本文選題：堆肥 + 纖維素降解��； 參考：《東北農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：堆肥環(huán)境能富集一個(gè)高效而龐大的降解木質(zhì)纖維素的微生物群落,對(duì)其中參與纖維素降解的功能性微生物的系統(tǒng)性研究一直是環(huán)境微生物學(xué)的研究熱點(diǎn)之一,對(duì)揭示全球碳循環(huán)及生物質(zhì)能利用研究具有重要的意義。在堆肥中木質(zhì)纖維素的降解速率在很大程度上影響著堆肥的腐熟進(jìn)程。在纖維素的酶解過(guò)程中,β-葡萄糖苷水解酶(β-glucosidase)容易受到終產(chǎn)物即葡萄糖的反饋抑制,是酶解途徑中影響纖維素降解的限速酶。為了揭示堆肥過(guò)程中產(chǎn)β-glucosidase的微生物群落功能和纖維素降解之間的關(guān)系,本實(shí)驗(yàn)構(gòu)建了自然堆肥和接種菌劑堆肥,比較不同堆肥中底物纖維素的降解狀況,纖維素酶活變化和與其相對(duì)應(yīng)的產(chǎn)β-glucosidase微生物群落生態(tài)功能的改變,從而深入地探究復(fù)雜堆肥過(guò)程中功能性微生物群落結(jié)構(gòu)及功能與纖維素降解之間的關(guān)系。實(shí)驗(yàn)設(shè)置了自然堆肥和接種菌劑DN-1堆肥兩組,分別對(duì)堆體的溫度及環(huán)境溫度進(jìn)行測(cè)定;對(duì)堆肥物料中纖維素、木質(zhì)素和半纖維素的相對(duì)含量進(jìn)行測(cè)定;利用高效液相色譜法(High pressure liquid chromatography,HPLC)測(cè)定代謝產(chǎn)物葡萄糖和纖維二糖的含量;測(cè)定羧甲基纖維素酶(CMC酶)活性和β-glucosidase活性;采用PCR-DGGE方法分析堆肥中產(chǎn)β-glucosidase基因GH1、GH3家族中微生物群落組成;利用實(shí)時(shí)熒光定量PCR(Real-time Fluorescent Quantitative PCR,q-PCR)和q-RT-PCR技術(shù),使用設(shè)計(jì)的通用引物和單一基因的特異性引物,分別在群體及個(gè)體水平上檢測(cè)產(chǎn)β-glucosidase基因GH1、GH3家族中微生物種屬功能基因拷貝數(shù)的變化以及功能基因的轉(zhuǎn)錄表達(dá)效率。通過(guò)分析自然堆肥與接種菌劑堆肥進(jìn)程中生產(chǎn)β-glucosidase的功能性微生物群落的演替、優(yōu)勢(shì)微生物種屬功能基因轉(zhuǎn)錄活性的變化規(guī)律與β-glucosidase活性變化規(guī)律的相關(guān)性的差異,進(jìn)一步揭示含β-glucosidase基因功能性微生物的群落結(jié)構(gòu)和功能與纖維素類物質(zhì)降解的潛在關(guān)系,為揭示堆肥過(guò)程中木質(zhì)纖維素的降解機(jī)制提供理論依據(jù)。研究結(jié)果如下:在堆肥過(guò)程中,自然堆肥在第5d進(jìn)入高溫期(45℃),高溫期持續(xù)13d,堆肥溫度在46d后降低至環(huán)境溫度;接種菌劑堆肥在第3d進(jìn)入高溫期,高溫期持續(xù)19d,堆肥溫度在34d后降低至環(huán)境溫度。纖維素和半纖維素的降解主要發(fā)生堆肥的高溫期,在接種菌劑堆肥中尤其明顯。在堆肥的高溫期,接種菌劑堆肥中的纖維素降解速率,β-glucosidase和CMC酶活性均較自然堆肥中高。自然堆肥進(jìn)入腐熟期后,仍有20%的可降解纖維素剩余,葡萄糖明顯積累(約78.70 mmol/kg),而CMCase和β-glucosidase的活性呈下降趨勢(shì)。自然堆肥中高濃度的葡萄糖與β-glucosidase活性的降低有關(guān),這可能是葡萄糖附屬性的反饋抑制的結(jié)果,當(dāng)葡萄糖累積到一定濃度時(shí)可以抑制β-glucosidase的活性,從而限制纖維素降解速率。隨著自然堆肥中葡萄糖濃度的增加,GH1細(xì)菌家族編碼葡萄糖耐受β-glucosidase的基因比例在第22~46 d從56%增加到77.78%。在本實(shí)驗(yàn)條件下,GH3真菌家族基因可能比GH1家族基因發(fā)生更多的突變以適應(yīng)高葡萄糖環(huán)境。這一現(xiàn)象表明,由于微生物對(duì)環(huán)境條件的良好的適應(yīng)性,在足夠高的葡萄糖濃度下,功能性微生物群落結(jié)構(gòu)和組成發(fā)生改變,使功能性微生物群落能夠適應(yīng)環(huán)境而做出相應(yīng)的變化以維持環(huán)境體系中酶的活性。β-glucosidase GH1家族基因在自然堆肥高溫期表現(xiàn)較低的基因豐度和高水平的轉(zhuǎn)錄效率,但在自然堆肥后期表現(xiàn)較高的基因豐度和較低的轉(zhuǎn)錄效率。β-glucosidase GH1家族基因豐度和轉(zhuǎn)錄效率在接種菌劑堆肥中與自然堆肥中的結(jié)果正相反。β-glucosidase GH3真菌家族基因豐度和轉(zhuǎn)錄效率在兩組堆肥的高溫期均表現(xiàn)為低水平。分析結(jié)果發(fā)現(xiàn)在接種菌劑堆肥高溫后期,GH1家族β-glucosidase基因比GH3真菌家族β-glucosidase基因在β-glucosidase活性和降解纖維素中發(fā)揮更重要作用。高β-glucosidase基因豐度低轉(zhuǎn)錄效率的功能微生物在纖維素降解中具有比高轉(zhuǎn)錄效率低基因豐度的微生物更大的生物學(xué)效應(yīng)。而且纖維二糖作為β-glucosidase的底物能夠在某一濃度條件下誘導(dǎo)β-glucosidase編碼基因的轉(zhuǎn)錄。通過(guò)對(duì)上述結(jié)果的系統(tǒng)分析表明,接種菌劑引起兩組堆肥中微生物群落的代謝差異,以及堆肥過(guò)程中纖維素的降解效率和功能微生物活性的差異。產(chǎn)β-glucosidase功能性微生物的群落結(jié)構(gòu)和功能與堆肥過(guò)程中堆體的溫度,纖維素含量以及葡萄糖和纖維二糖濃度等因素密切相關(guān)。
