CN108046441B - Method for degrading petroleum and phenanthrene by using strain P51 - Google Patents
Method for degrading petroleum and phenanthrene by using strain P51 Download PDFInfo
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F2101/327—Polyaromatic Hydrocarbons [PAH's]
Abstract
The divisional application relates to a method for degrading petroleum and phenanthrene by using a strain P51. The strain has a preservation number of CGMCC No.14411, and a 16SrRNA sequence of the strain is shown as SEQ ID No. 1. The strain P51 screened by the invention has the function of efficiently degrading petroleum, and the strain P51 has higher degradability to phenanthrene in polycyclic aromatic hydrocarbons while degrading petroleum, so that the characteristic solves the problem of marine pollution caused by petroleum pollution, the marine environment is completely recovered, and a foundation is laid for developing microbial remediation petroleum pollution products.
Description
The application is a divisional application with the application number of 2017110671186, the application date of 2017, 10 and 30 months and the invention name of 'petroleum and phenanthrene degradation strain P51 suitable for a seabed low-temperature environment'.
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a strain P51 with a petroleum degradation function and marine environment treatment by using the strain.
Background
With the development of socioeconomic, the demand of human beings on petroleum is continuously expanding. Petroleum products such as gasoline, diesel oil, lubricating oil, paraffin, asphalt and the like gradually come close to the life of human beings, and various pollution events accompanied with the petroleum products are also frequent. In 11 months 2007, a Russian tanker 'Volgar Petroleum No. 139' loaded with 4700 tons of heavy oil encounters stormy wind at the nicky-red strait, is disintegrated and sunk, and more than 3000 tons of heavy oil leaks, so that the marine area is seriously polluted; 20 days 4 months in 2010, the semi-submersible drilling platform explodes in a deep water horizon, sinks into the gulf of Mexico two days later, and about 1 million tons of crude oil leaks into the gulf of Mexico every day, so that very serious petroleum pollution is caused; in 17 days 7 and 2010, one oil pipeline of petroleum in the vicinity of the New Port in Dalian Liaoning explodes and fires, and although the fire is extinguished in 15 hours, the explosion causes a large amount of crude oil to leak into the sea; an oil leakage accident happens to an oil field of the middle sea oil Bohai Bay in 6 months in 2011, oil leakage lasts for several months, and the caused damage cannot be estimated. In 11 middle and late months in 2013, a middle petrochemical east China oil pipeline in the Qingdao economic development area is broken and exploded, so that the local sea area is polluted, and huge losses are caused to the life and property safety and the ecological environment of local residents. The damage caused by the burst of offshore oil wells and the rupture of oil pipelines has direct or indirect influence on the marine environment, the physical health and the sustainable development of human beings. According to statistics, about 600-1000 ten thousand tons of petroleum and products thereof flow into water every year all over the world, and most of the petroleum and products thereof flow into oceans.
The petroleum pollution is increasingly serious, which causes the attention of all social circles and the problem of treating the petroleum pollution, and becomes the task which is mainly solved by the current scholars. Currently, physical, chemical and biological treatments are commonly used. The physical treatment is to dilute, gather, migrate and the like the petroleum on the surface of the ocean in a physical mode, but cannot completely remove the petroleum dissolved in the seawater; the chemical treatment is to add a certain chemical reagent into seawater to achieve the purpose of degradation, but the mode can cause secondary pollution; the biological treatment is to degrade petroleum pollutants in the ocean by microbial metabolism by utilizing a bioremediation technology, has the advantages of good economic benefit, high treatment efficiency and small environmental pollution, and can fundamentally solve the problem of petroleum pollution. Compared with other treatment methods, the bioremediation technology has the advantages of low cost and good economic and environmental benefits, and becomes an effective method for solving the problem of complex environmental pollution.
Research has shown that there are 79 genera of microorganisms capable of degrading petroleum, more than 200 species of microorganisms, which belong to bacteria, actinomycetes, molds, yeasts and algae, respectively. Among the most common petroleum degrading bacteria in soil are: the most important degrading bacteria in the sea include, for example, Microbacterium (Microbacterium), Arthrobacter (Arthrobacter), Micrococcus (Micrococcus), and Streptomyces (Streptomyces): achromobacter (Achromobacter), Acinetobacter (Acinetobacter), Alcaligenes (Alcaligenes), and the like.
