整合宏组学方法揭示天然木质纤维素堆肥中的关键功能微生物群落
发布时间：2018-07-14 20:07
【摘要】：中国作为农牧业大国,每年会产生大量的农牧业废弃物。由于缺乏快速高效的处理方式,目前最常见的农业废弃物处理方式是堆积或焚烧,这会导致产生严重的土壤、水体和空气污染。此外,由于连年大规模集约化种植和养殖,同时缺乏轮作休耕制度,土壤肥力被严重透支,水土流失现象也日益严重。为了解决目前农牧业造成的环境问题,迫切需要一种合理环保的方式来针对性地处理农牧业废弃物,以实现绿色农业的可持续发展。堆肥工艺可以实现有机质的绿色转化,并且转化的产物可以成为优质的生物有机肥料,因此是无害化和资源化处理农牧业废弃物的最有前景的一种方式。堆肥过程主要由其中的微生物群落所驱动。堆肥可以完成不同有机质如农作物秸秆和畜禽粪便的生物转化,而对这些材料的降解需要多种微生物共同协作完成。但是迄今为止尚未有对堆肥微生物群落比较系统和全面的研究。针对这一问题,本文以天然农牧业废弃物堆制的堆肥为研究对象开展了系列研究,取得的主要成果如下：1.采用宏组学技术成功跟踪定位了天然玉米秸秆堆肥的微生物群落,确定了关键微生物群落的功能使用包括宏基因组学和宏蛋白组学在内的整合宏组学技术,跟踪了天然玉米秸秆堆肥中微生物的群落组成与结构随时空条件的动态变化,并对涉及木质纤维素降解的主导微生物群落的功能进行了鉴定。研究结果表明,在堆肥堆制过程中,天然玉米秸秆堆肥中形成了一个稳定的微生物群落结构,主要由嗜热丝孢菌属、喜热裂孢菌属和嗜热多孢菌属等微生物组成,并且这些菌属也是主导秸秆木质纤维素降解的功能微生物。其中真菌Thermomyces lanuginosus是堆肥中的主要半纤维素降解菌,细菌Thermobifida fusca是主要的纤维素降解菌。本研究采用的整合宏组学方法可以将微生物群落组成与功能的研究相结合,从而实现对堆肥生境关键功能微生物群落的准确定位。本项研究不仅从理论上对秸秆堆肥中微生物群落的结构功能与生态过程进行了研究,对丰富秸秆堆肥理论有重要意义,而且在该项研究中使用的整合宏组学方法也可以应用在其它生境的微生物群落研究中,具有广泛的普适性。2.对传统堆肥工艺进行了技术改造,成功实现了90 m3大型发酵罐农牧业废弃物堆肥的工业化应用使用工业规模的90m3大型发酵罐进行了高度达3.3 m的玉米芯牛粪混合堆肥发酵,借助宏组学技术对发酵罐堆肥的关键功能微生物群落进行了研究。实验表明通过发酵罐堆肥,可以将发酵周期从天然堆肥的42天缩短至11天,大大提高了堆肥发酵效率。此外,宏组学结果表明发酵罐堆肥中微生物群落随时空条件的变化而发生明显的群落演替现象。在发酵罐堆肥中,主导的微生物群落主要由细菌的喜热裂孢菌属、芽孢杆菌属等细菌属和真菌的嗜热丝孢菌属和曲霉属等真菌属所组成。对菌群的功能分析表明,喜热裂孢菌属是发酵罐堆肥中主要的纤维素降解者。本项研究成功进行了传统堆肥工艺的技术改造,实现了堆肥工艺技术的现代化,为农业废弃物的堆肥转化提供了一个成功的范例,极具工业化推广的价值,这对于实现微生物菌剂的复配利用也具有重要的指导意义。3.运用宏组学技术研究分析了小麦秸秆堆肥的微生物动态变化,证明由于原料中组分与结构的差异造成了小麦秸秆的堆肥效率低于玉米秸秆堆肥在天然玉米秸秆堆肥实验研究的基础上,使用整合宏组学技术跟踪调查了小麦秸秆堆肥微生物群落随时间的动态变化。发现相比于玉米秸秆堆肥,小麦秸秆堆肥中的微生物群落演替现象只有小幅变化,纤维素降解微生物未能形成主导的优势功能群落,因此纤维素底物降解效率低。检测发现小麦秸秆堆肥的内部温度低于60℃,因此也未能达到底物的无害化处理,使得富含动植物致病菌的拟杆菌门和变形菌门在整个堆肥过程都是主导的微生物。相比于可溶性糖分含量较高的玉米秸秆,小麦秸秆中具有更高的结构异质性,更高的硅含量和木质纤维素含量,这都导致相比之于玉米秸秆,小麦秸秆为底物时的堆肥降解效率明显低于玉米秸秆堆肥。4.对鸡粪堆肥中的微生物群落变化进行了宏组学分析,显示芽孢杆菌属是鸡粪堆肥中主导的功能微生物鸡粪作为一种高氮源和低含水率的畜禽粪便,非常适合堆肥工艺。采用肉鸡养殖场的鸡粪进行天然堆肥,通过宏组学技术对鸡粪堆肥中微生物群落随时空的动态变化进行了研究分析。研究发现鸡粪堆肥中微生物群落组成随时空变化发生了明显的群落演替现象。鸡粪堆肥中的微生物以细菌的厚壁菌门为主导,尤以属于该门的芽孢杆菌属为主要菌属。Native电泳蛋白酶谱分析也显示芽孢杆菌属细菌的蛋白酶为主要的酶谱条带,因此芽孢杆菌属是该类鸡粪堆肥中的主要功能菌属。该研究也显示不同的堆肥底物对堆肥微生物群落有显著的影响。该研究不仅在理论上对畜禽粪便堆肥中微生物群落的演替变化进行了分析,也对实现畜禽粪便的快速堆肥处理具有重要的现实指导意义。5.对堆肥主要降解真菌T. lanuginosus的表达谱进行了分析,证明天然复杂底物对该菌木聚糖酶的表达具有更高效的诱导作用T. lanuginosus是天然玉米秸秆堆肥中的主要半纤维素降解真菌,在本研究中对其胞外表达谱进行了分析。研究结果表明,在液体发酵时该菌可以利用木糖为底物进行快速生长,木糖和木聚糖都是木聚糖酶的诱导剂。相比于简单底物的液体培养,在进行固体发酵时,天然复杂底物更适合该菌的生长,而且玉米秸秆粉和小麦麸皮都能以更高的效率诱导该菌木聚糖酶的高效表达,说明天然底物是该菌木聚糖酶的优良诱导剂。这些结果为进一步研究T. lanuginosus在堆肥中的快速生长以及在天然纤维素的降解过程中的作用机制与功能提供了理论指导。
[Abstract]:As a big agriculture and animal husbandry country, China produces a large number of agricultural and animal husbandry wastes every year. Due to the lack of rapid and efficient treatment, the most common methods of disposal of agricultural waste are accumulation or incineration, which will lead to serious soil, water and air pollution. In addition, because of the large-scale intensive cultivation and cultivation, and the lack of wheels at the same time. In order to solve the environmental problems caused by agriculture and animal husbandry, a reasonable and environmentally friendly way is urgently needed to deal with the agricultural and animal husbandry wastes so as to realize the sustainable development of green agriculture. And the transformed product can be a high quality bio organic fertilizer, so it is the most promising way for the harmless and resourceful treatment of agricultural and animal husbandry waste. The composting process is mainly driven by the microbial community in which the composting can complete the biological transformation of different organic matter such as crop straw and livestock manure. The degradation needs a variety of microbes to work together. But so far, there has not been a systematic and comprehensive study of the microbial community of compost. In this paper, a series of studies have been carried out with the composting of the natural agricultural and animal husbandry waste heap as the research object. The main achievements are as follows: 1. the success of macroomics technology is followed. The microbial community of natural maize straw composting was located, and the function of key microbial communities was determined by integrated macroomics technology including macrogenomics and macroproteomics. The dynamic changes of microbial community composition and structure in natural maize straw composting were tracked and lignocellulose involved in the natural maize straw composting. The function of the degraded leading microbial community was identified. The results showed that in the composting process, a stable microbial community structure was formed in the composting process of natural corn stalk, mainly composed of thermophilic, thermophilic and thermophilic microorganism, and the genus were also the dominant straw wood. Thermomyces lanuginosus is the main hemicellulose