应用PCR-DGGE分析添加菌剂对堆肥微生物群落多样性的影响

发布时间：2018-01-24 08:18

本文关键词： 菌剂牛粪堆肥微生物群落多样性 PCR-DGGGE　出处：《东北农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：畜禽粪便含有大量的微生物,其中包含病源微生物,直接排放到环境中,会对环境造成污染,集约化、规模化处理粪便最好的处理方法是堆肥化处理,而堆肥原材料中含有大量的难降解的木质纤维素,向堆肥中添加菌剂是解决堆肥中难降解有机物是一种广泛采用的方法。本研究使用木质纤维素类复合菌剂作为外源菌剂进行堆肥,通过测定堆体理化指标、木质纤维素降解率、木质纤维素酶活研究添加外源菌剂对堆肥理化性质及木质纤维素的影响,并使用通用引物进行聚合酶链式反应-梯度变性凝胶电泳(PCR-DGGE)检测堆肥中的细菌、真菌和放线菌,探讨添加菌剂对堆肥微生物群落变化情况,研究结果如下:1.添加外源菌剂和未加菌剂的处理均在第2 d进入高温期(45℃),菌剂处理堆肥组于堆肥第3 d达到最高温72℃,高于对照组堆肥(55℃),且添加菌剂处理的堆体高温期持续21 d(2 d~22d),而对照组堆体仅持续15 d,而且高温期的平均温度菌剂堆肥明显高于对照组;两组堆肥的p H值均维持在7.8~9.0之间,在整个堆肥过程中菌剂处理堆肥的pH值均略低于对照堆肥,此结果显示加入菌剂可使堆肥的p H值降低;两种堆肥处理的含水率均呈下降趋势,堆肥第12 d含水率下降速率最大,堆肥结束后分别降至42%、48%;不同堆肥处理C/N均呈现下降趋势,菌剂堆肥在第31 d C/N达到19.5,率先达到腐熟标准,而对照堆肥在第46 d C/N才达到20以下。因此,添加外源菌剂能够提高堆肥温度,延长高温期时间,并能够促进堆肥腐熟。2.接外源菌剂处理堆肥的木质纤维素酶活在各个时期均高于对照堆肥,其中羧甲基纤维素钠酶(CMCase)、滤纸酶和半纤维素酶最大值分别为1120 U·L-1、268 U·L-1、1681 U·L-1,而对照堆肥的木质纤维素酶活最大值为959 U·L-1、209 U·L-1、1459 U·L-1;且木质纤维素降解速率随着酶活的变化而改变,除木质素外,其他两种木质纤维素降解速率和酶活力随堆肥p H值的减小而增大,当堆肥第12 d时,pH值(7.5)最小时,纤维素和半纤维素的降解速率最大(19.07%,24%)。而经过菌剂处理堆肥组的木质素降解率和木质素降解酶(漆酶)在堆肥过程中出现了双峰值,菌剂对木质纤维素中木质素的降解有强效作用,整体上可以提高木质纤维素降解速率和酶活。3.应用PCR-DGGE技术分析菌剂堆肥过程中细菌、真菌和放线菌群落的变化。结果表明,随着堆肥进程,DGGE条带表现出了明显的动态变化,高温期和腐熟期出现了新的优势条带。对DGGE条带进行UPGMA聚类分析结果表明:微生物群落多样性变化规律为细菌最大,放线菌次之、真菌最小;细菌样品之间的相似性在0.5~0.8之间,真菌样品之间的相似性在0.14~0.84之间,放线菌样品之间的相似性在0.5~0.9。对DGGE条带进行典型分析,提取两因子进行降维的主成成分分析,显示菌剂组高温期的条带与自然组分别聚于不同组,两者有明显差异,综合考虑,温度和添加菌剂对微生物的群落演替起到了重要的限制作用,其对真菌群落演替的筛选作用大于细菌,对细菌筛选作用大于放线菌。对优势条带回收测序的结果表明外源菌剂能够成功定殖,在堆肥高温期起主要优势细菌菌株可分为2类:厚壁菌门(Firmicutes)中的芽孢杆菌菌属(Bacillus)和变形菌门(Proteobacteria)中的α-变形菌纲(α-proteobacteria)、β-变形菌纲(β-proteobacteria)、γ-变形菌纲(γ-proteobacteria);检测到堆肥中真菌主要集中于2类,包括子囊菌门(Ascomycota)和担子菌门(Basidiomycota),其中堆肥高温期起主要优势作用的真菌菌株属于子囊菌门(Ascomycota)的曲霉属(Aspergillus)和短梗蠕孢属(Trichocladium)、担子菌门(Basidiomycota)的中的白腐真菌异担孔菌属(Heterobasidion);堆肥高温期起主要作用的放线菌群落可分为3类:拟诺卡氏放线菌属(Nocardiopsis sp.)、链霉菌属(Streptomyces sp.)和节杆菌属(Arthrobacter sp.)。检测到三类微生物中高温期优势菌株大部分为木质纤维素降解菌,因此,添加外源菌剂能够增加木质纤维素降解菌的数量和种类。
[Abstract]:Animal manure contains a large number of microorganisms, including pathogenic microorganisms, directly discharged into the environment, will cause pollution to environment, intensive, large-scale processing method is the best manure composting, compost and wood fiber raw materials containing refractory large amounts of nutrients, to add compost compost inoculum is solved refractory organics is a widely used method. This study uses lignocellulosic composite agents as exogenous bacteria compost, through the determination of pile physicochemical indexes, lignocellulose degradation rate, study the effects of exogenous microbial agents on physicochemical properties and lignocellulose live lignocellulose enzyme, and the use of universal primers polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) detection in composting of bacteria, fungi and actinomycetes, to investigate the effects of agents on composting microbial community changes, The results are as follows: 1. inoculant and without inoculum were second D into the period of high temperature (45 DEG C), treatment group in third compost compost inoculum D reached the highest temperature 72 degrees Celsius, higher than that of the control group (55 C), and the compost pile to add high temperature period agents processing for 21 d (2 D ~22d), while the control group pile lasted only 15 d, and the average temperature was high temperature composting period was significantly higher than the control group; the two group P compost H value was maintained at 7.8~9.0, in the whole composting process in composting agent treatment the pH value was slightly lower than that of the control compost, this results show that adding agents can make compost P H decreased; two aqueous composting rate decreased, compost twelfth D moisture content decreased the maximum rate, after composting were reduced to 42%, 48%; different composting C/N showed a downward