【作者】 喻曼；

【导师】 曾光明；

【作者基本信息】 湖南大学 ， 环境科学与工程， 2009， 博士

【摘要】 针对堆肥化环境微生物的复杂性以及木质纤维素难降解的特性,本文以农业秸秆为主要原料,采用好氧堆肥,从物理化学、酶学以及基于非培养生物技术的微生物群落结构演替等方面较系统地研究了接种黄孢原毛平革菌对堆肥发酵进程和木质纤维素降解的影响;并综合运用多种非培养生物研究方法与数学统计方法系统地研究堆肥化过程中微生物群落演替规律;基于上述规律研究其降解机理与特性并从堆肥化各阶段选育高效优势的降解菌株,为堆肥工艺的优化和接种剂的开发提供依据。在稻草固态发酵体系中同时接种土壤微生物和黄孢原毛平革菌,用磷脂脂肪酸(PLFA)谱图分析法研究发酵过程的微生物群落和生物量变化,同时监测木质纤维素降解率的变化。结果表明,复合微生物体系降解木质纤维素的效率明显高于单一菌种的效率。根据标志性脂肪酸的变化,在发酵第6d,革兰氏阳性菌、革兰氏阴性菌、真菌的含量都达到了最高值,其中,革兰氏阳性菌的含量较低;真菌和细菌的脂肪酸含量比值变化范围为0.2~0.5,说明真菌是降解木质纤维素的主要群落。主成分分析结果显示发酵后期以18C不饱和脂肪酸为主,与标志性脂肪酸分析结果一致,同时跟木质纤维素降解率的变化趋势对应,因此PLFA分析法可以较好地反映稻草固态发酵过程中的微生物群落结构和生物量的变化。P. chrysosporium对应单体PLFA与木质纤维素相关性分析显示革兰氏阳性菌和真菌在降解木质素上具有相同的效率,P. chrysosporium是降解木质素的主要真菌。用RFLP方法和酶活检测方法研究了在农业废物堆肥一次发酵和二次发酵期间添加黄孢原毛平革菌对微生物多样性和木质纤维素降解酶系的影响。结果表明,在不同发酵期接种P. chrysosporium对堆肥进程的影响不同:(1)三种典型的限制性内切酶Alu I、Hae III和Taq I在分析堆肥细菌微生物多样性的灵敏性上,Hae III效果最好,Alu I次之,Taq I则不是很适用于分析堆肥细菌微生物多样性。添加的P. chrysosporium对堆肥系统中某些微生物更具有一定的选择作用,使其成为优势物种以促进堆肥物质的降解,多样性有所降低,二次接种能够巩固一次接种效果;(2)接种P. chrysosporium能够促进堆肥体系中的木质纤维素降解,实验结果显示接种P. chrysosporium的堆体中木质素降解率要明显高于未接种的堆体。接种P. chrysosporium对木聚糖酶活和CMC酶活的分泌没有明显影响,对Lip和Mnp酶活产生有促进作用,但是对Lac的产生有一定抑制作用。组合应用了三类非培养生物技术,即PLFA法、RFLP法、DGGE法,分析农业废物堆肥化中微生物群落和多样性的变化。结果表明三类方法显示的信息并不完全一致:(1)PLFA法检测的样点数据PCA分布比较离散,说明每个样点都具有其特征图谱,样点选取具有代表性。三类方法中以PLFA数据指示的微生物多样性最高,同时PLFA数据可以表征较完整的微生物群落结构变化并较为明确的表征微生物生物量的变化。(2)RFLP法检测的样点和条带信息PCA分布都比较集中,数据还显示在堆肥后期的细菌群落具有不稳定性,RFLP法指示的微生物多样性较低;(3)DGGE方法检测的数据显示堆肥过程中细菌群落结构至少经历了三个阶段的演替,即嗜温细菌群落,高温细菌群落和腐熟期细菌群落。根据从农林废物堆肥中筛选得到的木质素降解优势土著微生物枯草芽孢杆菌、铜绿假单孢菌、黑曲霉、简青霉、栗褐链霉菌,依据PLFA-PLS定量分析所得堆肥化二次发酵期有效的木质素降解微生物群落组成比例混合接种至稻草基质发酵瓶中,并通过正交实验L9(34)优化混合比例,以期研究开发一种基于木质素降解的高效堆肥化接种剂。结果表明,混合菌剂具有较强的木质素降解能力,其对木质素降解是木质素过氧化物酶、锰过氧化物酶、漆酶、纤维素酶和半纤维素酶共同作用的结果;当细菌:放线菌:真菌个数比为85:5:10,枯草芽孢杆菌:铜绿假单孢菌为55:25,黑曲霉:简青霉为2:1时,木质素、纤维素、半纤维素降解率最高,分别达到22.13%、48.97%、55.93%。比空白样高出19.16%、38.25%、46.30%。更多还原