degrading bacteria in the composting, and the bacterial Thermobifida fusca is the main cellulose degrading bacteria. The integrated macroomics method used in this study can combine the study of microbial community composition and function, thus realizing the composting habitat. This study not only theoretically studies the structural function and ecological process of microbial community in straw composting, but also enriches the theory of straw composting, and the integrated macroomics method used in this study can also be applied to the research of microbial communities in other habitats. In the study, the traditional compost technology was transformed with a wide range of universal.2., the industrial application of 90 m3 large fermenting tank farm animal waste composting was successfully implemented. The large-scale 90m3 fermenting tank of industrial scale was carried out with a height of 3.3 m of corncob cow dung mixed composting. The microorganism community of the key function was studied. The experiment showed that the fermentation cycle could be shortened from 42 days to 11 days by the fermentation tank composting, and the efficiency of the composting fermentation was greatly improved. In addition, the macrostructure results showed that the microbial community in the fermenting tank composted with the spatiotemporal conditions. In the tank composting, the dominant microbial communities are mainly composed of bacteria like thermophilia, bacillus and fungi of the genus thermophilia and Aspergillus. The functional analysis of the flora shows that the genus thermophilia is the main fibrinolytic degrading in the fermentor. This study successfully carried out the traditional heap The technological transformation of fertilizer technology has realized the modernization of the composting technology. It provides a successful example for the transformation of the composting of agricultural wastes, which is of great value to the popularization of industrialization, which is also of great guiding significance to the realization of the compound utilization of microbial bacteria.3.. On the basis of the experimental study on the composting of the corn straw composting, the dynamic change of biological dynamic changes caused the wheat straw composting efficiency to be lower than that of the corn straw composting. The dynamic change of the microbial community of the wheat straw composting was investigated by the integrated macroomics technology. There is only a small change in microbial community succession in the composting of straw and wheat straw composting, and cellulose degradation microorganisms fail to form dominant dominant functional communities. Therefore, the degradation efficiency of cellulose substrate is low. The internal temperature of the wheat straw composting is lower than 60 C, so the harmless treatment of the substrate can not be reached, which makes it rich. The bacteriobacteria and deformable bacteria are the dominant microorganisms in the whole composting process. Compared to the corn straw with higher soluble sugar content, the wheat straw has higher structural heterogeneity, higher silicon content and lignocellulose content, which results in comparison to corn straw and wheat straw as the substrate. The degradation efficiency of compost was significantly lower than that of maize straw composting.4.. The microbiological analysis of microbial community in chicken manure composting was analyzed. It showed that Bacillus is a functional microbial manure dominated by chicken manure compost as a kind of high nitrogen source and low water content of livestock and poultry manure, which is very suitable for composting. The dynamic change of microbial community in chicken manure composting was studied and analyzed by macro composting technology. It was found that the microbial community composition in chicken manure composting occurred obvious community succession with temporal and spatial variation. The microorganism in chicken manure composting was dominated by the bacteria's thick wall bacteria gate, especially the door. The.Native electrophoresis protease analysis of Bacillus genus also showed that the protease of bacillus was the main enzyme band, so bacillus was the main functional genus in the chicken manure compost. The study also showed that different composting substrates had significant influence on the microbial community of compost. The evolution of microbial community in livestock manure composting was analyzed, and it also had important practical significance for the rapid composting treatment of livestock and poultry manure.5.. The expression of T. lanuginosus, the main