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, identification and enzyme characterization of a thermophilic cellulolytic anaerobic bacterium

Objective] To identify a thermophilic bacterium from horse manure to degrade cellulose efficiently,and to enrich microbial resources producing cellulolytic ethanol by co-culturing with thermophilic ethanol producing bacterium.Methods] We used Hungate anaerobic technique to isolate a strain named as HCp from horse manure mixed culture;its phylogeny was identified through 16S rDNA sequencing.Enzymatic assays were determined using DNS method.Results] The isolated HCp cells were straight with rods size of(0.35-0.50)μm ×(2.42-6.40)μm,in the form of single or paring.This strain belongs to a strictly anaerobic Gram-negative bacterium,it is able to form spores,shows motile ability and resistance to neomycin.The strain could degrade filter paper cellulose,cellulose powder,microcrystalline cellulose,cotton wool,rice straw and gelatin,and it was also able to utilize abundant saccharides as substrates such as cellobiose,glucose,xylose,xylan,raffinose,maltose,sorbose,fructose and galactose.The growth pH ranges from 6.5 to 8.5,temperature from 35 to 70℃ and concentration of NaCl on cellulose from 0% to 1.0%,while the optima of pH6.85,60℃ and 0.2% NaCl.Under the optimal growth conditions,the filter paper cellulose degradation rate was up to 90.40% after 10 days.The optimum temperatures for FPA,CMCase,β-glucosidase and xylanase were 70℃,70℃,70℃,and 60℃ respectively.CMCase activity was found with high thermal stability.The phylogenetic analysis based on partial 16S rDNA revealed that HCp was close to Acetivibrio cellulolyticus and A.cellulosolvens with 97.5% sequence similarities.Conclusion] Strain HCp is thermophilic,efficiently cellulolytic anaerobe.It is able to utilize vast substrates and produce highly thermostable enzymes.It is a potential bacterium that can be used for cellulolytic ethanol production.