堆肥过程中参与C、N代谢部分功能性微生物群落动态的研究

发布时间：2018-02-09 02:37

本文关键词： 堆肥 C代谢循环 N代谢循环 DGGE 分子标记基因　出处：《东北农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：高温好氧堆肥是禽畜类粪便以及各种秸秆等农业固体废弃物的资源化处理方式之一,堆肥过程中微生物利用粪便作为氮源进行氮(N)素代谢,利用秸秆等木质纤维素类物质作为碳源参与碳(C)素代谢。本实验分A、B两组设计牛粪与稻草秸秆的堆垛式高温好氧堆肥,两组处理堆肥初始条件均相同,A组进行自然堆肥,B组添加外源菌剂DN-1进行菌剂堆肥。采用PCR-DGGE技术以部分功能基因(碳代谢循环:β-葡萄糖苷水解酶基因的配糖水解酶1家族GH1、配糖水解酶3细菌家族GH3B、配糖水解酶3真菌家族GH3E;氮代谢循环:氨单加氧酶基因amo A、氧化亚氮还原酶基因nos Z)为分子标记基因,设计参与碳、氮代谢功能性标记基因的通用简并引物来研究堆肥过程中碳、氮代谢功能性微生物群落结构的动态变化。同时测定堆肥过程中温度、全碳、全氮、硝态氮(NO3--N)、铵态氮(NH4+-N)、纤维素降解率、羧甲基纤维素酶(CMC)活性以及β-葡萄糖苷水解酶酶活性的变化,来研究堆肥进程中菌剂及酶活等理化指标与微生物群落结构动态变化的相关性。研究结果如下:1 A组处理0~3d为堆肥的升温期,3~18d为堆肥的高温期,最高温度在堆肥第10d达到59℃,18~28d为堆肥的降温期,28d之后进入堆肥腐熟期。B组处理0~2d为堆肥的升温期,2~22d为堆肥的高温期,最高温度在堆肥第5d达到70℃,22~28d为堆肥的降温期,28d之后为堆肥的腐熟期。A组处理在堆肥第31d全C含量为346.5g/kg,全N含量为18.5g/kg,C/N为19.5,NH4+-N含量为0.28g/kg,NO3--N含量为1.35g/kg,NH4+-N/NO3--N为0.34,纤维素降解率为31.27%。B组处理在堆肥第31d全C含量为360.8g/kg,全N含量为19.4g/kg,C/N为18.5,NH4+-N含量为0.19g/kg,NO3--N含量为1.43g/kg,NH4+-N/NO3--N为0.13,纤维素降解率为62.57%。添加外源菌剂能促进堆体温度升高,提高纤维素的降解速率,促进堆肥更快达到腐熟。2 A、B两种处理CMC酶活性的峰值分别在堆肥第26d和第12d,为12.44U/L和14.81U/L,在堆肥结束时CMC酶活性降到最低,为6.43U/L和6.71U/L。A、B两组β-葡萄糖苷水解酶酶活性分别在堆肥的第20d和12d达到最高,为0.64U/L和0.66U/L,在堆肥的第8d两种处理β-葡萄糖苷水解酶的活性降到最低分别为0.45U/L及0.48U/L。分析β-葡萄糖苷水解酶功能性基因的PCR-DGGE结果,A组含β-葡萄糖苷水解酶功能性基因的微生物多样性高于B组。GH3真菌家族微生物多样性高于GH1家族和GH3细菌家族。堆肥的升温期和高温前期(0~8d)GH1、GH3细菌家族在降解纤维素过程中发挥主要作用,优势菌种是枯草芽孢杆菌属(Bacillus subtilis)、链霉菌属(Streptomyces)、纤维弧菌属(cellvibrio gilvus)。堆肥的中后期,β-葡萄糖苷水解酶活性增强,GH3真菌家族成为主要的功能性群落组成,优势菌种是木霉属(Trichoderma)和黑曲霉属(Aspergillus)。从聚类分析来看,GH1家族B5、B6泳道的相似性达72%,说明B组堆肥高温后期和降温期GH1家族微生物群落结构较为相似。GH3细菌家族泳道A2、A4的相似率为68%,表明A组堆肥高温前期GH3细菌家族微生物群落结构相似性较高。GH3家族菌剂堆肥高温后期泳带B5、B6相似性较高为67%,表明B组堆肥降温期GH3真菌家族微生物群落结构相似性较高。3在本实验堆肥过程中,反硝化细菌微生物的多样性高于硝化细菌,A组微生物多样性高于B组。从聚类分析来看,反硝化细菌B4、B6泳道的相似达到71%,表明反硝化细菌在B组的高温后期和降温期群落组成最为相似。大部分堆肥样品集中在第一主成份的正端,表明A组在堆肥各时期反硝化细菌微生物群落结构较为相似。该堆肥实验中反硝化细菌的优势菌属为假单孢杆菌属(Pseudomonas)。硝化细菌DGGE图谱A6和B1泳道的相似性达到70%,表明硝化细菌在A组的后期和B组的前期群落结构较为相似。硝化细菌在该堆肥中的优势菌属为根瘤菌属(Sinorhizobium fredii)和亚硝化螺菌属(Nitrosospira multiformis)。
[Abstract]:High temperature aerobic composting treatment is one of the resources of livestock and poultry manure and various straw and other agricultural solid waste, composting process using microbial manure as a nitrogen source of nitrogen (N) metabolism, use of straw lignocellulose as the carbon source in the carbon metabolism (C). The experiment was divided into A, stacker high temperature aerobic composting B two group design of cow dung and rice straw compost, two groups of initial conditions are the same, A group of natural compost, B group of inoculant DN-1 microbial compost. Using PCR-DGGE technology to functional genes (glycoside hydrolase carbon Xie Xunhuan: beta glucoside hydrolase gene family GH1 1. Glycosidic hydrolase 3 GH3B glycoside hydrolase family of bacteria, 3 fungi family GH3E; nitrogen metabolism: ammonia monooxygenase gene amo A, Nitrous Oxide NOS Z) reductase gene as molecular marker gene, involved in the design of carbon and nitrogen metabolism. To study the composting process of carbon in general marker genes and Jane primers, the dynamic changes of nitrogen metabolism of the microbial community structure. The simultaneous determination of temperature during composting, total carbon, total nitrogen, nitrate nitrogen (NO3--N), ammonium nitrogen (NH4+-N), the degradation rate of cellulose, carboxymethyl cellulose (CMC) base changes of enzyme activity of grape glycoside hydrolase activity and beta, to study the correlation between agents and enzyme activities in composting process, physicochemical and microbial index dynamic changes of community structure. The results are as follows: 1 the group of A 0~3d for the composting heating period, 3~18d during high-temperature composting, the highest temperature reached 59 degrees in the compost 10d, 18~28d for the cooling period