【作者】 邓辉；

【导师】 吴伟祥； 王飞儿；

【作者基本信息】 浙江大学 ， 环境工程， 2013， 硕士

【摘要】 全球富含纤维素的农林废弃物资源产量巨大且缺乏相应的资源化利用手段,随意丢弃、野外焚烧秸秆等现象普遍存在,造成诸多环境问题。发掘高效的纤维素酶菌株对于富纤维素废弃物的资源化利用对解决这些环境问题、实现农业的可持续发展意义深远。由于在高温、碱性环境下放线菌有更好的纤维素降解活性而受到越来越多的关注。富纤维素堆肥环境中蕴藏着包括放线菌在内的丰富的微生物种质资源,论文选择对猪粪与木屑混合堆肥过程中放线菌的多样性与群落结构变化展开研究并发掘其中的纤维素降解放线菌资源。主要研究结果如下：(1)利用实时荧光定量PCR对不同时期堆肥的细菌与放线菌进行了定量研究,发现在该堆肥体系中,放线菌占据非常重要的比例。PCR-DGGE与克隆文库结合16S rRNA基因系统发育分析结果显示,堆肥体系中放线菌分布于放线菌目5个亚目中的9个科,共24个属,包括糖单孢菌属,微球菌属,红球菌属,拟诺卡氏菌属,棒状杆菌属,库克菌属,普氏菌属,迪茨菌属,乔治菌属,短杆菌属,杆菌属,假诺卡氏菌属,链霉菌属菌属,嗜热裂孢菌属,丙酸杆菌属等。堆体理化性质的变化使得不同堆肥时期放线菌菌的群落结构存在非常显著的差异,其中堆体温度对耐热放线菌具有强烈的筛选作用。棒状杆菌是堆肥初期的主要放线菌群,占据库容的35%,Actinotalea占据库容的18%；堆体温度升高后,放线菌从常温菌群向耐热菌群过渡,此时糖单孢菌属,乔治菌属,嗜热裂孢菌属三者在高温期共占据库容的80%以上；而短杆菌属,糖单孢菌属,杆菌属是堆肥降温腐熟期的优势放线菌,共占据了库容的70%。多样性指数分析表明堆肥初期和后期的多样性高于高温期。(2)纯培养的获取是研究微生物功能及进行开发利用的基本前提。本研究通过可培养的方法从堆肥中分离得到了103株放线菌纯培养物。采用纤维素刚果红平板对所得菌株进行纤维素水解能力初步测试,发现A值(透明圈直径：菌落直径)大于5的放线菌占据21%,其中9株放线菌A值大于7。结果表明猪粪、木屑混合堆肥体系中分离得到的放线菌绝大多数都具有一定的纤维素分解能力。通过对初筛表现较好的68株放线菌进行摇瓶发酵试验以对其纤维素酶活进行定量研究,CMC酶活力最高达到0.16U/mL,FPA酶活最高0.15U/mL最后,对CMC酶活性最高的3株放线菌进行16S rDNA水平鉴定,通过BLAST序列比对发现均属于链霉菌属。更多还原

【Abstract】 Celloluse is the major component of plant biomass and a potentially utilisable source of glucose, which would cause environmental problems if dose not treat properly. The major bottleneck in conversion technology is the recalcitrance of these rawmaterials. Actinomycetes form an imoportant part of the microbial community responsible for nutrient recycling in natural substrates and the specific contribution to cellulose degradation in the environment have been relatively little studied. As a semi-nature environment which contains rich cellulose biomass, compost is an excellent source for trapping novel cellulose-degrading bacterium especially actinobacteria and their secreted enzymes that more rubst than those from fungi could provide a means to upgrade feasibility for cellulosic biomass conversion, ultimately providing means to a ’greener’technology.In this study, compost samples from different composting periods were collected. The changes of community structure during composting were detected by a conbined molecular microbiology method include real-time quantitative PCR, PCR-DGGE and clone library. The results showed that actinomycetes formed an important part even the dominant group of the pig manure and sawdust compost system which contained abundant cellulose. Bioinformatics analysis of the16S rDNA sequences from the isolates could be assigned to24genus include Saccharomonospora, Micrococcus, Rhodococcus, Nocardia, Corynebacterium, Kocuria, Prauserella, Dietz, George, Brevibacterium, Pseudonocardia, Streptomyces, Propionibacterium and so on. Temperature has a strong effect on the diversity and structure of actinomycetes community in compost. When the pile temperature rose above45℃, actinomycetes flora varied from mesophlic group to thermotolerant flora. Saccharomonospora and George were the dominant groups in the high temperature stage.Aditionally,103actinomycetal strains were isolated from compost and divided into9groups roughly by Spore color. Cellulose degrading ability of the strains were tested by Cellulose Congo red plate and cellulase activity test. The result showed that cellulose-degrading ability are widely distibuted among actinomycetes. Value A (ratio of diameter of hydrolysis cycle and the conoly on congo red plate) of the strains were mesured,9%of which reached7and30%greater than5. The cellulase activity of the crude enzyme from fermentation broth reached0.16U/mLFinally,3strains with the highest CMCase activity were identified on the16S rDNA level, BLAST results for the16S rDNA sequence of strain showed that its closest relatives were members of genera Strepnomycetes.更多还原