Isolation and characterization of an anaerobic chlorophenol-transforming bacterium.

An obligately anaerobic bacterium which transforms several chlorinated phenols was isolated. Dechlorination of the substituents ortho to the phenolic OH group was preferred, while removal of a meta-substituted chlorine was observed only with 3,5-dichlorophenol. The bacterium was a gram-positive, endospore-forming, motile, slightly curved rod. Sulfate was not reduced. Nitrate was reduced via nitrite to ammonium. The bacterium is related to the genus Clostridium. The highest growth rate was obtained in a medium containing pyruvate and yeast extract. Pyruvate supported growth as the sole source of carbon, and the fermentation of pyruvate produced almost equimolar amounts of acetate.     

Isolation and characterization of an anaerobic dehydrodivanillin-degrading bacterium.

A novel, strictly anaerobic, gram-negative, non-spore-forming, fusiform, rod-shaped bacterium having high dehydrodivanillin (DDV)-degrading activity was isolated from cow ruminal fluid. This strain degraded a range of six main lignin-related compounds such as DDV, ferulic acid, dehydrodiisoeugenol, guaiacoxyacetic acid, vanillin, and veratrylglycerol-beta-guaiacyl ether to the extent of 14 to 83% within 2 days under strictly anaerobic conditions. As DDV degradation intermediates, three aromatic compounds (dehydrodivanillic acid, vanillic acid, and 5-carboxyvanillic acid) and two alicyclic compounds (cyclohexanecarboxylic acid and cyclohexanol) were detected by thin-layer, high-performance liquid, and gas chromatography and mass spectrometry. The addition of 1% glucose and peptone in a synthetic medium stimulated growth of the strain but slowed down DDV degradation. The presence of 0.1% yeast extract increased both cell growth and DDV degradation. The growth yield in defined medium was 151.5 g (dry weight) of cells per mol of DDV utilized. Characterization of the strain indicated that it was distinct from known Fusobacterium and Clostridium species. The bacterium was easily induced to form protoplasts after treatment with either penicillin or lysozyme. The frequencies of protoplast formation and regeneration in the strain were 94 and 18%, respectively.     

Isolation and characterization of an anaerobic dehydrodivanillin-degrading bacterium

A novel, strictly anaerobic, gram-negative, non-spore-forming, fusiform, rod-shaped bacterium having high dehydrodivanillin (DDV)-degrading activity was isolated from cow ruminal fluid. This strain degraded a range of six main lignin-related compounds such as DDV, ferulic acid, dehydrodiisoeugenol, guaiacoxyacetic acid, vanillin, and veratrylglycerol-beta-guaiacyl ether to the extent of 14 to 83% within 2 days under strictly anaerobic conditions. As DDV degradation intermediates, three aromatic compounds (dehydrodivanillic acid, vanillic acid, and 5-carboxyvanillic acid) and two alicyclic compounds (cyclohexanecarboxylic acid and cyclohexanol) were detected by thin-layer, high-performance liquid, and gas chromatography and mass spectrometry. The addition of 1% glucose and peptone in a synthetic medium stimulated growth of the strain but slowed down DDV degradation. The presence of 0.1% yeast extract increased both cell growth and DDV degradation. The growth yield in defined medium was 151.5 g (dry weight) of cells per mol of DDV utilized. Characterization of the strain indicated that it was distinct from known Fusobacterium and Clostridium species. The bacterium was easily induced to form protoplasts after treatment with either penicillin or lysozyme. The frequencies of protoplast formation and regeneration in the strain were 94 and 18%, respectively.     

