本文關(guān)鍵詞： 堆肥 C代謝循環(huán) N代謝循環(huán) DGGE 分子標(biāo)記基因　出處：《東北農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：高溫好氧堆肥是禽畜類糞便以及各種秸稈等農(nóng)業(yè)固體廢棄物的資源化處理方式之一,堆肥過(guò)程中微生物利用糞便作為氮源進(jìn)行氮(N)素代謝,利用秸稈等木質(zhì)纖維素類物質(zhì)作為碳源參與碳(C)素代謝。本實(shí)驗(yàn)分A、B兩組設(shè)計(jì)牛糞與稻草秸稈的堆垛式高溫好氧堆肥,兩組處理堆肥初始條件均相同,A組進(jìn)行自然堆肥,B組添加外源菌劑DN-1進(jìn)行菌劑堆肥。采用PCR-DGGE技術(shù)以部分功能基因(碳代謝循環(huán):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細(xì)菌家族GH3B、配糖水解酶3真菌家族GH3E;氮代謝循環(huán):氨單加氧酶基因amo A、氧化亞氮還原酶基因nos Z)為分子標(biāo)記基因,設(shè)計(jì)參與碳、氮代謝功能性標(biāo)記基因的通用簡(jiǎn)并引物來(lái)研究堆肥過(guò)程中碳、氮代謝功能性微生物群落結(jié)構(gòu)的動(dòng)態(tài)變化。同時(shí)測(cè)定堆肥過(guò)程中溫度、全碳、全氮、硝態(tài)氮(NO3--N)、銨態(tài)氮(NH4+-N)、纖維素降解率、羧甲基纖維素酶(CMC)活性以及β-葡萄糖苷水解酶酶活性的變化,來(lái)研究堆肥進(jìn)程中菌劑及酶活等理化指標(biāo)與微生物群落結(jié)構(gòu)動(dòng)態(tài)變化的相關(guān)性。研究結(jié)果如下:1 A組處理0~3d為堆肥的升溫期,3~18d為堆肥的高溫期,最高溫度在堆肥第10d達(dá)到59℃,18~28d為堆肥的降溫期,28d之后進(jìn)入堆肥腐熟期。B組處理0~2d為堆肥的升溫期,2~22d為堆肥的高溫期,最高溫度在堆肥第5d達(dá)到70℃,22~28d為堆肥的降溫期,28d之后為堆肥的腐熟期。A組處理在堆肥第31d全C含量為346.5g/kg,全N含量為18.5g/kg,C/N為19.5,NH4+-N含量為0.28g/kg,NO3--N含量為1.35g/kg,NH4+-N/NO3--N為0.34,纖維素降解率為31.27%。B組處理在堆肥第31d全C含量為360.8g/kg,全N含量為19.4g/kg,C/N為18.5,NH4+-N含量為0.19g/kg,NO3--N含量為1.43g/kg,NH4+-N/NO3--N為0.13,纖維素降解率為62.57%。添加外源菌劑能促進(jìn)堆體溫度升高,提高纖維素的降解速率,促進(jìn)堆肥更快達(dá)到腐熟。2 A、B兩種處理CMC酶活性的峰值分別在堆肥第26d和第12d,為12.44U/L和14.81U/L,在堆肥結(jié)束時(shí)CMC酶活性降到最低,為6.43U/L和6.71U/L。A、B兩組β-葡萄糖苷水解酶酶活性分別在堆肥的第20d和12d達(dá)到最高,為0.64U/L和0.66U/L,在堆肥的第8d兩種處理β-葡萄糖苷水解酶的活性降到最低分別為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結(jié)果,A組含β-葡萄糖苷水解酶功能性基因的微生物多樣性高于B組。GH3真菌家族微生物多樣性高于GH1家族和GH3細(xì)菌家族。堆肥的升溫期和高溫前期(0~8d)GH1、GH3細(xì)菌家族在降解纖維素過(guò)程中發(fā)揮主要作用,優(yōu)勢(shì)菌種是枯草芽孢桿菌屬(Bacillus subtilis)、鏈霉菌屬(Streptomyces)、纖維弧菌屬(cellvibrio gilvus)。堆肥的中后期,β-葡萄糖苷水解酶活性增強(qiáng),GH3真菌家族成為主要的功能性群落組成,優(yōu)勢(shì)菌種是木霉屬(Trichoderma)和黑曲霉屬(Aspergillus)。從聚類分析來(lái)看,GH1家族B5、B6泳道的相似性達(dá)72%,說(shuō)明B組堆肥高溫后期和降溫期GH1家族微生物群落結(jié)構(gòu)較為相似。GH3細(xì)菌家族泳道A2、A4的相似率為68%,表明A組堆肥高溫前期GH3細(xì)菌家族微生物群落結(jié)構(gòu)相似性較高。GH3家族菌劑堆肥高溫后期泳帶B5、B6相似性較高為67%,表明B組堆肥降溫期GH3真菌家族微生物群落結(jié)構(gòu)相似性較高。3在本實(shí)驗(yàn)堆肥過(guò)程中,反硝化細(xì)菌微生物的多樣性高于硝化細(xì)菌,A組微生物多樣性高于B組。從聚類分析來(lái)看,反硝化細(xì)菌B4、B6泳道的相似達(dá)到71%,表明反硝化細(xì)菌在B組的高溫后期和降溫期群落組成最為相似。大部分堆肥樣品集中在第一主成份的正端,表明A組在堆肥各時(shí)期反硝化細(xì)菌微生物群落結(jié)構(gòu)較為相似。該堆肥實(shí)驗(yàn)中反硝化細(xì)菌的優(yōu)勢(shì)菌屬為假單孢桿菌屬(Pseudomonas)。硝化細(xì)菌DGGE圖譜A6和B1泳道的相似性達(dá)到70%,表明硝化細(xì)菌在A組的后期和B組的前期群落結(jié)構(gòu)較為相似。硝化細(xì)菌在該堆肥中的優(yōu)勢(shì)菌屬為根瘤菌屬(Sinorhizobium fredii)和亞硝化螺菌屬(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).

【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S141.4

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