[Abstract]:The composting environment can enrich a highly efficient and huge microbial community to degrade lignocellulose. The systematic study of functional microorganisms involved in cellulose degradation has been one of the hotspots of environmental microbiology. It is of great significance to reveal the global carbon cycle and the utilization of biomass in the composting. In the enzymatic hydrolysis process of cellulose, beta glucoside hydrolase (beta -glucosidase) is susceptible to the feedback inhibition of the final product, that is, glucose, and is the speed limiting enzyme that affects the degradation of cellulose in enzymatic hydrolysis. It is a microorganism to reveal the middle class beta -glucosidase in the composting process. The relationship between community function and cellulose degradation, this experiment constructed natural compost and inoculant compost, compared the degradation of substrate cellulose in different composts, the change of cellulase activity and its corresponding ecological function of producing beta -glucosidase microbial community, so as to further explore the function of complex composting process. The relationship between microbial community structure and function and cellulose degradation. Two groups of natural compost and inoculant DN-1 composting were set up to determine the temperature and ambient temperature of the heap; the relative content of cellulose, lignin and hemicellulose in the compost materials was measured; High pressure Li (high performance liquid chromatography) was used. Quid chromatography, HPLC) determine the content of glucose and two sugar of metabolites; determine the activity of carboxymethyl cellulase (CMC enzyme) and beta -glucosidase activity; use PCR-DGGE to analyze the beta -glucosidase gene GH1 in the compost and the microbial community in the GH3 family; and use real time fluorescent quantitative PCR (Real-time Fluorescent) E PCR, q-PCR) and q-RT-PCR technology, using the designed universal primers and the specific primers of a single gene, to detect the production of beta -glucosidase gene GH1, the changes in the copy number of the functional genes of the microbial species in the GH3 family and the efficiency of the transcriptional table of the functional genes at the population and individual levels, respectively, and by analyzing the compost of natural compost and inoculant inoculant. The succession of functional microbial communities producing beta -glucosidase, the variation of functional gene transcriptional activity of dominant microbes and the variation of beta -glucosidase activity, and further reveal the potential of the community structure and function of functional microorganisms containing beta -glucosidase gene and the potential of cellulose degradation. It provides a theoretical basis for revealing the degradation mechanism of lignocellulose in the process of composting. The results are as follows: during the composting process, the natural compost enters the high temperature period (45 C) at 5D, and keeps 13D at high temperature. The composting temperature is reduced to the ambient temperature after 46d; the inoculant composting fertilizer enters the high temperature period in 3D, the high temperature period continues 19d, and the composting temperature is at the temperature. 