利用PCR-DGGE技术分析病死猪堆肥过程中的微生物群落结构和种类特征

发布时间：2018-02-14 05:39

本文关键词： 病死猪尸体堆肥 PCR-DGGE 多样性微生物发酵　出处：《华中农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：集约化养殖业迅猛发展的同时,也造成来自畜禽粪便、污水以及病死畜禽残体的严重污染。相对于畜禽粪便对环境的污染,死畜禽由于携带大量的病原微生物,如果不及时处理,对人和动物的危害更大更直接。据统计,养殖场的畜禽死亡率在5%~10%之间,而生猪的死亡率在8%~12%之间,遇到重大疫情,死亡率还会升高。因此,对死畜禽进行无害化处理是解决环境污染,实现畜牧业良性发展的必由之路。堆肥是一个可被生物降解的有机质堆,通过好氧微生物分解成稳定的、类似土壤的腐殖质过程。病死猪堆肥相对于传统的死猪处理方法,如,掩埋、焚烧、高温熬煮等,具有更加安全和环保的优势。堆肥过程的物质转化归根到底是由堆肥体系中微生物的代谢活动来完成。因此充分了解病死猪堆肥体系中微生物的菌群种类和动态分布,挖掘病死猪堆肥体系的优势微生物,为开发病死猪堆肥微生物菌剂,促进腐熟进程,缩短堆肥周期奠定基础。本研究通过开展6个月为一个堆肥周期的病死猪尸体自然发酵试验,检测了堆肥过程不同阶段的理化参数,利用PCR-DGGE技术分析了堆肥过程不同阶段的微生物种类,构建了系统树。分离和筛选出5株高温优势菌,并通过生理生化实验和16s r RNA测序对这5株优势菌进行了初步鉴定。主要研究结果如下:(1)实时监测病死猪堆肥不同阶段不同高度的温度变化,对不同阶段的样品含氮量、含水率、p H值、有机质含量、有效活菌数、蛔虫死亡率、重金属含量和类大肠杆菌数理化参数进行测定和分析。堆肥试验中堆体温度的变化经历了典型的升温期、高温期、降温期三个阶段,且堆体上、中、下三层温度的变化趋势基本是一致的,前3个月是有机质降解阶段,微生物活动频繁,中层温度近70℃;p H变化遵循先下降-再升高-再下降的进程趋势。堆肥结束,病死猪尸体软组织全部降解,堆料腐熟,外观成黑色颗粒如腐殖质,依稀可见骨头和牙齿,但这些难降解物质很脆,容易捏碎。氮含量为5.3%左右,有机质含量为53%,大肠杆菌值低于100个/g,蛔虫卵杀灭率接近于100%,有效活菌数接近于0.35亿/g,堆体周围没有活的蛆、蛹或新羽化的成蝇,达到了《粪便无害化卫生标准(GB7959-87)》的规定。(2)提取了微生物基因组总DNA,完成了16s r RNA V6~V8区片段的PCR扩增。通过变形梯度凝胶电泳,得到了18个样品的DGGE图谱。通过Quantity One软件分析DGGE图谱发现,各个时期的细菌丰富度都比较高。前三个月的细菌的分布比较凌乱,后三个月的细菌分布趋于稳定。致病菌只在前三个月存在,后三个月致病菌被杀死。分别对切胶回收后的45条优势条带测序,并与已知序列在NCBI上进行同源性比对,得到了这45条序列的详细结果并完成了它们的系统发育分析。结果发现,堆肥过程中存在大量未知细菌,它们在堆肥过程中的作用还有待探究。堆肥过程的优势菌为枯草芽孢杆菌(Bacillus subtillis)、地衣芽孢杆菌(Bacillus lincheniformis)、枝芽孢杆菌(Virgibacillus)和苏云金芽孢杆菌(Bacillus thuringiensis)。(3)传统培养并结合高温处理,分离获得了5株优势菌。通过生理生化鉴定、显微镜形态观察和16s r RNA测序,结果显示,J1为未知菌与已知的Uncultured bacterium clone(JF155193)相似率为98%,J2、J3和J5为Bacillus subtillis strain,J4为Bacillus thuringiensis strain。产淀粉酶酶活性由强至弱依次为J4,J1,J2,J3,J5,产蛋白酶活性由强至弱依次为J1,J5,J2,J4,J3,产脂肪酶活性由强至弱依次为J3,J2,J5,其中J1和J4不产生脂肪酶。
[Abstract]:At the same time, the rapid development of intensive livestock farming, also caused from livestock manure, sewage and serious pollution of dead livestock and poultry residues. Compared with the pollution of livestock and poultry manure on the environment, dead livestock because carrying a lot of pathogens, if not timely treatment of human and animal harm more directly. According to statistics, farms livestock and poultry mortality between 5%~10% and mortality in pig 8%~12%, encountered a major epidemic, the mortality rate is also increased. Therefore, for the harmless treatment of dead livestock is to solve the environmental pollution, the only way which must be passed to achieve healthy development of animal husbandry. Composting is a organic matter biodegradation reactor, through aerobic microbial decomposition into stable the process is similar, humus soil. Compared with the dead pigs composting treatment methods, such as traditional, buried, burning, high-temperature boiling, with more safety and environmental protection. The advantages of composting The process of material transformation after all is by the metabolic activity of microorganisms in composting system to complete. Therefore fully understand microbial dead pig composting system bacteria species and the dynamic distribution of mining microorganisms pigs composting system, for the development of a dead pig compost microorganism, promote the composting process, lay the foundation for the study by shorten the composting period. Carry out 6 months for a composting cycle dead pig bodies test natural fermentation, detect the physicochemical parameters of different stages of the composting process, analysis of microbial species in different stages of the