堆肥中产β-glucosidase微生物群落功能与纤维素降解互作机制的研究

发布时间：2018-04-25 07:20

本文选题：堆肥 + 纤维素降解　；参考：《东北农业大学》2017年硕士论文

【摘要】：堆肥环境能富集一个高效而庞大的降解木质纤维素的微生物群落,对其中参与纤维素降解的功能性微生物的系统性研究一直是环境微生物学的研究热点之一,对揭示全球碳循环及生物质能利用研究具有重要的意义。在堆肥中木质纤维素的降解速率在很大程度上影响着堆肥的腐熟进程。在纤维素的酶解过程中,β-葡萄糖苷水解酶(β-glucosidase)容易受到终产物即葡萄糖的反馈抑制,是酶解途径中影响纤维素降解的限速酶。为了揭示堆肥过程中产β-glucosidase的微生物群落功能和纤维素降解之间的关系,本实验构建了自然堆肥和接种菌剂堆肥,比较不同堆肥中底物纤维素的降解状况,纤维素酶活变化和与其相对应的产β-glucosidase微生物群落生态功能的改变,从而深入地探究复杂堆肥过程中功能性微生物群落结构及功能与纤维素降解之间的关系。实验设置了自然堆肥和接种菌剂DN-1堆肥两组,分别对堆体的温度及环境温度进行测定;对堆肥物料中纤维素、木质素和半纤维素的相对含量进行测定;利用高效液相色谱法(High pressure liquid chromatography,HPLC)测定代谢产物葡萄糖和纤维二糖的含量;测定羧甲基纤维素酶(CMC酶)活性和β-glucosidase活性;采用PCR-DGGE方法分析堆肥中产β-glucosidase基因GH1、GH3家族中微生物群落组成;利用实时荧光定量PCR(Real-time Fluorescent Quantitative PCR,q-PCR)和q-RT-PCR技术,使用设计的通用引物和单一基因的特异性引物,分别在群体及个体水平上检测产β-glucosidase基因GH1、GH3家族中微生物种属功能基因拷贝数的变化以及功能基因的转录表达效率。通过分析自然堆肥与接种菌剂堆肥进程中生产β-glucosidase的功能性微生物群落的演替、优势微生物种属功能基因转录活性的变化规律与β-glucosidase活性变化规律的相关性的差异,进一步揭示含β-glucosidase基因功能性微生物的群落结构和功能与纤维素类物质降解的潜在关系,为揭示堆肥过程中木质纤维素的降解机制提供理论依据。研究结果如下:在堆肥过程中,自然堆肥在第5d进入高温期(45℃),高温期持续13d,堆肥温度在46d后降低至环境温度;接种菌剂堆肥在第3d进入高温期,高温期持续19d,堆肥温度在34d后降低至环境温度。纤维素和半纤维素的降解主要发生堆肥的高温期,在接种菌剂堆肥中尤其明显。在堆肥的高温期,接种菌剂堆肥中的纤维素降解速率,β-glucosidase和CMC酶活性均较自然堆肥中高。自然堆肥进入腐熟期后,仍有20%的可降解纤维素剩余,葡萄糖明显积累(约78.70 mmol/kg),而CMCase和β-glucosidase的活性呈下降趋势。自然堆肥中高浓度的葡萄糖与β-glucosidase活性的降低有关,这可能是葡萄糖附属性的反馈抑制的结果,当葡萄糖累积到一定浓度时可以抑制β-glucosidase的活性,从而限制纤维素降解速率。随着自然堆肥中葡萄糖浓度的增加,GH1细菌家族编码葡萄糖耐受β-glucosidase的基因比例在第22~46 d从56%增加到77.78%。在本实验条件下,GH3真菌家族基因可能比GH1家族基因发生更多的突变以适应高葡萄糖环境。这一现象表明,由于微生物对环境条件的良好的适应性,在足够高的葡萄糖浓度下,功能性微生物群落结构和组成发生改变,使功能性微生物群落能够适应环境而做出相应的变化以维持环境体系中酶的活性。β-glucosidase GH1家族基因在自然堆肥高温期表现较低的基因丰度和高水平的转录效率,但在自然堆肥后期表现较高的基因丰度和较低的转录效率。β-glucosidase GH1家族基因丰度和转录效率在接种菌剂堆肥中与自然堆肥中的结果正相反。β-glucosidase GH3真菌家族基因丰度和转录效率在两组堆肥的高温期均表现为低水平。分析结果发现在接种菌剂堆肥高温后期,GH1家族β-glucosidase基因比GH3真菌家族β-glucosidase基因在β-glucosidase活性和降解纤维素中发挥更重要作用。高β-glucosidase基因丰度低转录效率的功能微生物在纤维素降解中具有比高转录效率低基因丰度的微生物更大的生物学效应。而且纤维二糖作为β-glucosidase的底物能够在某一浓度条件下诱导β-glucosidase编码基因的转录。通过对上述结果的系统分析表明,接种菌剂引起两组堆肥中微生物群落的代谢差异,以及堆肥过程中纤维素的降解效率和功能微生物活性的差异。产β-glucosidase功能性微生物的群落结构和功能与堆肥过程中堆体的温度,纤维素含量以及葡萄糖和纤维二糖浓度等因素密切相关。
[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.

【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S141.4

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