Abstract：In the face of the inefficiency of bioconversion of agricultural biomass waste in low temperature environment in the winter, this study was aimed at enrichment culture and community structure analysis of a cold-adapted cellulose degrading microflora A25-3, which was isolated from the soil in alpine region. The microflora A25-3 could grow rapidly by enrichment culture at 15 ℃, with 10% cow dung and 10% LB sodium salt, and the weight loss ratio of cow dung was 48.28% in 72 h. The carboxymethyl cellulose (CMCase) activity of the supernatant of microflora culture at 15 ℃, with a peak of 42.37 U in 120 h was higher than that at 25 ℃. Total microbial DNA was directly extracted from the microflora cultured at different temperatures of 15 and 25 ℃. The clone library of 16S rRNA genes was amplified using PCR with universal bacterial primer sets. Each unique restriction fragment polymorphism pattern, which was created with 2 restriction endonucleases (HinfⅠand Csp6Ⅰ), was designated as an operational taxonomic unit (OTU). Amplified DNA was used for diversity analysis. The microflora phylogenetic trees of bacterial clone library at different temperatures of 15 and 25 ℃ was constructed. Community structure analysis revealed that the genera Brevundimonas, Pseudomonas, Chryseobacterium, Acinetobacte, Sphingobacterium and Bacillus were dominant in the clone library from microflora cultured at 25 ℃, while the genera Acinetobacte, Psychrobacillus and Bacillus were dominant at 15 ℃. The results suggested that the dominant population of cold-adapted cellulose degrading bacteria important species, especially Acinetobacte, provided the effect of increasing the CMCase activity at 15 ℃. The result will provide a scientific basis for the next study to separate active bacteria strains from the bacterial community and to optimize cellulase synthesis conditions.
Amann R I, Ludwig W, Schleifer K H, et al. Phylogenetic identification and in situ detection of individual microbial cells without cultivation[J]. Microbiological Review, 1995, 59(1):143-169.Banerjee G, Car S, Scott-Craig J S, et al.Synthetic multi-component enzyme mixtures for deconstruction of lignocellulosic biomass [J]. Bioresource Technology, 2010, 101: 9097-9105. Beer H, Hugo C J, Jooste P J, et al. Chryseobacterium piscium sp. nov., isolated from fish of the South Atlantic Ocean off SouthAfrica[J]. International Journal of Systematic and Evolutionary Microbiology, 2006,56(6): 1317-1322.Feller G, Gerday C. Psychrophilic enzymes : molecular basis of cold adaptation [J] . cellular and molecular life sciences, 1997, 53 : 830-841.Krishnamurthi S, Ruckmani A, Pukall R, et al. Psychrobacillus gen. nov. and proposal for reclassification of Bacillus insolitus Larkin & Stokes, 1967, B. psychrotolerans Abd-El Rahman et al., 2002 and B. psychrodurans Abd-El Rahman et al., 2002 as Psychrobacillus insolitus comb. nov., Psychrobacillus psychrotolerans comb. nov. and Psychrobacillus psychrodurans comb. nov. Systematic and Applied Microbiology, 2010,33(7):367-73.Santiago-Hernández A, Vega-Estrada J, Montes-Horcasitas MC, et al. Purification and characterization of two sugarcane bagasse-absorbable thermophilic xylanases from the mesophilic Cellulomonas Xavigena. Journal of Industrial Microbiology & Biotechnology, 2007,34:331–338.Whiting R C．Buchanan R L. A classification of modles for predicrive[J]．Food Microbiology，1993，10：175-177．Yu, H.，Zeng, G.