一株嗜热厌氧杆菌的分离、鉴定及其代谢特征

【目的】分离、保护油藏嗜热微生物资源,解析其主要的代谢特征。【方法】利用Hungte厌氧分离技术从大港油田埕海一区油层采出液中分离出厌氧菌株BF1。通过生理生化特征分析、16S rRNA基因序列比对与电化学分析,确定BF1的分类地位及其S元素代谢对腐蚀电流的影响。【结果】菌株BF1为严格嗜热厌氧革兰氏阴性杆菌,顶端产芽孢、不运动,菌体大小为0.42μm×(1.6 5.4)μm,单生、成对或成串生长。其温度生长范围为45°C 75°C(最适温度60°C);pH生长范围在4.5 8.5(最适pH 6.5)之间,比生长速率(μm)0.99 h 1,倍增时间为42 min。能利用葡萄糖、松三糖、棉子糖、甘露糖、乳糖、纤维二糖、果糖、核糖等碳水化合物,利用葡萄糖发酵的产物是乙醇、乙酸、CO2及少量的H2。菌株BF1能还原亚硫酸盐与硫代硫酸盐产生H2S,其耐受上限分别为50 mmol/L和75 mmol/L;还原硫代硫酸钠(50 mmol/L)后其极化电阻由2 099/cm2降低至776/cm2,腐蚀电流由9.936e-006 A提高至3.25e-005 A。细胞膜脂肪酸主要由高级饱和脂肪酸组成,含量最丰富的为十五烷酸占70.6%。菌株BF1的DNA(G+C)mol%含量为34.0%,其16S rRNA与Thermoanaerobacter pseudethanolicus DSM 2355T相似性最高,为98.3%,与T.brockii subsp.brockii DSM 1457T次之,为98.0%。菌株BF1的许多生理、生化特征与T.pseudethanolicus DSM 2355T和T.brockii subsp.brockii DSM 1457T有着明显的差别,如倍增时间、最适生长温度及底物利用等;而菌株BF1的细胞膜脂肪酸组成与T.pseude-thanolicus DSM 2355T也不相同。【结论】菌株BF1可能是Thermoanaerobacter属中的一个新种,其确切分类地位还需要进一步进行DNA分子杂交;其代谢元素硫提高腐蚀电流密度,可能会对油田管道与设备造成腐蚀。     

Isolation and characterization of an anaerobic ruminal bacterium capable of degrading hydrolyzable tannins.

An anaerobic diplococcoid bacterium able to degrade hydrolyzable tannins was isolated from the ruminal fluid of a goat fed desmodium (Desmodium ovalifolium), a tropical legume which contains levels as high as 17% condensed tannins. This strain grew under anaerobic conditions in the presence of up to 30 g of tannic acid per liter and tolerated a range of phenolic monomers, including gallic, ferulic, and p-coumaric acids. The predominant fermentation product from tannic acid breakdown was pyrogallol, as detected by high-performance liquid chromatography and mass spectrometry. Tannic acid degradation was dependent on the presence of a sugar such as glucose, fructose, arabinose, sucrose, galactose, cellobiose, or soluble starch as an added carbon and energy source. The strain also demonstrated resistance to condensed tannins up to a level of 4 g/liter.     

Isolation and characterization of an anaerobic syntrophic benzoate-degrading bacterium from sewage sludge

An anaerobic, motile, gram-negative, rod-shaped bacterium is described which degrades benzoate in coculture with an H₂-utilizing organism and in the absence of exogenous electron acceptors such as O₂, SO ₄ ⁼ or NO ₃ ^- . The bacterium was isolated from a municipal primary, anaerobic sewage digestor using anaerobic roll-tube medium with benzoate as the main energy source and in syntrophic association with an H₂-utilizing sulfate-reducing Desulfovibrio sp. which cannot utilize benzoate or fatty acids apart from formate as energy source. The benzoate utilizer produced acetate (3 mol/mol of substrate degraded) and presumably CO₂ and H₂, or formate from benzoate. In media without sulfate and with Methanospirillum hungatei (a methanogen that utilizes only H₂–CO₂ or formate as the energy source) added, 3 mol of acetate and 0.7 mol of methane were produced per mol of benzoate and CO₂ was probably formed. Low numbers of Desulfovibrio sp. were present in the methanogenic coculture and a pure coculture of the benzoate utilizer with M. hungatei was not obtained. The generation times for growth of the sulfate-reducing and methanogenic cocultures were 132 and 166h, respectively. The benzoate utilizer did not utilize other common aromatic compounds, C ₃ ^- –C₇ monocarboxylic acids, or C₄-C₆ dicarboxylic acids for growth, nor did it appear to use SO ₄ ⁼ , NO ₃ ^- or fumarate as alternative electron acceptors. Addition of H₂ inhibited growth and benzoate degradation.     