At present, more microorganisms reported in literature are mainly used for treating environmental petroleum pollution and mainly focus on land petroleum pollution. For example, just dawn[1]3 strains which take crude oil as a unique carbon source are separated from petroleum-polluted seawater, and the 3 strains are identified to belong to Gamma-Proteobacteria and Actinomycetes respectively, wherein the degradation rate of the Gamma-Proteobacteria is the highest and can reach 30.52 percent. When the three bacteria are respectively combined and cultured in pairs, the degradation rate of the combined bacteria to crude oil is higher than that of the single bacterial strain and is not equal to the sum of the degradation rates of the corresponding single bacteria. Therefore, the combined bacteria can degrade petroleum hydrocarbon more effectively than single bacteria, and the degradation rate of the mixed bacteria is formed among the bacterial strains due to the effects of various factors such as competition, antagonism, promotion and the like. Guoqian yoga and the like[2]2 dominant oil reducing bacteria are obtained by screening, and one strain is preliminarily identified to be bacillus, and the other strain is also identified to be acinetobacter. And finally obtaining the optimal degradation conditions of the two strains by changing different conditions such as temperature, pH, inoculation amount and the like. Wangchana et al[3]6 strains of petroleum degrading bacteria are separated from petroleum polluted soil of an Tianjin Hongkong oilfield and oil washing sewage of a Bohai sea offshore drilling platform and are respectively identified to belong to the genus Bacillus, the genus Pseudomonas and the genus Xanthium, wherein the genus Bacillus S3 has the highest alkane (41.3%) and arene (30.9%) degradation rates.
Although a great deal of research on the biological method for treating petroleum-polluted environments has been carried out, most of the degrading bacteria applied to the petroleum-polluted treatment at present come from soil, and relatively few reports of degrading bacteria derived from marine habitation exist. Corresponding to the soil environment, the marine environment has the characteristics of low temperature, low oxygen and oligotrophy, the feeding of high-efficiency degrading bacteria is often accompanied by the feeding of nutrient sources and the like, whether the exogenous microorganisms can maintain competitive advantages on indigenous microorganisms for a long time or not can threaten the indigenous organisms, whether the feeding of nutrient substances can influence the ecological environment or not can not be the same as the opinion of a few scholars. The problems are solved to a great extent by screening the marine inhabitant degrading bacteria. Therefore, the treatment of marine oil pollution by screening marine microorganisms has become the direction of research today.
Disclosure of Invention
In order to solve the problem of treatment of the marine petroleum pollution in the prior art, the inventor obtains a microbial strain with petroleum degradation performance by screening a petroleum-polluted sea area, determines the classification status of the microbial strain and evaluates the petroleum degradation performance of the microbial strain, and meanwhile, researches show that the strain P51 also has the function of degrading phenanthrene while degrading petroleum, thereby laying a foundation for degrading polycyclic aromatic hydrocarbon pollutants.
In order to achieve the purpose, the invention adopts the following technical scheme:
a strain P51 with petroleum degradation function, which is named Tessaracoccocus flavescens in Latin literature, and the 16S rRNA sequence thereof is shown as SEQ ID No. 1. The strain P51 is collected from seabed sediments in the petroleum-polluted sea area of the Xingang, and is obtained by enrichment and separation.
The strain P51 is submitted for preservation, and the specific preservation information is as follows:
the name of the depository: china general microbiological culture Collection center (CGMCC);
the address of the depository: beijing, Chaoyang, Chenlu No.1 institute No. 3, institute of microbiology, China academy of sciences;
the preservation date is as follows: 7, 7 and 12 in 2017;
the preservation number is: CGMCC No. 14411;
the strain P51 has the morphological and physicochemical characteristics that:
the strain P51 is subjected to underline culture on a solid LB culture medium at 28 ℃ until a single colony is obtained, the colony morphology of the strain P51 is observed, the surface of the bacterial body is smooth and convex, the edge is neat, and the bacterial body is mostly circular, opaque and orange yellow.