degradation fungus, was analyzed. It was proved that the natural complex substrate was more efficient in the expression of the xylanase. The inducible T. lanuginosus is the main hemicellulose degrading fungus in the natural corn straw composting. In this study, the extracellular expression profiles are analyzed. The results show that the fungus can be used as a substrate for rapid growth during liquid fermentation. Xylose and xylan are both xylanase inducers. Compared with simple bottom, the xylose and xylan are the inducers of xylanase. In liquid culture, natural complex substrates are more suitable for the growth of the bacteria in solid fermentation, and both corn straw powder and wheat bran can induce high efficiency expression of the xylanase by higher efficiency, indicating that the natural substrate is a good inducer for the xylanase of the bacteria. These results can be used to further study the compost of T. lanuginosus. It provides a theoretical guide for the rapid growth and the mechanism and function of natural cellulose degradation.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S141.4
，
本文编号：2122806
[Abstract]:As a big agriculture and animal husbandry country, China produces a large number of agricultural and animal husbandry wastes every year. Due to the lack of rapid and efficient treatment, the most common methods of disposal of agricultural waste are accumulation or incineration, which will lead to serious soil, water and air pollution. In addition, because of the large-scale intensive cultivation and cultivation, and the lack of wheels at the same time. In order to solve the environmental problems caused by agriculture and animal husbandry, a reasonable and environmentally friendly way is urgently needed to deal with the agricultural and animal husbandry wastes so as to realize the sustainable development of green agriculture. And the transformed product can be a high quality bio organic fertilizer, so it is the most promising way for the harmless and resourceful treatment of agricultural and animal husbandry waste. The composting process is mainly driven by the microbial community in which the composting can complete the biological transformation of different organic matter such as crop straw and livestock manure. The degradation needs a variety of microbes to work together. But so far, there has not been a systematic and comprehensive study of the microbial community of compost. In this paper, a series of studies have been carried out with the composting of the natural agricultural and animal husbandry waste heap as the research object. The main achievements are as follows: 1. the success of macroomics technology is followed. The microbial community of natural maize straw composting was located, and the function of key microbial communities was determined by integrated macroomics technology including macrogenomics and macroproteomics. The dynamic changes of microbial community composition and structure in natural maize straw composting were tracked and lignocellulose involved in the natural maize straw composting. The function of the degraded leading microbial community was identified. The results showed that in the composting process, a stable microbial community structure was formed in the composting process of natural corn stalk, mainly composed of thermophilic, thermophilic and thermophilic microorganism, and the genus were also the dominant straw wood. Thermomyces lanuginosus is the main hemicellulose degrading bacteria in the composting, and the bacterial Thermobifida fusca is the main cellulose degrading bacteria. The integrated macroomics method used in this study can combine the study of microbial community composition and function, thus realizing the composting habitat. This study not only theoretically studies the structural function and ecological process of microbial community in straw composting, but also enriches the theory of straw composting, and the integrated macroomics method used in this study can also be applied to the research of microbial communities in other habitats. In the study, the traditional compost technology was transformed with a wide range of universal.2., the industrial application of 90 m3 large fermenting tank farm animal waste composting was successfully implemented. The large-scale 90m3 fermenting tank of industrial scale was carried out with a height of 3.3 m of corncob cow dung mixed composting. The microorganism community of the key function was studied. The experiment showed that the fermentation cycle could be shortened from 42 days to 11 days by the fermentation tank composting, and the efficiency of the composting fermentation