Isolation and characterization of an extremely thermophilic,cellulolytic, anaerobic bacterium

Summary A cellulolyticm obligately anaerobic, extreme thermophile (strain NA10) was isolated from an alkaline hot spring in Nagano Prefecture, Japan. The microorganism was a non-spore-forming, flagellated rod which had a negative reaction to Gram stain, and occurred singly or in pairs. The growth temperature was between 50° C and 85° C with the optimum at 75° C, and the growth pH was between 6.0 and 9.5 with the optimum at 8.1. The anaerobe characteristically fermented cellulose, and produced acetic acid, H₂, CO₂ (main products) and lactic acid (minor product). The DNA had a base composition of 37.7 mol% guanine+cytosine content.     

Isolation and characterization of a furfural degrading sulfate-reducing bacterium from an anaerobic digester

A sulfate-reducing bacterium (SRB) was isolated from a continuous anaerobic digester, which converted the furfural-containing wastewater to methane and CO₂. This SRB isolate could use furfural, furfuryl alcohol, and 2-furoic acid as sole source of carbon and energy in a defined mineral sulfate medium. Acetic acid was the major end product of furfural degradation. This organism also used wide varieties of other carbon sources, including ethanol, pyruvate, lactate, succinate, propanol, formate, and malate. The SRB isolate contained the electron carrier desulfoviridin. It used SO₄, NO₃, and thiosulfate as electron acceptors. This isolate used ammonium chloride, nitrate and glutamate as nitrogen source. The characteristics of the SRB isolate were closely similar toDesulfovibrio sp.     

Isolation and characterization of an anaerobic benzoate-degrading spore-forming sulfate-reducing bacterium, Desulfotomaculum sapomandens sp. nov.

Abstract Spore-forming sulfate-reducing bacteria (SRB) were enriched selectively from various kinds of aerobic soils with fatty acids as the sole carbon and energy source. A Gram-negative motile rod-shaped bacterium, which produced gas vacuoles during sporulation was isolated. It degraded alcohols, aromatic and n-fatty acids (up to C₁₈) except for propionate, completely to CO₂. Sulfate, sulfite, thiosulfate or elemental sulfur served as electron acceptors. Because of its sensitivity to H₂S, the isolate never produced more than 8 mM dissolved sulfide at pH 7.0. G + C-content of the DNA was 48.0 mol %. The isolated strain Pato is described as a new species Desulfotomaculum sapomandens .     

Isolation and characterization of an N-methylcarbamate insecticide-degrading methylotrophic bacterium.

A gram-negative bacterium which hydrolyzed aryl N-methylcarbamate insecticides was isolated from an agricultural soil which quickly degraded these pesticides. This organism, designated strain ER2, grew on carbofuran as a sole source of carbon and nitrogen with a doubling time of 3 h in a mineral salts medium. The aromatic nucleus of the molecule was not metabolized, and carbofuran 7-phenol accumulated as the end product of metabolism. The insecticides carbaryl, bendiocarb, and propoxur were similarly hydrolyzed, with each yielding the corresponding phenol. Strain ER2 contained two plasmids (120 and 130 kb). A probe cloned from the pDL11 plasmid of Achromobacter sp. strain WM111, which encodes the carbofuran hydrolase (mcd) gene (P. H. Tomasek and J. S. Karns, J. Bacteriol. 171:4038-4044, 1989), hybridized to the 120-kb plasmid. Restriction fragment profiles of pDL11 and strain ER2 plasmid DNAs suggested that the 120-kb plasmid of strain ER2 is very similar to pDL11. On the basis of the results of biochemical tests, 16S rRNA sequence analysis, and membrane lipid analyses, strain ER2 was found to be a phylogenetically unique type II methylotroph. The constitutive carbofuran hydrolase activity in glucose-grown cells increased sevenfold when strain ER2 was grown in the presence of 100 mg of carbofuran per liter as the sole source of carbon and nitrogen or as the sole nitrogen source in the presence of glucose. Growth on carbofuran resulted in the induction of enzymes required for methylamine-dependent respiration and the serine pathway of formaldehyde assimilation. These results indicate that the carbofuran hydrolase mcd gene is conserved on a plasmid found in organisms from different geographic areas and that the specific activity of carbofuran degradation may increase in response to carbofuran treatment.     