，Huang, H., et al. Microbial community succession and lignocellulose degradation during agricultural waste composting[J]. Biodegradation, 2007,18(6): 793-802.Zhou J Z, Bruns M A, Tiedje J M. DNA recovery from soils of diverse composition. Applied and Environmental Microbiology, 1996, 62: 316-322.曾胤新,俞勇,蔡明宏,等.低温微生物及其酶类的研究概况[J].微生物学杂志, 2004, 24(5)：83-88.（Zeng Y X,Yu Y,Cai M H,et al. A Survey on Cold-adapted Microorganisms and their Enzymes [J].Journal of Microbiology, 2004.,24(5)：83-88.）陈亮, 迟乃玉, 张庆芳.低温纤维素酶菌株CNY086 选育及发酵培养基优化(Ⅰ) [J]. 微生物学通报, 2009,36 (10): 1547-1552 （Chen L, ChiN Y, Zhang Q F. Breeding and fermentation medium optimization of cold-active cellulase strain CNY086 (Ⅰ) [J]. Bulletin of Microbiology, 2009,36 (10):1547-1552）戴国俊,王金玉,杨建生,等.应用统计软件SPSS拟合生长曲线方程[J].畜牧与兽医, 2006, 38(9):28-30.（ Dai G J,Wang J Y,Yang J SH, et al. The fitness of growth curve equation used by statistical software SPSS[J]. Animal Husbandry & Veterinary Medicine, 2006, 38(9):28-30.）董硕,迟乃玉,王鑫,等.低温纤维素酶的研究进展[J].生物技术通报，2011, (2):37-41.（Dong SH, Chi N Y, Wang X,et al. Research Progress of Cold-adapted Cellulase[J]. Biotechnology Bulletin，2011, (2):37-41.）林艳梅, 生吉萍, 申琳,等. 适冷纤维素降解微生物研究进展[J].生物技术, 2010,20 (2): 95-98（Lin Y M, Sheng J P, Shen L, et al.Trends in cold-adapted and cellulose-degrading microorganisms. Biotechnology, 2010, 20 (2): 95-98）. 刘玮琦,茆振川,杨宇红,等. 应用16S rRNA基因文库技术分析土壤细菌群落的多样性[J].微生物学报, 2008,48(10):1344-1350.（Liu W Q, Mao ZH CH, Yang Y H,et al. Analysis of soil bacterial diversity by using the 16S rRNA gene Library. Acta Microbiologica Sinica, 2008, 48(10):1344-1350.）卢月霞,吕志伟,袁红莉,等. 纤维素降解菌的筛选及其混合发酵研究[J].安徽农业科学, 2008, 36(10):3952-3953.（Lu Y X, Lv ZH W, Yuan H L, et al. Research on the Screening and Mixed Fermentation of Cellulose-decomposing Microorganisms[J]. Journal of Anhui Agricultural Sciences, 2008, 36(10):3952-3953.）孟建宇，李蘅，樊兆阳,等. 低温纤维素降解菌的分离与鉴定[J]. 应用与环境生物学报, 2014, 20(1): 152-156.（Meng J Y, LI H, FAN ZH Y，et al. Isolation and identification of cellulose-degrading bacteria under low temperature[J]. Journal of applied and Environmental Biology，2014, 20(1): 152-156.）邱春生,孙力平.木质纤维素类农业废弃物生物转化资源化研究进展[J].安徽农业科学, 2014, (18):5928-5932.（Qiu CH SH, Sun L P. Research Progress on Resource Utilization of Lignocellulose Agricultural Wastes Based on Biotransformation. Journal of Anhui Agricultural Sciences，2014, (18):5928-5932.）唐景春. 生物质废弃物堆肥过程与调控[M]. 北京：中国环境科学出版社 , 2010, pp.17.（Tang J CH. Biomass waste composting process and regulation[M]. Beijing: China Environmental Science Press, 2010, pp.17.）田伟,王磊,李刚,等. 高温纤维素降解菌的筛选及内切酶基因的克隆表达[J].农业环境科学学报, 2014, (4):788-793.（Tian W, Wang L, LI G, et al. Isolation of Thermophilic Cellulose-Decomposing Bacteria and Cloning and Expression of an Endoglucanase Gene[J]. Journal of Agro-Environment Science, 2014, (4):788-793.）王继元,周育,赖仞,等. 1株金黄杆菌分离、鉴定及其胞外蛋白酶活性的初步研究[J].微生物学杂志, 2010, 30(1):21-26.（Wang J Y, Zhou Y, Lai R, et al. Isolation and Identification of a Chryseobacterium Strain and Its Extracellular Protease Activity[J]. Journal of Microbiology, 2010,30(1):21-26.）王世杰,王翔,卢桂兰,等. 低温微生物修复石油烃类污染土壤研究进展[J].应用生态学报, 2011,22(4):1082-1088.(Wang SH J，Wang X，Lu G L, et al. 2011.Bioremediation of petroleum hydrocarbon-contaminated soils by cold-adapted microorganisms:Research advance, Chinese Journal of Applied Ecology, 2011,22(4): 1082-1088.)张俙何,洪春来,朱凤香,等. 农业废弃物资源化利用现状与前景展望[J].现代农业科技, 2013, (20):209-209,218.（zhang X H, Hong CH L, Zhu F X, et al. Agricultural Waste Recycling Use Present Situation and Prospect[J]. Modern Agricultural Science and Technology, 2013, (20):209-209,218.）