34d decreased to ambient temperature. The degradation of cellulose and hemicellulose mainly occurred in the high temperature period of compost, especially in the inoculant compost. In the high temperature period of the composting, the cellulose degradation rate in the inoculant compost, the activity of beta -glucosidase and CMC enzyme were higher than that in the natural composting. After the natural compost entered the decayed period, it still had 20% The glucose accumulation (about 78.70 mmol/kg) was obvious and the activity of CMCase and beta -glucosidase decreased. The high concentration of glucose in the natural composting was related to the decrease of beta -glucosidase activity, which may be the result of the feedback inhibition of the glucose attachment properties, which could inhibit the beta -gluco when the glucose was accumulated to a certain concentration. With the increase of glucose concentration in natural compost, the proportion of GH1 bacteria family encoding glucose tolerance beta -glucosidase increased from 56% to 77.78%. from 56% to 77.78%. in this experimental condition, and the GH3 fungus family gene may have more mutations than GH1 family genes to adapt to the high Portuguese, as the glucose concentration in natural compost increases. This phenomenon shows that, due to the good adaptability of microbes to environmental conditions, the structure and composition of functional microbial communities are changed at high enough glucose concentration, and the functional microbial communities can adapt to the environment and make corresponding changes in the activities of enzymes in the environmental system. Beta -glucosidase GH1 family The gene abundance and high level of transcriptional efficiency were low in the high temperature period of natural compost, but higher gene abundance and lower transcriptional efficiency were shown in the late stage of natural composting. The gene abundance and transcription efficiency of beta -glucosidase GH1 family were opposite to the result of natural composting. Beta -glucosidase GH3 was true The gene abundance and transcriptional efficiency of the bacteria family were low in the high temperature period of the two groups. The results showed that the GH1 family beta -glucosidase gene was more important than the GH3 fungus family beta -glucosidase gene in the beta -glucosidase activity and cellulose degradation in the late period of high temperature inoculation. The abundance of high beta -glucosidase gene was more important. The functional microorganisms with low transcriptional efficiency have greater biological effects in the degradation of cellulose than those with high transcriptional efficiency and low gene abundance. And the fiber two sugar, as a substrate of beta -glucosidase, can induce the transcription of the beta -glucosidase encoding gene at a certain concentration. The metabolic difference of microbial community in two groups of compost was caused by the bacteria, and the difference of cellulose degradation efficiency and functional microbial activity during the composting process. The community structure and function of producing beta -glucosidase functional microorganisms were closely related to the temperature of the heap, the content of cellulose and the concentration of glucose and fiber two sugar during the composting process. Close.

【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】：2017
【分類號(hào)】：S141.4

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