trend in the thirty-first D C/N, microbial compost reached 19.5, has reached maturity and standard. The control was achieved at forty-sixth D C/N compost below 20. Therefore, inoculant can improve the composting temperature, extend the period of high temperature time, and.2. can promote the maturity of the compost treated with exogenous microbial agents compost wood cellulase activity in each period were higher than the control compost, the sodium carboxymethyl cellulose enzyme (CMCase), fpase and half the maximum value of cellulase were 1120 U - L-1268 U - L-11681 U - L-1, while the control compost wood cellulase maximum is 959 U - L-1209 U - L-11459 U - L-1; and lignocellulose degradation rate with the changes of enzyme activity change, in addition to lignin, the other two kinds of lignocellulose degradation rate and the enzyme activity decreases with the increase of P value with compost H, when compost twelfth D, pH value (7.5) of the hour, the cellulose and hemicellulose degradation rate (19.07%, 24%) maximum. After inoculated with compost group lignin degradation rate And the lignin degrading enzymes (Qi Mei) the Shuangfeng values appear in the process of composting inoculants have potent effects on lignin degradation of lignocellulose, the overall analysis could increase the activity of bacteria in composting process.3. the application of PCR-DGGE technology in lignocellulose degradation and enzyme, fungi and Actinomyces community changes. The results showed that and with the composting process, DGGE bands showed obvious dynamic change, high temperature period and maturity period showed an advantage of new belt. UPGMA cluster analysis results show that the DGGE band: the microbial community diversity of bacteria, actinomycetes and fungi bacteria samples between the minimum; the similarity between 0.5~0.8 fungi, the similarity between the samples between 0.14~0.84, put the similarity between actinomycetes samples were typical analysis of DGGE bands in 0.5~0.9. dimension extracted two principal component factor Analysis shows, agents group during the high temperature period bands and natural group were classified into different groups, there was significant difference between two groups, considering the temperature and additive limited effects on microbial community succession plays its role, screening of fungi community succession than bacteria, the bacteria screening effect than actinomycetes the dominant bands sequenced. Results show that exogenous microbial agents can successfully colonize in the high temperature period the main advantage of bacterial strains can be divided into 2 categories: Firmicutes (Firmicutes) in the genus Bacillus (Bacillus) and Proteobacteria (Proteobacteria) in alpha Proteobacteria (alpha -proteobacteria), beta Proteobacteria (beta -proteobacteria), gamma Proteobacteria (gamma -proteobacteria); detection of fungi in composting mainly concentrated in 2 categories, including Ascomycota and Basidiomycota (Ascomycota) (Basidiomycota), the main advantage of the high temperature period The role of fungi belonging to the Ascomycota (Ascomycota) Aspergillus (Aspergillus) and trichocladium (Trichocladium), Basidiomycota (Basidiomycota) of white rot fungi in the genus of bear hole (Heterobasidion); high temperature composting period plays a major role in the actinomycetes communities can be divided into 3 categories: quasi Nocardia Actinomyces (Nocardiopsis sp.), Streptomyces (Streptomyces sp.) and Arthrobacter sp. (Arthrobacter sp.). To detect three kinds of microbes in the high temperature period of dominant strains for the majority of lignocellulose degrading bacteria, therefore, exogenous agents could increase the number and types of lignocellulose degrading bacteria.

【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S141.4

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