Strain P51 was short rod-shaped, nonfilamentous, approximately 0.74-0.84 μm long and approximately 0.30-0.37 μm wide.
The method for separating the strain P51 comprises the following steps: inoculating strain P51 into enrichment medium according to 1% inoculum size, and culturing at 15 deg.C with strain P51 suspension concentration of 108cfu/ml, and the composition of the enrichment medium is as follows: using petroleum as the onlyThe carbon source is added into the inorganic salt culture medium, and the petroleum adding amount is 0.5% (v/v) of the total amount of the inorganic salt culture medium.
The inorganic salt culture medium comprises the following components: MgSO (MgSO)4·7H2O 0.7g，NH4NO31g，KCl 0.7g，KH2PO42g，Na2HPO43g, 1000mL of natural seawater, pH7.5, and supplementing 10mL of microelement mixed solution after sterilization.
The second purpose of the invention requests to protect the application of the strain P51, namely the treatment of petroleum pollution in a petroleum degradation and phenanthrene degradation mode.
The third purpose of the invention requests to protect the method for degrading petroleum by the strain P51, which comprises the following steps:
the bacterial suspension is concentrated to 108cfu/ml strain P51 was inoculated to enrichment medium at 1% inoculum size and shaken on a shaker at 150r/min at 15 ℃ for 15 d.
The method for degrading phenanthrene by using the strain P51 comprises the following steps: the bacterial suspension is concentrated to 108cfu/ml strain P51 was inoculated into enrichment medium at an inoculum size of 1% and cultured at 15 ℃ for 7 d.
Compared with the prior art, the invention has the beneficial effects that: the invention screens out a strain P51 which has petroleum degradation and phenanthrene degradation simultaneously from the submarine sediments for the first time. After the strain is shaken in a shaking table at 15 ℃ for 15 days, the petroleum degradation rate can reach 28.90 percent. Through GC-MS detection and analysis, the degradation rate of phenanthrene can reach 61.60% after the strain P51 is subjected to static culture at 15 ℃ for 7 d. In the screening process of the strain, the strain P51 has higher degradability on phenanthrene in polycyclic aromatic hydrocarbons while degrading petroleum, the characteristic solves the problem of marine pollution caused by petroleum pollution, the marine environment is completely recovered, and a foundation is laid for developing microbial remediation petroleum pollution products.
Drawings
FIG. 1 is a photograph of the colony morphology of strain P51;
FIG. 2 is a photograph showing the form of a cell of the strain P51;
FIG. 3 is a phylogenetic tree of strain P5116 SrRNA;
FIG. 4 is a photograph of the degradation of petroleum by strain P51;
FIG. 5 is a photograph of the degradation of phenanthrene by strain P51.
Detailed Description
The technical solutions of the present invention are further described below with reference to specific examples, but the present invention is not limited to the contents of the examples in any way. In the examples, unless otherwise specified, the experimental methods are all conventional methods; unless otherwise specified, the reagents and biomaterials are commercially available.
Example 1
Collection and isolation of Strain P51
Samples are collected from seabed sediments in the petroleum-polluted sea area of the Xingang great harbor, and the collected samples are stored in an ice box and quickly transported back to a laboratory for enrichment and separation of petroleum-degrading microorganisms.
About 10g of newly collected sediment sample is weighed and added into a 250mL triangular flask containing 100mL of enrichment medium, and the mixture is enriched for 7d under the condition of static culture at 15 ℃, and then separated by a gradient dilution separation method to obtain the petroleum degrading strain.
The culture medium comprises the following components:
inorganic salt culture medium: MgSO (MgSO)4·7H2O 0.7g，NH4NO31g，KCl 0.7g，KH2PO42g，Na2HPO43g, 1000mL of natural seawater, pH7.5, and supplementing 10mL of microelement mixed solution after sterilization.
And (3) mixing trace element liquid: CaCl22mg，FeCl3·6H2O 50mg，CuSO40.5mg，MnCl2·4H2O0.5mg，ZnSO4·7H2O10 mg, distilled water 1000 mL.