of composting, composting period after entering the 28d group of.B 0~2d for the composting heating period, 2~22d during high-temperature composting, the highest temperature reached 70 degrees in the compost 5D, 22~28d as the cooling period of compost, compost for composting 28d Group.A in total C content 31d 346.5g/kg composting, total N content is 18.5g/kg, C/N is 19.5, the content of NH4+-N was 0.28g/kg, NO3--N was 1.35g/kg, NH4+-N/NO3--N was 0.34, the rate of cellulose degradation for the treatment of 31.27%.B group in the C content of 31d compost was 360.8g/kg, the content of N is 19.4g/kg, C/N is 18.5 the content of NH4+-N, 0.19g/kg, NO3--N content of 1.43g/kg, NH4+-N/NO3--N was 0.13, the rate of cellulose degradation of 62.57%. inoculant can promote the stack temperature, improve the degradation rate of cellulose, promote faster compost reached maturity.2 A B two CMC, the peak of enzyme activity in compost 26D and 12D, 12.44U/L and 14.81U/L, at the end of the compost CMC activity to a minimum, 6.43U/L and 6.71U/L.A, B two groups of beta glucoside hydrolase enzyme activity respectively in the 20d and 12D compost was the highest, 0.64U/L and 0.66U/L, 8D in the compost Two kinds of beta glucosidase enzyme activity decreased to the lowest were 0.45U/L and 0.48U/L. analysis of beta glucosidase enzyme functional gene PCR-DGGE results, A group containing beta glucoside hydrolase genes in microbial diversity of.GH3 fungi was higher than that of B group family of microbial diversity is higher than that of the GH1 family and the GH3 family of bacteria. The compost temperature increasing stage and pre - (0~8d) GH1, GH3 family of bacteria play a major role in cellulose degradation process, dominant bacteria is Bacillus subtilis (Bacillus subtilis), Streptomyces (Streptomyces), cellvibrio (cellvibrio gilvus). In the late period of composting, enhanced beta glucoside hydrolase activity, GH3 the fungi family becoming part of functional community, dominant strains of Trichoderma and Aspergillus niger (Trichoderma) (Aspergillus). From clustering analysis, GH1 family B5, B6 up to 72% Lane similarity, B group during the high temperature and cooling period of late GH1 family of microbial community structure is similar to the.GH3 family of bacteria Lane A2, A4 is similar to the rate of 68%, showed that higher.GH3 family agents during the high temperature band of B5 A was similar to the late high temperature composting prophase GH3 family of bacteria microbial community structure, B6 high similarity was 67%, that in B group during the cooling period of the GH3 family of fungi microbial community structure similarity higher.3 in the process of the composting, microbial diversity of denitrifying bacteria was higher than that of nitrifying bacteria, microbial diversity in A group than B group. From clustering analysis, denitrifying bacteria B4, similar to 71% B6 lanes, indicating that the late denitrifying bacteria in high temperature B group and community composition during cooling is most similar. Most compost samples concentrated in the positive end of the first principal component, A group showed in each period of composting of denitrifying bacteria in the microbial community structure is similar. The dominant bacteria denitrifying bacteria in the composting experiment in genus Pseudomonas (Pseudomonas). The similarity of nitrifying bacteria DGGE map A6 and B1 Lane reached 70%, that the community structure of nitrifying bacteria in the late B group and A group were very similar. The advantages of bacteria nitrifying bacteria in the compost in the genus for the genus Rhizobium (Sinorhizobium fredii) and nitrosospira (Nitrosospira multiformis).

【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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