composting process by using PCR-DGGE technology, constructed a phylogenetic tree. Isolated and screened 5 strains of dominant bacteria in high temperature, and through physiological and biochemical tests and 16S R RNA sequencing of the 5 bacterial strains were identified. The main results are as follows: (1) temperature real-time monitoring of pigs in different composting stages In the different stages of the sample, nitrogen content, water content, P, H value, organic matter content, the effective number of living bacteria, Ascaris mortality, heavy metal content and coliform mathematical parameters were determined and analyzed. The changes of composting pile temperature experienced a typical heating period, high temperature period and cooling period in three stages, and the pile body, and the change trend of three layer temperature is basically the same, 3 months before the stage of organic matter degradation, microbial activity, middle temperature near 70 DEG C; P H changes follow the first drop process trend of increase decrease. The end of compost, mortality pig carcasses all soft tissue degradation, compost maturity, the appearance of black particles such as humus, faintly visible bones and teeth, but these refractory materials are brittle, easy to crush. The nitrogen content is about 5.3%, the content of organic matter was 53%, the Escherichia coli value below 100 / g, the killing rate of Ascaris eggs close to 100%, The effective number of living bacteria close to 35 million /g, no live maggot fly around the pile, or the newly emerged pupae, reached "manure harmless sanitary standard (GB7959-87) > rules. (2) the extraction of microbial genomic DNA, 16S R RNA completed the V6~V8 fragment by PCR amplification. The shape gradient gel electrophoresis, DGGE spectra of 18 samples were obtained by Quantity One software. DGGE atlas analysis showed that bacterial richness in each period are relatively high. The distribution of the first three months of the bacteria is messy, three months after the bacterial distribution tends to be stable. The pathogenic bacteria exist only in the first three months after. Three months of pathogens were killed. On gel cut after the 45 dominant bands of sequencing, homology and NCBI with known sequences, obtained the detailed results of the 45 sequences and the phylogenetic analysis of their systems. The results showed that there are large in the composting process The amount of unknown bacteria, their role in the composting process remains to be explored. The dominant bacteria during composting for Bacillus subtilis (Bacillus subtillis), Bacillus licheniformis (Bacillus lincheniformis), branch (Virgibacillus) and Bacillus thuringiensis (Bacillus thuringiensis). (3) the traditional culture combined with high temperature treatment isolated 5 strains of dominant bacteria. Through physiological and biochemical identification, microscopic observation and 16S R RNA sequencing results show that J1 is an unknown bacteria with known Uncultured bacterium clone (JF155193) similar rate was 98%, J2, J3 and J5 Bacillus subtillis strain J4 Bacillus thuringiensis strain., amylase enzyme activity is strong to the weak are J4, J1, J2, J3, J5, protease activity egories as J1, J5, J2, J4, J3, lipase activity from high to low according to the times of J3, J2, J5, J1 and J4 do not produce lipase.

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

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