℃ after 7 days of incubation. It could remove 100% of fluorene, 98.93% of three-ring phenanthrene and 65.73% of fourring pyrene within 3, 7 and 9 days, respectively. Twelve strains from six different genera were isolated from KO5-2 and only eight ones were able to utilize the TPH. Compared with the twelve isolates, KO5-2 showed the fastest degradation rate and the best degradation efficiency. The community compositions of KO5-2 in carbon source of TPH and saturated hydrocarbons, respectively, were roughly the same, but Bacillus sp. was probably the key saturates degrading bacterium. The community structures of KO5-2 were distinct in the three different carbon sources of PAHs. Sphingomonas sp. likely played a key role in the PAHs degradation. Ochrobactrum sp. might be the functional bacterial strain capable of degrading phenanthrene.

Acknowledgements: This research was supported by the Scientific Research Fund of Liaoning Provincial Education Department (L2014148).

[1] Kerstin E S, Tserennyam L, Viviana K, et al. Changes in bacterial communities from anaerobic digesters during petroleum hydrocarbon degradation [J]. Journal of Biotechnology, 2012, 157(4): 564-572

[2] Rahman K S M, Rahman T J, Lakshmanaperumalsamy P, et al. Towards efficient crude oil degradation by a mixed bacterial consortium [J]. Bioresource Technology, 2002, 85(3): 257-261

[3] Yu C, Liao X Q, Liu Z G, et al. Research progress on microbial remediation of petroleum contaminated soil [J]. Hubei Agricultural Sciences, 2009, 48(5): 1260-1263 (in Chinese)

[4] Margesin R, Schinner F. Bioremediation (natural attenuation and biostimulation) of diesel-oil-contaminated soil in an alpine glacier skiing area [J]. Applied and EnvironmentalMicrobiology, 2001, 67(7): 3127-3133

[5] Liu Chunshuang, Zhao Dongfeng, Zhang Yunbo. Effects of temperature, acetate and nitrate on methane generation from petroleum hydrocarbons[J]. China Petroleum Processing and Petrochemical Technology, 2014, 16(4): 24-31

[6] Li H H, Shan A Q, Zhou H X. Study on the screening and function of degrading petroleum bacteria [J]. Pollution Control Technology, 2008, 21(5): 10-12 (in Chinese)

[7] Andrew S W, Mark J B. Bacterial community structure and physiological state within an industrial phenol bioremediation system [J]. Applied and Environmental Microbiology, 2000, 66(6): 2400-2407

[8] Sheng K T, Lee J T, Mario V, et al. Array-based identification of species of the genera Abiotrophia, Enterococcus, Granulicatella, and Streptococcus [J]. Journal of Clinical Microbiology,2006, 44(12): 4414-4424

[9] Leaw S N, Chang H C, Barton R, et al. Identification of medically important Candida and non-Candida yeast species by an oligonucleotide array [J]. Journal of Clinical Microbiology, 2007, 45(7): 2220-2229

[10] Muckian L, Grant R, Doyle E, et al. Bacterial community structure in soils contaminated by polycyclic aromatic hydrocarbons [J]. Chemosphere, 2007, 68(8): 1535-1541

[11] Liu P W G, Wang S Y, Huang S G, et al. Effects of soil organic matter and ageing on remediation of diesel oilcontaminated soil and on the microbial ecology [J]. Environmental Technology, 2012, 33(23): 2661-2672 (in Chinese)

[12] Muyzer G, Dewaal E C, Uitterlinden A G. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reactionamplified genes coding for 16S rRNA [J]. Applied and Environmental Microbiology, 1993, 59(3): 695-700

[13] Ke S Z, Zhang M, Shi Z, et al. Phenol degradation and microbial characteristics in upflow anaerobic sludge blanket reactors at ambient and mesophilic temperatures [J]. International Journal of Environment and Pollution, 2008, 32(1): 68-77

[14] Watanabe K, Kodama Y, Harayama S. Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting [J]. Journal of Microbiological Methods, 2001, 44(3): 253-262

[15] Cao Q M, Chen G Z, Huang X F, et al. Analysis of characteristics of petroleum hydrocarbons in zoobenthos from Shantou mangroves [J]. Environmental Chemistry, 2009, 28(4): 547-552 (in Chinese)

[16] Ghazali F M, Rahman R N Z A, Salleh A B, et al. Biodegradation of hydrocarbons in soil by microbial consortium [J]. International Biodeterioration & Biodegradation, 2004, 54(1): 61-67

[17] Cha?neau C H, Rougeux G, Yéprémian C, et al. Effects of nutrient concentration on the biodegradation of crude oil and associated microbial populations in the soil [J]. Soil Biology and Biochemistry, 2005, 37(8): 1490-1497

[18] Marcon R, Bestetti G, Frati F, et al. Naphthalene and biphenyl oxidation by two marine Pseudomonas strains isolated from Venice Lagoon sediment [J]. International Biodeterioration & Biodegradation, 2007, 59(1): 25-31

[19] Saadoun I. Isolation and characterization of bacteria from crude petroleum oil contaminated soil and their potential to degrade diesel fuel [J]. Journal of Basic Microbiology, 2002, 42(6): 420-428

[20] Li G, Huang W, Lerner D N, et al. Enrichment of degrading microbes and bioremediation of petrochemical contaminants in polluted soil [J]. Water Research, 2000, 34(15): 3845-3853