嗜热厌氧纤维素分解菌的分离、鉴定及其酶学特性

【目的】分离高效降解纤维素的嗜热厌氧菌,通过与嗜热产乙醇菌株联合培养的方式,为生产纤维素乙醇提供微生物资源。【方法】利用厌氧分离技术从降解纤维素的马粪富集物中分离到一株嗜热厌氧细菌HCp。采用形态学观察、生理生化鉴定、结合16S rDNA序列的系统发育学分析确定该菌株的分类地位,利用DNS酶活分析方法测定此分离菌株的酶学性质。【结果】分离菌株HCp革兰氏染色阴性,直杆,细胞单个或成对出现,菌体大小为(0.35-0.50)μm×(2.42-6.40)μm,严格厌氧,形成芽胞,能运动,对新霉素有一定的抗性。此菌能利用滤纸纤维素、纤维素粉、微晶纤维素、脱脂棉和水稻秸秆、明胶等,还可以利用葡萄糖、纤维二糖、木糖、木聚糖、果糖、蔗糖、核糖、半乳糖、麦芽糖、山梨糖、海藻糖、蜜二糖、甘露糖等。该菌株在pH6.5-8.5、温度35-70℃、盐浓度0%-1.0%范围内利用纤维素生长,最适pH为6.85,最适温度为60℃,最适NaCl浓度为0.2%,最佳生长条件下,在10 d内滤纸纤维素降解率可达90.40%。在HCp的纤维小体中,滤纸酶、羧甲基纤维素酶、β-葡萄糖苷酶、木聚糖酶的最适作用温度分别为70℃、70℃、70℃、60℃,并且羧甲基纤维素酶具有较高的热稳定性。部分长度的16S rDNA序列分析表明,分离菌株HCp与Acetivibrio cellulolyticus、A.cellulosolvens相似性为97.5%。【结论】分离菌株HCp是从马粪富集物中分离到的一株嗜热厌氧细菌,该菌具有较强的降解纤维素能力,生长温度范围广,酶的热稳定性好,纤维素底物利用广泛等特性,为纤维素降解产乙醇提供了良好的材料。     

Isolation and characterization of a methyl chloride utilizing,strictly anaerobic bacterium

From sludge obtained from the sewage digester plant in Stuttgart-Möhringen a strictly anaerobic bacterium was enriched and isolated with methyl chloride as the energy source. The isolate, which was tentatively called strain MC, was nonmotile, gram-positive, and occurred as elongated cocci arranged in chains. Cells of strain MC formed about 3 mol of acetate per 4 mol of CH₃Cl consumed, indicating that the organism was a homoacetogenic bacterium fermenting methyl chloride plus CO₂ according to: The organism grew with 2–3% methyl chloride in the gas phase at a doubling time of near 30 h. Dichloromethane was not utilized. The bacterium also grew on carbon monoxide, H₂ plus CO₂, and methoxylated aromatic compounds. Optimal growth with methyl chloride was observed at 25°C and pH 7.3–7.7. The G+C-content of the DNA was 47.5±1.5%. The methyl chloride conversion appeared to be inducible, since H₂ plus CO₂-grown cells lacked this ability. From the morphological and physiological characteristics, the isolate could not be affiliated to a known species.     

Isolation of an anaerobic bacterium which reductively dechlorinates tetrachloroethene and trichloroethene

Strain TEA, a strictly anaerobic, motile rod with one to four lateral flagella and a crystalline surface layer was isolated from a mixed culture that completely reduces chlorinated ethenes to ethene. The organism coupled reductive dehalogenation of tetrachloroethene or trichloroethene to cis-1,2-dichloroethene to growth, using molecular hydrogen as the electron donor. It was unable to grow fermentatively or in the presence of tri- or tetrachloroethene with glucose, pyruvate, lactate, acetate or formate. The 16S rDNA sequence of strain TEA was 99.7% identical to that of Dehalobacter restrictus. The two organisms thus are representatives of the same species or the same genus within the Bacillus/Clostridium subphylum of the gram-positive bacteria.     

Isolation and characterization of a new bacterium carboxylating phenol to benzoic acid under anaerobic conditions.

