整合宏組學方法揭示天然木質纖維素堆肥中的關鍵功能微生物群落
發(fā)布時間：2018-07-14 20:07
【摘要】：中國作為農牧業(yè)大國,每年會產生大量的農牧業(yè)廢棄物。由于缺乏快速高效的處理方式,目前最常見的農業(yè)廢棄物處理方式是堆積或焚燒,這會導致產生嚴重的土壤、水體和空氣污染。此外,由于連年大規(guī)模集約化種植和養(yǎng)殖,同時缺乏輪作休耕制度,土壤肥力被嚴重透支,水土流失現象也日益嚴重。為了解決目前農牧業(yè)造成的環(huán)境問題,迫切需要一種合理環(huán)保的方式來針對性地處理農牧業(yè)廢棄物,以實現綠色農業(yè)的可持續(xù)發(fā)展。堆肥工藝可以實現有機質的綠色轉化,并且轉化的產物可以成為優(yōu)質的生物有機肥料,因此是無害化和資源化處理農牧業(yè)廢棄物的最有前景的一種方式。堆肥過程主要由其中的微生物群落所驅動。堆肥可以完成不同有機質如農作物秸稈和畜禽糞便的生物轉化,而對這些材料的降解需要多種微生物共同協作完成。但是迄今為止尚未有對堆肥微生物群落比較系統和全面的研究。針對這一問題,本文以天然農牧業(yè)廢棄物堆制的堆肥為研究對象開展了系列研究,取得的主要成果如下：1.采用宏組學技術成功跟蹤定位了天然玉米秸稈堆肥的微生物群落,確定了關鍵微生物群落的功能使用包括宏基因組學和宏蛋白組學在內的整合宏組學技術,跟蹤了天然玉米秸稈堆肥中微生物的群落組成與結構隨時空條件的動態(tài)變化,并對涉及木質纖維素降解的主導微生物群落的功能進行了鑒定。研究結果表明,在堆肥堆制過程中,天然玉米秸稈堆肥中形成了一個穩(wěn)定的微生物群落結構,主要由嗜熱絲孢菌屬、喜熱裂孢菌屬和嗜熱多孢菌屬等微生物組成,并且這些菌屬也是主導秸稈木質纖維素降解的功能微生物。其中真菌Thermomyces lanuginosus是堆肥中的主要半纖維素降解菌,細菌Thermobifida fusca是主要的纖維素降解菌。本研究采用的整合宏組學方法可以將微生物群落組成與功能的研究相結合,從而實現對堆肥生境關鍵功能微生物群落的準確定位。本項研究不僅從理論上對秸稈堆肥中微生物群落的結構功能與生態(tài)過程進行了研究,對豐富秸稈堆肥理論有重要意義,而且在該項研究中使用的整合宏組學方法也可以應用在其它生境的微生物群落研究中,具有廣泛的普適性。2.對傳統堆肥工藝進行了技術改造,成功實現了90 m3大型發(fā)酵罐農牧業(yè)廢棄物堆肥的工業(yè)化應用使用工業(yè)規(guī)模的90m3大型發(fā)酵罐進行了高度達3.3 m的玉米芯牛糞混合堆肥發(fā)酵,借助宏組學技術對發(fā)酵罐堆肥的關鍵功能微生物群落進行了研究。實驗表明通過發(fā)酵罐堆肥,可以將發(fā)酵周期從天然堆肥的42天縮短至11天,大大提高了堆肥發(fā)酵效率。此外,宏組學結果表明發(fā)酵罐堆肥中微生物群落隨時空條件的變化而發(fā)生明顯的群落演替現象。在發(fā)酵罐堆肥中,主導的微生物群落主要由細菌的喜熱裂孢菌屬、芽孢桿菌屬等細菌屬和真菌的嗜熱絲孢菌屬和曲霉屬等真菌屬所組成。對菌群的功能分析表明,喜熱裂孢菌屬是發(fā)酵罐堆肥中主要的纖維素降解者。本項研究成功進行了傳統堆肥工藝的技術改造,實現了堆肥工藝技術的現代化,為農業(yè)廢棄物的堆肥轉化提供了一個成功的范例,極具工業(yè)化推廣的價值,這對于實現微生物菌劑的復配利用也具有重要的指導意義。3.運用宏組學技術研究分析了小麥秸稈堆肥的微生物動態(tài)變化,證明由于原料中組分與結構的差異造成了小麥秸稈的堆肥效率低于玉米秸稈堆肥在天然玉米秸稈堆肥實驗研究的基礎上,使用整合宏組學技術跟蹤調查了小麥秸稈堆肥微生物群落隨時間的動態(tài)變化。發(fā)現相比于玉米秸稈堆肥,小麥秸稈堆肥中的微生物群落演替現象只有小幅變化,纖維素降解微生物未能形成主導的優(yōu)勢功能群落,因此纖維素底物降解效率低。檢測發(fā)現小麥秸稈堆肥的內部溫度低于60℃,因此也未能達到底物的無害化處理,使得富含動植物致病菌的擬桿菌門和變形菌門在整個堆肥過程都是主導的微生物。相比于可溶性糖分含量較高的玉米秸稈,小麥秸稈中具有更高的結構異質性,更高的硅含量和木質纖維素含量,這都導致相比之于玉米秸稈,小麥秸稈為底物時的堆肥降解效率明顯低于玉米秸稈堆肥。4.對雞糞堆肥中的微生物群落變化進行了宏組學分析,顯示芽孢桿菌屬是雞糞堆肥中主導的功能微生物雞糞作為一種高氮源和低含水率的畜禽糞便,非常適合堆肥工藝。采用肉雞養(yǎng)殖場的雞糞進行天然堆肥,通過宏組學技術對雞糞堆肥中微生物群落隨時空的動態(tài)變化進行了研究分析。研究發(fā)現雞糞堆肥中微生物群落組成隨時空變化發(fā)生了明顯的群落演替現象。雞糞堆肥中的微生物以細菌的厚壁菌門為主導,尤以屬于該門的芽孢桿菌屬為主要菌屬。Native電泳蛋白酶譜分析也顯示芽孢桿菌屬細菌的蛋白酶為主要的酶譜條帶,因此芽孢桿菌屬是該類雞糞堆肥中的主要功能菌屬。該研究也顯示不同的堆肥底物對堆肥微生物群落有顯著的影響。該研究不僅在理論上對畜禽糞便堆肥中微生物群落的演替變化進行了分析,也對實現畜禽糞便的快速堆肥處理具有重要的現實指導意義。5.對堆肥主要降解真菌T. lanuginosus的表達譜進行了分析,證明天然復雜底物對該菌木聚糖酶的表達具有更高效的誘導作用T. lanuginosus是天然玉米秸稈堆肥中的主要半纖維素降解真菌,在本研究中對其胞外表達譜進行了分析。研究結果表明,在液體發(fā)酵時該菌可以利用木糖為底物進行快速生長,木糖和木聚糖都是木聚糖酶的誘導劑。相比于簡單底物的液體培養(yǎng),在進行固體發(fā)酵時,天然復雜底物更適合該菌的生長,而且玉米秸稈粉和小麥麩皮都能以更高的效率誘導該菌木聚糖酶的高效表達,說明天然底物是該菌木聚糖酶的優(yōu)良誘導劑。這些結果為進一步研究T. lanuginosus在堆肥中的快速生長以及在天然纖維素的降解過程中的作用機制與功能提供了理論指導。
[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.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S141.4
，
本文編號：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.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S141.4
，
本文編號：2122806
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