[21] Sathishkumar M, Binupriya A R, Baik S H, et al. Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium isolated from hydrocarbon contaminated areas [J]. Clean- Soil, Air, Water, 2008, 36(1): 92-96

[22] Lechner U, Baumbach R, Becker D, et al. Degradation of 4-chloro-2-methylphenol by an activated sludge isolate and its taxonomic description [J]. Biodegradation, 1995, 6(2): 83-92

[23] Meintanis C, Chalkou K I, Kormas K A, et al. Biodegradation of crude oil by thermophilic bacteria isolated from a volcano island [J]. Biodegradation, 2006, 17(2): 105-111

[24] Song B, Palleroni N J, H?ggblom M M. Isolation and characterization of diverse halobenzoate-degrading denitrifying baeteria from soils and sediments [J]. Applied and Environmental Microbiology, 2000, 66(8): 3446-3453

[25] Pornsunthorntawee O, Wongpanit P, Chavadej S, et al. Structural and physicochemical characterization of crude biosurfactant produced by Pseudomonas aeruginosa SP4 isolated from petroleum-contaminated soil [J]. Bioresource Technology, 2008, 99(6): 1589-1595

[26] Ito H, Hosokawa R, Morikawa M, et al. A turbine oildegrading bacterial consortium from soils of oil fields and its characteristics [J]. International Biodeterioration & Biodegradation, 2008, 61(3): 223-232

[27] Das K, Mukherjee A K. Crude petroleum-oil biodegradation efficiency of Bacillus subtilis and Pseudomonas aeruginosastrains isolated from a petroleum-oil contaminated soil from North-East India [J]. Bioresource Technology, 2007, 98(7): 1339-1345

[28] Muyzer G. DGGE/TGGE a method for identifying genes from natural ecosystems [J]. Current Opinion in Microbiology, 1999, 2(3): 317-322

[29] Katsivela E, Moore E R B, Maroukli D, et al. Bacterial community dynamics during in-situ bioremediation of petroleum waste sludge in landfarming sites [J]. Biodegradation, 2005, 16(2): 169-180

[30] Pasternak G, Rutkowski P, ?liwka E, et al. Broad coal tar biodegradative potential of Rhodococcus erythropolis B10 strain isolated from former gasworks site [J]. Water Air & Soil Pollution, 2011, 214(1-4): 599-608

[31] Story S P, Parker S H, Kline J D, et al. Identification of four structural genes and two putative promoters necessary for utilization of naphthalene, phenanthrene, and fluoranthene by Sphingomonas paucimobilis var. EPA505 [J]. Gene, 2000, 260(1-2): 155-169

[32] Zylstra G J, Kim E. Aromatic hydrocarbon degradation by Sphingomona yanoikuyae B1 [J]. Journal of Industrial Microbiology & Biotechnology, 1997, 19: 408-414

Sinopec’s Project “Key Process, Technology and Equipment for Manufacture of Low-Carbon Olefins and Derivatives” Passed Technical Acceptance Tests

The project “Key process, technology and equipment for manufacture of low-carbon olefins and derivatives”envisaged in the National “863’ Program and undertaken by Sinopec’s Shanghai Petrochemical Research Institute (SPRI) has passed the technical acceptance tests organized by the High-tech Research and Development Center of the Ministry of Science and Technology.

This task was cooperatively implemented by 18 institutions with SPRI assuming the leading role. The researchers focusing on three directions, viz.: “the high-efficiency utilization of byproduct resources’, “the technology for synthesis of high-performance derivatives from lowcarbon olefins” and “the domestic technology for fabrication of key large equipment for production of low-carbon olefins and derivatives”, have been engaging in the study of nine key techniques. After jointly tackling key problems in three years the research teams have developed 4 new technologies for manufacture of low-carbon olefins and their derivatives (among which three technologies have been verified in pilot scale tests), and 2 package process technologies. Three novel high-performance olefin polymerization catalysts and three new olefin-derived products have been developed. In the same time there key large pieces of equipment, including the ethylene oxide reactor, the recycle gas compressor for olefin polymerization unit and the extruding-pelleting machine have been applied in the industry.

These research achievements, covering the development of catalytic materials, green production processes, and design and fabrication of large equipment that have the capabilities of competing with major multinational companies, are currently being applied effectively in the relevant facilities at the Fujian United Petrochemical Company, the Qilu Petrochemical Company, the Wuhan Petrochemical Company, the Yangzi Petrochemical Company, the Shanghai Petrochemical Company, and the Sinopec Catalyst Company, Ltd.

In the course of implementing the research work SPRI has successfully developed the super-hydrophobic titaniumcontaining mesoporous SiO2-based catalyst for synthesis of propylene oxide and two new techniques, viz.: “disproportionation of C4olefins” and “green technology for synthesis of propylene oxide”, and also has completed the PDP of a 200-kt/a unit for manufacture of propylene via olefins disproportionation and the PDP of an 100-kt/a unit for manufacture of propylene oxide via the cumene hydroperoxide (CHP) method. Meanwhile, SPRI has also implemented the pilot-scale testing of green CHP process for synthesis of propylene oxide, and has applied for 84 invention patents to satisfactorily meet the requirements specified in the R&D program.

date: 2014-12-06; Accepted date: 2015-04-09.

Li Zheng, E-mail: lizheng.zz@163. com; Telephone: +86-24-56860968.