A consortium of spore-forming bacteria transforming phenol to benzoic acid under anaerobic conditions was treated with antibiotics to eliminate the four Clostridium strains which were shown to be unable to accomplish this reaction in pure culture and coculture. Clostridium ghonii was inhibited by chloramphenicol (10 micrograms/ml), whereas Clostridium hastiforme (strain 3) and Clostridium glycolicum were inhibited by clindamycin (20 micrograms/ml), without the transformation of phenol being affected. Electron microscopic observations of resulting liquid subcultures revealed the presence of two different bacilli: a dominant C hastiforme strain (strain 2) (width, 1 micron) and an unidentified strain 6 (width, 0.6 micron) which was not detected on solid medium. Bacitracin (0.5 U/ml) changed the ratio of the strains in favor of strain 6. C hastiforme 2 was eliminated from this culture by dilution. The isolated strain 6 transformed phenol to benzoic acid and 4-hydroxybenzoic acid to phenol and benzoic acid in the presence of proteose peptone. Both of these activities are inducible. This strain is a gram- variable, flagellated rod with a doubling time of 10 to 11 h in the presence of phenol. It has a cellular fatty acid composition like that of C. hastiforme. However, strain 6 does not hydrolyze gelatin or produce indole. The 16S rRNA sequence of strain 6 was found to be most similar to that of some Clostridium species, with homology ranging from 80 to 86%. Tbe evolutionary relationships of strain 6 to different groups of Clostridium and Clostridium-related species revealed that it does not emerge from any of these groups. Strain 6 most likely belongs to a new species closely related to Clostridium species.     

Isolation and characterization of a pentachlorophenol-degrading bacterium.

With a new enrichment protocol, pentachlorophenol (PCP)-degrading bacteria were isolated from soil, water, and sewage. When characterized, all isolates were related and shared characteristics of the genus Arthrobacter. Growth rates for strain NC were determined for a number of substrates, including PCP and 2,4,6-trichlorophenol. Changes in PCP concentration affected growth rate and length of the lag phase but not cell yield. Increasing the pH from 6.8 to 7.8 decreased the length of the lag phase for growth on PCP. Cessation of growth, upon incremental addition of PCP, was found to be related to a decrease in pH rather than to a buildup of a toxic metabolite. Degradation of PCP by strain NC was shown to be complete.     

Isolation and characterization of an active mannanase-producing anaerobic bacterium, Clostridium tertiumKT-5A, from lotus soil

Of 10 strains of mannanase-producing anaerobicbacteria isolated from soils and methanogenic sludges, Clostridium tertium KT-5A,which was isolated from lotus soil, produced high amounts of extracellular β-1,4-mannanase. The isolate was an aerotolerant anaerobe without quinon systems; the cell growthcultivated with no addition of reducing agents was also stable. High yields of mannanasewere obtained by inducing enzyme production with galactomannan guar gum and beef extract/peptone as carbon and nitrogen sources, respectively. Fermentation endproducts on galactomannan fermentation were formate, acetate, lactate, butyrate, carbondioxide and hydrogen. The extracellular mannanase displayed high activity ongalactomannans of locust bean gum galactose/mannose (G/M) ratio 1:4 and spinogum (G/M 1:3), but weak activity on guar gum galactomannan (G/M 1:2) and konjac glucomannan. As far as is known, this is the first report on the isolation of an activemannanase-producing anaerobic bacterium from natural environments.     

一株油藏嗜热厌氧杆菌的分离鉴定和代谢特征的分析

摘要：【目的】从高温油藏中发掘新的微生物种质资源。【方法】采用 Hungate 厌氧操作技术从大港油田采出水中分离到一株厌氧杆菌 HL-3。通过生理生化特征比较和16S rRNA序列比对,确定HL-3的分类地位。【结果】菌株HL-3为严格厌氧的革兰氏阴性杆菌。生长温度范围 40℃－75℃（最适温度 60℃）;pH 范围 5.0－8.0（最适 pH 6.5）;NaCl 浓度范围 0%－3.2%（最适NaCl浓度0.25%）。能够利用葡萄糖、核糖、甘露糖、木糖、纤维二糖等多种碳水化合物,发酵葡萄糖的产物是乙醇、乙酸、CO2及少量丙酸和丁醇。菌株HL-3的（G+C）mol%含量为 33.9%,与Thermoanaerobacter（嗜热厌氧杆菌属）中模式菌株T.uzonensis DSM18761T (EF530067)的16SrRNA 序列相似性为98.8%,与T.sulfurigignens DSM17917T (AF234164)的相似度次之为98.1％。菌株能够耐受浓度较高的亚硫酸根（0.1 mol/L）离子和浓度极高的硫代硫酸根（0.8 mol/L）。当硫代硫酸根浓度高于0.075 mol/L时,菌体内产生硫单质颗粒;同时,在培养血清瓶顶空中检测到硫化氢气体。菌株 HL-3与T.uzonensis DSM18761T对硫代硫酸根和亚硫酸根的耐受程度有很大不同。菌株HL-3对硫代硫酸根和亚硫酸根耐受程度及对硫代硫酸根的代谢机制与T.sulfurigignens DSM17917T(AF234164)极为相似,但二者代谢葡萄糖的产物却极不相同。【结论】所以菌株HL-3可能是Thermoanaerobacter属中的一个新种,其确切分类地位还有待用DNA分子杂交[1]的技术手段做进一步的鉴定。     