【Abstract】 Since microorganism, and lignocellulose, which is difficult to be degraded, are of great importance of technique improvement and efficiency enhancement during agricultural waste composting. The relationships between the succession of microbial community and degradation of lignocellulose were studied by analyzing in physico-chemical properties, enzymology, microbial community based on culture-independent method during agricultural waste composting. Kinds of culture-independent methods and statistical method were synthetically used for analyzing microbial community. Based on the characteristic and rules in composting, preponderant microorganisms selected in different stage during composting were developed to compound microorganism.To investigate the changes of microbial communities and influence of Phanerochaete chrysosporium during solid-state fermentation (SSF) of rice straw, phospholipid fatty acids (PLFA) and lignocellulose components were measured with periodical sampling. The results showed that the lignocellulose degrading ratios in SSF which was inoculated by P. chrysosporium and soil microorganisms were higher than those degraded by culturing a single species. The total amount of PLFAs, as an indicator of microbial biomass, reached the peak on day 6. Principal component analysis (PCA) of the PLFA compositions revealed that P. chrysosporium was well responsible for the succession of microbial community and showed that fungi were the predominant species at the end of the process. The correlation analysis between lignocellulose degrading ratio and PLFA profile in P. chrysosporium suggested that P. chrysosporium promoted lignin degrading as the main fungi with gram-positive bacteria.The lignocellulolytic microorganism, P. chrysosporium, was inoculated during different phases of agricultural waste composting and the effects on the diversity of microbial community and the enzyme activities were observed. The results of the present investigation revealed that inoculation during different phases had different effects on these parameters. (1) Three kinds of restriction enzymes, Alu I, Hae III and Taq I were used to analyse the change of microbial community diversity. The results showed that restriction enzyme Hae III was the most sensitive to the change of microbial community, restriction enzyme Alu I the second, and restriction enzyme Taq I the last. Restriction enzyme Taq I was not much suitable for analyzing composting samples. P. chrysosporium inoculated to composting system acted on certain microorganisms selectively and made them be the dominant species in order to promote the degradation of compost materials. However, the diversity of microorganism decreased after the first inoculation, the inoculation in the second fermentation was able to advance the effects of the inoculation in the first fermentation; (2) Inoculating P. Chrysosporium to composting did not induce much apparent changes on xylanase and CMCase activities. Whereas, P. Chrysosporium had significant stimulative effect on the secreting of MnP, LiP and certain prohibitive influence on the secreting of Lac when inoclated during the second fermentation phase.Three types of non-cultivate biology techniques, PLFA method, RFLP method and DGGE method were synthetically applicated to analyze changes of microbial community diversity during agricultural waste composting. The results showed that the three types of methods did not reveal the same relative similarities of composting samples based on PCA analysis of the biological characteristics entirely. (1) Data from PLFA profile showed discrete, it suggested each sample had its characteristic pattern, samples selected were representative. Microbial diversity of PLFA data was the highest among three types of methods. And data from PLFA profile was comprehensive to characterization of microbial community structure changes and more specific to characterization of microbial biomass changes. (2) Data from RFLP profile was more concentrated. The data also showed that bacteria community was instable at later stage during composting. And diversity of microbial community shown by RFLP method was lower; (3) Data from DGGE analysis showed that the succession of the microbial community included at least three different stages: mesophilic microbial community, thermophilic microbial community and maturation microbial community. The difference indicated that observing biological characteristics in composting with just one method should not be considered absolute, since the use of another method may lead to another interpretation of the relative composting similarities.Five strains of microorganisms were isolated from of agricultural waste composting. They were identified to be Bacillus subtilis, Pseudom onas aeruginosa, Aspergillusniger, Penicillium sim plicissim, and S treptom yces badius. To develop a high-efficient microbial inocula for composting, these strains of different ratios designed by orthogonal test L9 (34) were inoculated to natural rice straw during solid-state fermentation for 30 days. The strain mixture showed ligninolytic ability, which was further proved by enzyme secreting. When the ratio of bacteria:actinomycete:fungus was 85:5:10; B acillussubtilis:Pseudom onas aeruginosa, 55:25; and Aspergillus niger:Penicillium simplicissim, 2:1, the degradation rate of lignin, cellulose and hemicellulose was the highest, being 22.13%, 48.97% and 55.93%, respectively, after 30 day incubation. It was increased 19.16, 38.25 and 46.30 percent points, respectively, compared with the control.更多还原