Enrichment culture medium: in the inorganic salt medium, petroleum was added as a sole carbon source, and the petroleum was added in an amount of 1.5% (v/v) based on the total amount of the inorganic salt medium.
Separating a culture medium: 1.5 percent (mass percent) of agar coagulant is added into the enrichment medium.
Example 2
Morphological observation and identification of the strains:
the strain P51 was underlined at 15 ℃ to form a single colony on an enrichment medium and an LB medium each using petroleum as a sole carbon source, and the colony morphology of the strain P51 was observed. Meanwhile, selecting bacterial colonies, fixing the bacterial colonies for 1-2 hours by using a 2.5% glutaraldehyde solution, then dripping the bacterial suspension on a silicon wafer, naturally airing to a slightly humid and semi-dry state, washing the silicon wafer for 10 minutes by using a phosphoric acid buffer solution with the pH of 7.2, after washing for 3 times, performing gradient dehydration by using 30%, 50%, 70%, 85%, 95% and 100% ethanol respectively, dripping isoamyl acetate on the silicon wafer for fixing, and standing overnight. And coating the prepared sample on the next day, and observing by using a scanning electron microscope. As shown in figure 1, the bacterial strain P51 on the solid LB medium has smooth and convex surface, regular edges, round shape, opacity and orange yellow color. As shown in FIG. 2, the strain P51 was in the form of a short rod, without flagella, about 0.74 to 0.84 μm in length and about 0.30 to 0.37 μm in width.
Duplicate strains were excluded according to strain morphology and culture characteristics, and the obtained strains were subjected to 16S rRNA gene sequence analysis. Extracting and separating the obtained microbial genome DNA by a microwave method, performing PCR amplification by using a bacterial universal primer F27:5'-AGAGTTTGATCCT GGCTCAG-3', 5'-TACCTTGTTACGAC TT-3' (Shanghai synthetic), submitting the sequencing result of the PCR amplification product to a GeneBank database of VCBI for comparison analysis, constructing the obtained microbial strain phylogenetic tree by using Blast software and MEGA software, and performing culturable microbial population diversity analysis. As shown in FIG. 3, 16S rRNA sequence analysis of strain P51, combined with morphological structure observation, identified strain P51 as Tessaracoccocus flavescens.
Example 3
Determination of petroleum degradation Performance of Strain P51
The petroleum degradation performance evaluation of the strain P51 adopts an ultraviolet light spectrum division method to measure the petroleum degradation rate of the strain, uses an extracted petroleum ether mixture as a sample, and uses a U-5100 type ultraviolet light spectrum division meter to analyze the petroleum content change before and after the strain is degraded, and the petroleum degradation rate of the strain is calculated according to the formula of η (n is equal to n)0-n1)/n0× 100%, wherein η is the petroleum degradation rate, n0As a blank control, n1For cultivation of inoculated strainsResidual petroleum content in the extract of nutrient solution.
150 mu L of petroleum is added into the enrichment medium as a unique carbon source to carry out petroleum degradation performance analysis of the strain. The strain (bacterial suspension concentration is 10)8cfu/ml) is inoculated into an enrichment medium containing 100ml according to the inoculation amount of 1 percent, after respectively standing at 15 ℃ and shaking for 15d in a shaking table at 150r/min, 300ml of petroleum ether is used for extraction, and the degradation condition of each component of petroleum hydrocarbon in the extraction liquid is determined by adopting a GC-MS method. The experiment was repeated 3 times with enriched medium without added microbial strain as a control.
Under the condition of shake culture at 15 ℃, the degradation rate of the strain P51 to petroleum is the highest, and 15d can reach 48.90%; under the condition of static culture at 15 ℃, the degradation rate of the strain P51 is obviously lower than that of shake culture, and the degradation rate is only 41.32% in 15 d. The degradation capability of the strain P51 to petroleum is obviously higher than that of the strain obtained by static culture during shaking culture, and oxygen is necessary for the degradation of petroleum.