浮霉菌门严格厌氧产氢细菌(Thermopirellula anaerolimosa)的分离及其生理特性

[目的]厌氧颗粒污泥中含有大量未知微生物资源,利用低浓度底物及添加抗生素的培养基进行厌氧发酵细菌的筛选,并对分离菌株进行生理生化特性研究.[方法]利用系列稀释法及亨盖特厌氧滚管技术从制糖废水厌氧处理反应器的颗粒污泥中分离到一株高温厌氧产氢细菌VM20-7T,通过16S rRNA基因序列同源性确定其系统发育地位.[结果]菌株VM20-7T为高温、严格厌氧、革兰氏阴性梨形细菌,细胞大小为(0.7-2.0)μm×(0.7-2.0) μm,不运动,不产芽胞.其生长温度范围为35℃-50℃(最适温度45℃),pH范围为6.0-8.3(最适pH7.0-7.5),NaCl耐受范围为0％-0.5％(w/v,最适浓度0％).菌株VM20-7T可利用葡萄糖、麦芽糖、核糖等多种糖类为唯一碳源生长,葡萄糖发酵终产物是乙酸和H2.该菌株不利用硝酸盐、硫酸盐等作为电子受体生长.G+C含量为60.9 mol％,16S rRNA基因序列同源性显示菌株属于浮霉菌门,但与已培养菌株的同源性较低,与梨形菌属一红小梨形菌属-芽殖小小梨形菌属(Pirellula-Rhodopirellul -Blastopirellula,PRB)分支的亲缘关系最近,但序列相似性也仅为82.7％-84.3％.[结论]利用低浓度糖类并添加抗生素分离厌氧颗粒污泥中的微生物,获得了浮霉菌门首例严格厌氧细菌VM20-7T.生理生化特性和系统发育分析显示,菌株VM20-7T为浮霉菌目的新属新种,命名为Thermopirellula anaerolimosa.该菌株的菌种保藏号为CGMCC 1.5169T=JCM 17478T=DSM 24165T.     

阿特拉津降解菌SA1的分离鉴定及其降解特性研究

为进行阿特拉津(AT)污染的生物修复,从AT降解混合菌群中,经长期的交替液体摇瓶培养和平板划线分离,筛选到一株能完全降解AT的菌株SA1。经生理生化特征及16S rDNA序列分析,将该菌鉴定为假单胞菌属(Pseudomonas sp.)。与已报道的AT降解菌Pseudomonas sp.ADP不同,SA1能以AT为唯一碳源、氮源和能源生长,培养基中添加铵盐不抑制SA1的降解功能,而添加葡萄糖时,累积的氰尿酸会被快速降解。SA1生长的最适温度为37℃,最适pH值为7.0。SA1的静息细胞在10℃~40℃或pH值4~11时均能高效降解AT,比ADP降解具有更广的pH和温度范围,表明SA1降解菌株具有广阔的应用前景。SA1中AT降解基因为保守的atzABCD,并含有IS1071的tnpA基因片段,传代过程中降解基因会以一定频率丢失。     

厌氧丁草胺降解菌BAD-20的分离鉴定及降解特性研究

[目的]分离并鉴定能够降解除草剂丁草胺的厌氧微生物菌株,研究其厌氧降解丁草胺的特性和代谢途径,为深入研究丁草胺厌氧降解机制提供依据.[方法]以丁草胺为碳源作为选择压力从水稻田土壤中富集驯化丁草胺降解菌,利用16SrRNA基因系统发育分析结合菌株培养特征对降解菌株进行初步鉴定,利用液相色谱-时间飞行质谱(LC-TOF-M...