was greatly improved. In addition, the macrostructure results showed that the microbial community in the fermenting tank composted with the spatiotemporal conditions. In the tank composting, the dominant microbial communities are mainly composed of bacteria like thermophilia, bacillus and fungi of the genus thermophilia and Aspergillus. The functional analysis of the flora shows that the genus thermophilia is the main fibrinolytic degrading in the fermentor. This study successfully carried out the traditional heap The technological transformation of fertilizer technology has realized the modernization of the composting technology. It provides a successful example for the transformation of the composting of agricultural wastes, which is of great value to the popularization of industrialization, which is also of great guiding significance to the realization of the compound utilization of microbial bacteria.3.. On the basis of the experimental study on the composting of the corn straw composting, the dynamic change of biological dynamic changes caused the wheat straw composting efficiency to be lower than that of the corn straw composting. The dynamic change of the microbial community of the wheat straw composting was investigated by the integrated macroomics technology. There is only a small change in microbial community succession in the composting of straw and wheat straw composting, and cellulose degradation microorganisms fail to form dominant dominant functional communities. Therefore, the degradation efficiency of cellulose substrate is low. The internal temperature of the wheat straw composting is lower than 60 C, so the harmless treatment of the substrate can not be reached, which makes it rich. The bacteriobacteria and deformable bacteria are the dominant microorganisms in the whole composting process. Compared to the corn straw with higher soluble sugar content, the wheat straw has higher structural heterogeneity, higher silicon content and lignocellulose content, which results in comparison to corn straw and wheat straw as the substrate. The degradation efficiency of compost was significantly lower than that of maize straw composting.4.. The microbiological analysis of microbial community in chicken manure composting was analyzed. It showed that Bacillus is a functional microbial manure dominated by chicken manure compost as a kind of high nitrogen source and low water content of livestock and poultry manure, which is very suitable for composting. The dynamic change of microbial community in chicken manure composting was studied and analyzed by macro composting technology. It was found that the microbial community composition in chicken manure composting occurred obvious community succession with temporal and spatial variation. The microorganism in chicken manure composting was dominated by the bacteria's thick wall bacteria gate, especially the door. The.Native electrophoresis protease analysis of Bacillus genus also showed that the protease of bacillus was the main enzyme band, so bacillus was the main functional genus in the chicken manure compost. The study also showed that different composting substrates had significant influence on the microbial community of compost. The evolution of microbial community in livestock manure composting was analyzed, and it also had important practical significance for the rapid composting treatment of livestock and poultry manure.5.. The expression of T. lanuginosus, the main degradation fungus, was analyzed. It was proved that the natural complex substrate was more efficient in the expression of the xylanase. The inducible T. lanuginosus is the main hemicellulose degrading fungus in the natural corn straw composting. In this study, the extracellular expression profiles are analyzed. The results show that the fungus can be used as a substrate for rapid growth during liquid fermentation. Xylose and xylan are both xylanase inducers. Compared with simple bottom, the xylose and xylan are the inducers of xylanase. In liquid culture, natural complex substrates are more suitable for the growth of the bacteria in solid fermentation, and both corn straw powder and wheat bran can induce high efficiency expression of the xylanase by higher efficiency, indicating that the natural substrate is a good inducer for the xylanase of the bacteria. These results can be used to further study the compost of T. lanuginosus. It provides a theoretical guide for the rapid growth and the mechanism and function of natural cellulose degradation.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S141.4
，
本文编号：2122806
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