Example 4
Determination of degradation Properties of Strain P51 phenanthrene
Evaluation of degradation performance of phenanthrene of strain P51 petroleum degradation rate of the strain is determined by adopting a gas chromatography, the extracted dichloromethane mixture is used as a sample, a GC-MS-QP2010 type gas chromatography-mass spectrometer is used for analyzing the content change of phenanthrene before and after the strain degradation, the model of a chromatographic column is HP-5MS (30m × 0.25.25 nm × 0.25.25 mu m), the sample amount is 1 mu L, and the degradation rate of the strain to the phenanthrene is calculated according to the formula of η ═ (n is η ═ and (n is 1 mu L)0-n1)/n0× 100%, wherein η is the degradation rate of phenanthrene, n0Peak area of blank control, n1Peak area of experimental group for inoculated strain.
150 mu L of petroleum is added into the enrichment medium as a unique carbon source to carry out petroleum degradation performance analysis of the strain. The strain (bacterial suspension concentration is 10)8cfu/ml) is inoculated into an enrichment medium containing 100ml according to the inoculation amount of 1 percent, after respectively standing at 15 ℃ and shaking for 7d in a shaking table at 150r/min, 30ml of dichloromethane is used for extraction, and the degradation condition of phenanthrene in the extraction liquid is determined by adopting a GC-MS method. The experiment was repeated 3 times with enriched medium without added microbial strain as a control.
The chromatographic conditions for analyzing phenanthrene by GC-MS are as follows: the temperature of a sample inlet is 200 ℃; the carrier gas is helium, the initial temperature of the column box is 40 ℃, the temperature rising program is set to 40 ℃ and kept for 5min, then the temperature rises to 230 ℃ at the speed of 10.00 ℃/min and is kept for 17 min; the ion source temperature was 230 ℃ and the scan range was 50-600 amu.
When the strain is cultured under the standing condition of 15 ℃, the degradation rate of the strain P51 to phenanthrene is the highest, and the 7d can reach 61.60 percent; under the condition of shake culture at 15 ℃, the degradation rate of the strain P51 is obviously lower than that of static culture, and the degradation rate is only 52.62% after 7 d. The degradation capability of the strain P51 on phenanthrene is obviously higher than that of aerobic shake culture during low-oxygen culture, so that the strain is suitable for phenanthrene degradation in a seabed low-temperature low-oxygen environment.
The degradation performance of the strain P51 on petroleum and phenanthrene is evaluated, and the strain P51 has better degradation performance on both the petroleum and the phenanthrene. Wherein, the degradation rate of the strain P51 to petroleum is 28.90 percent when the strain is cultured for 15 days under the condition of a shaker at 15 ℃; when the strain is cultured for 7d under the standing condition at 15 ℃, the degradation rate of the strain P51 to phenanthrene is 61.60 percent.
Reference to the literature
[1] Separation and diversity analysis of petroleum degrading bacteria in the original Yan coast zone [ D ]. Shandong: qingdao university of justice 2015:46-47
[2] Screening of petroleum degrading bacteria in treatment of oil-containing wastewater by Qian Yu, Zhangjiamin, Hrong and J, basic science of textile university, 2016,29(2): 269)
[3] Separation and identification of Wangchan, Shiyanyun, Zhengli and petroleum degrading bacteria and interspecific effect of 4 strains of bacillus [ J ]. environmental science 2016,36(6): 2245-supplement 2251.
