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【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。
【Objective】 The objective of this study is to isolate the thermophilic anaerobic bacteria that can effectively degrade cellulose and provide microbial resources for the production of cellulosic ethanol through co-culture with thermophilic ethanologenic strains. 【Method】 An anaerobic anaerobic bacterium HCp was isolated from the horse manure enrichment of cellulose by anaerobic separation. The morphological, physiological and biochemical identification, combined with 16S rDNA sequence phylogenetic analysis to determine the taxonomic status of the strain, the use of DNS enzyme activity analysis of the isolated strains of the enzymatic properties. 【Result】 The results showed that Gram stain of the isolated strain HCp was negative, and the cells were single or in pairs. The cell size was (0.35-0.50) μm × (2.42-6.40) μm, strictly anaerobic and sporulated, Neomycin has some resistance. The bacteria can use filter paper cellulose, cellulose powder, microcrystalline cellulose, absorbent cotton and rice straw, gelatin, etc., but also glucose, cellobiose, xylose, xylans, fructose, sucrose, ribose, galactose, Maltose, sorbose, trehalose, melibiose, mannose and the like. The strain grew at pH 6.5-8.5, temperature 35-70 ℃ and salt concentration 0% -1.0%, the optimum pH was 6.85, the optimal temperature was 60 ℃, the optimum NaCl concentration was 0.2% Under the optimal growth conditions, the degradation rate of filter paper cellulose reached 90.40% within 10 days. In the HCp fiber body, the optimum temperature of filter paper, carboxymethyl cellulose, β-glucosidase and xylanase were 70 ℃, 70 ℃, 70 ℃, 60 ℃, respectively, Cellulase has high thermal stability. A partial 16S rDNA sequence analysis showed that the similarity between isolates HCp and Acetivibrio cellulolyticus and A. cellulosolvens was 97.5%. 【Conclusion】 Isolate strain HCp is a thermophilic anaerobic bacterium isolated from horse manure enrichment. It has strong ability of degrading cellulose, wide range of growth temperature, good thermal stability of the enzyme, Substrates such as the use of a wide range of characteristics, for the degradation of ethanol produced ethanol provides a good material.