Sequence listing
SEQ ID No.1 (16S rRNA sequence of Strain P51 (Tessaracococcus flavescens)):
CGGGTGTTACCGACTTTCATGACTTGACGGGCGGTGTGTACAAGCCCCGGGAACGTATTCACCGCAGCGTTGCTGATCTGCGATTACTAGCGACTCCGACTTCATGGGGTCGAGTTGCAGACCCCAATCCGAACTGAGACCGGCTTTCTGAGATTCGCTCACCCTCACAGGCTCGCAGCTCTTTGTACCGGCCATTGTAGCATGCGTGAAGCCCTGGACATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGAGTTGACCCCGGCGGTCTCCAATGAGTCCCCGGCATTACCCGCTGGCAACATTGGACGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTATACCGACCAAAAAGGGGCACCTATCTCTAGATGTTTCCGGCATATGTCAAACCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGGCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGTACTTAATGCGTTAGCTGCGGCACGGAGAACGTGGAATGTCCCCCACACCTAGTACCCACCGTTTACGGCGTGGACTACCAGGGTATCTAAGCCTGTTTGCTCCCCACGCTTTCGCTTCTCAGCGTCAGGAAAGGTCCAGAGATCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTCCACCGCTCCACCAGGAATTCCGATCTCCCCTACCTTCCTCAAGTCTGCCCGTATCGGAAGCAGGCTCAGTGTTGAGCACTGAGTTTTCACTCCCGACGTGACAAACCGCCTACAAGCTCTTTACGCCCAATAAATCCGGACAACGCTCGCACCCTACGTATCACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTCTTCTCCCACTACCGTCACGTTAGCTTCGTCATGGGTGAAAGCGGTTTACAACCCGAAGGCCGTCATCCCGCACGCGGCGTTGCTGCATCAGGCTTTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTTTGGGCCGTATCTCAGTCCCAATGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGAAGCCTTGGTGAGCCATTACCTCACCAACAAGCTGATAGGCCGCGAGTCCATCCATGACCGCCGGAGCTTTCCAACCCCAAACATGAGTCCAGGGTTCATATCCGGTATTAGCACCTGTTTCCAGATGTTATCCCAGAGTCAAGGGCAGGTTACTCACGTGTTACTCACCCGTTCGCCACTCGTG
Claims (1)
1. the method for degrading petroleum and phenanthrene by using the strain P51 is characterized in that the preservation number of the strain P51 is CGMCC No.14411, the 16SrRNA sequence is shown as SEQ ID No.1, and the degradation method comprises the following steps: inoculating the strain P51 into an enrichment medium according to the inoculation amount of 1%, and culturing at 15 ℃; the method for separating the strain P51 comprises the following steps: collecting a sample from the submarine sediment of the petroleum-polluted sea area of the Xingang harbor, storing the collected sample in an ice box, and quickly transporting the ice box back to a laboratory for enrichment and separation of petroleum-degrading microorganisms; weighing a newly collected sediment sample, adding the sediment sample into a triangular flask containing an enrichment medium, enriching for 7 days under the condition of static culture at 15 ℃, and then separating by a gradient dilution separation method to obtain a strain; the enrichment medium comprises the following components: adding petroleum serving as a unique carbon source into an inorganic salt culture medium, wherein the adding amount of the petroleum is 0.5% (v/v) of the total amount of the inorganic salt culture medium; the inorganic salt culture medium comprises the following components: MgSO (MgSO)4·7H2O 0.7g，NH4NO31g，KCl 0.7g，KH2PO42g，Na2HPO43g, 1000mL of natural seawater and pH7.5, and supplementing 10mL of microelement mixed solution after sterilization; the mixed solution of the trace elements comprises: CaCl22mg，FeCl3·6H2O50mg，CuSO40.5mg，MnCl2·4H2O 0.5mg，ZnSO4·7H2O10 mg, distilled water 1000 mL.
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| CN110117559B (en) * | 2019-04-29 | 2022-12-02 | 北京润世能源技术有限公司 | Microbacterium W-Y13 and application thereof |
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| CN106190923A (en) * | 2016-08-11 | 2016-12-07 | 中山大学 | New sphingolipid bacterium (Novosphingobium sp.) 1MP25 bacterial strain application in degrading polycyclic aromatic hydrocarbons |
| CN106315868A (en) * | 2016-10-25 | 2017-01-11 | 山东大学 | Application of degrading bacterium capable of metabolizing various hydrocarbons to petroleum pollutant disposal |
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| CN106190923A (en) * | 2016-08-11 | 2016-12-07 | 中山大学 | New sphingolipid bacterium (Novosphingobium sp.) 1MP25 bacterial strain application in degrading polycyclic aromatic hydrocarbons |
| CN106315868A (en) * | 2016-10-25 | 2017-01-11 | 山东大学 | Application of degrading bacterium capable of metabolizing various hydrocarbons to petroleum pollutant disposal |
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| Characterization of aliphatic hydrocarbon degrading anaerobes isolated from sand dune soils;Marsh, N. A.等;《Abstracts of the General Meeting of the American Society for Microbiology》;20011231;第101卷;第651页摘要 * |
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