农业废物堆肥中微生物总DNA提取方法的研究及其应用
摘要
堆肥是利用自然界中广泛存在的微生物,在人工控制的条件下,将可生物降解的有机物转化为肥料的过程。然而,可培养微生物仅占自然界微生物的0.1%~10%,不能很好地反映自然环境中微生物多样性的原始状态。随着分子生物学和系统发育分析方法的发展,基于16S rDNA基因的分子生物学方法逐渐被用来分析复杂的生态系统。本文对农业废物垃圾进行堆肥处理,并采用聚合酶链式反应-变性梯度凝胶电泳(Polymerase Chain Reaction-Denature Gradient Gel Electrophoresis,PCR-DGGE)技术对堆肥过程中微生物群落与木质纤维素降解的关系进行了研究。
首先在固态发酵培养条件下,研究了用于其微生物群落多样性及演替情况分析的微生物基因组提取方法。对比3种(溶壁酶法,超声波法,液氮研磨+CTAB法)可同时从固态发酵中提取细菌和真菌DNA的方法。使用紫外分光光度计测定DNA产量与纯度;使用细菌16S rDNA基因通用引物(341F和907R)和真菌18S rDNA基因通用引物(NU-SSU-0817和NU-SSU-119)对DNA进行PCR扩增;采用变性梯度凝胶电泳(DGGE)法对纯化后的PCR产物进行多样性分析。结果显示,三种方法得到的粗提和纯化DNA长度都约为23kb;细菌和真菌PCR产物长度分别约为586bp和422bp;DGGE分析表明3种方法提取的DNA反映的微生物多样性比较一致;但通过紫外分光光度计的测定表明:溶壁酶法提取固态发酵中微生物总DNA产量最高,超声波法次之,液氮研磨+CTAB法最差。所以综合以上各结果,溶壁酶法最优。
采用筛选到的最佳提取DNA方法,从农业废物垃圾好氧堆肥样品中提取出堆肥混合菌群的总DNA,粗提DNA经过纯化后,采用GC-341F/907R引物进行PCR扩增16SrDNA基因段,用DGGE进行分离和鉴定PCR扩增产物,从而得出农业废物垃圾堆肥过程中微生物种群多样性的信息。DGGE图谱显示,堆肥初期有较多的微生物多样性,随着堆肥温度的升高,一些中温期的微生物被淘汰,高温期微生物多样性减少,同时噬热菌出现,并成为优势菌群。高温期过后,微生物多样性进一步锐减。从而表明堆肥的温度是影响堆体中微生物生长的重要因素,不同的温度,堆体具有适应这个温度的不同的优势菌种。同时,对几种特征堆肥参数进行了测定,结果显示,经过39d的堆肥,堆肥产物基本达到要求。而通过对木质纤维素降解以及各酶活(Lac、LiP、MnP)的测定,可以发现在微生物种群数增加阶段,纤维素、半纤维素降解速度和酶活量都有增加。而在微生物数递减阶段,木质纤维素的降解速度有所缓慢且酶活量有下降趋势。从而看出,微生物的种群数和木质纤维素的降解有一定的相关关系。从以上研究表明,将溶壁酶法用于研究堆肥过程中菌落的多样性、演替情况以及群落与生物降解关系是可行的。此研究对于堆肥菌剂的开发提供了很有意义的方法借鉴,有助于解决在菌剂开发中选择菌株缺乏依据的问题,还可以为其他环境体系的菌落研究提供参考。
Composting takes the advantage of natural microorganisms, under the manual control condition, it is a process which will transform biodegradable organic to fertilizer. However, the microbes can be cultured, only 1% to 10% of the microbes from natural habitats, so it can’t well reflect the microbial diversity in the natural environment of the original state. With the development of molecular biology and phylogenetic analysis, based on 16SrDNA gene molecular biology methods are gradually being used to analyze complex ecosystems. In this paper, composting used polymerase chain reaction - denaturing gradient gel electrophoresis (PCR-DGGE) technology to study the relationship between colonies and lignocellulose degradation during the composting process.
Lyticase lysis, ultrasonic lysis and grinding lysis in liquid nitrogen with CTAB as genomic DNA extraction methods for bacteria and fungi were studied to analyze the diversity and succession of the microbes during solid-state fermentation. Quantity and purity of DNA were measured by ultraviolet spectrophotometer. PCR amplification was carried out with a eubacterial 16S rDNA targeted primer pair (341F and 907R) and a fungi 18S rDNA targeted primer pair (NU-SSU-0817 and NU-SSU-1196). The bacterial and fungal diversity during solid-state fermentation was analyzed by denaturing gradient gel electrophoresis (DGGE). The results showed that the rude and purified DNA fragments extracted during solid-state fermentation have a length of about 23 kb. The length of DNA fragments after bacterial and fungal PCR amplification is about 586 bp and 422 bp, respectively. DNA band profiles by DGGE analysis for either bacterial or fungal PCR products testify the similar microbial diversity for three DNA extract methods. The results by the ultraviolet spectrophotometer showed that lyticase lysis yielded the highest quantity of DNA, ultrasonic lysis followed and grinding lysis in liquid nitrogen with CTAB produced the lowest quantity of DNA. Therefor, Lyticase lysis was best.
According to the screening method of DNA extraction, we choosed lyticase lysis by which the total DNA of the organic solid waste composting microorganisms was extracted. After purification, the total DNA was amplified with PCR using the GC-341F/907R primers to obtain the 16S rDNA genes. The PCR amplified products were separated and identified by DGGE,which could show the information of organic solid waste composting microorganisms. It showed that there was more microbial diversities in the initial stage of composting. Some mesothermal microorganisms were eliminated with the rising of the temperature, and the microbial diversities of high temperature stage decreased. The thermophilic bacterias emergence became to the preponderant microorganisms. After the high temperature stage, the diversity of microorganisms still dereased. The temperature of the composting was an important factor effecting the microorganisms of the pile. And the pile might have different preponderant microorganisms in different temperature. At the same time, several characteristics of composting parameters were measured. After 39 days compost, the results showed that the product of compost met the basic requirements. Measurement of the lignocellulose degradation as well as the enzyme activity of Lac, Lip, Mnp showed that when microbial populations increased, cellulose, hemicellulose degradation rate and volume of activity also increased. When the number of microbial decreased, the rate of lignocellulose degradation and enzyme activity would slow. So we could see that the number of microbial populations and lignocellulose degradation have a certain relationship.
In conclusion, Lyticase lysis which was used to study diversity and succession of the microbes and the relationship between communities and biodegradation was feasible. These methods would be helpful for solving the problem of subjectivity about strains selecting, they could provide useful information for the researching of the environment systematic communities.
首先在固态发酵培养条件下,研究了用于其微生物群落多样性及演替情况分析的微生物基因组提取方法。对比3种(溶壁酶法,超声波法,液氮研磨+CTAB法)可同时从固态发酵中提取细菌和真菌DNA的方法。使用紫外分光光度计测定DNA产量与纯度;使用细菌16S rDNA基因通用引物(341F和907R)和真菌18S rDNA基因通用引物(NU-SSU-0817和NU-SSU-119)对DNA进行PCR扩增;采用变性梯度凝胶电泳(DGGE)法对纯化后的PCR产物进行多样性分析。结果显示,三种方法得到的粗提和纯化DNA长度都约为23kb;细菌和真菌PCR产物长度分别约为586bp和422bp;DGGE分析表明3种方法提取的DNA反映的微生物多样性比较一致;但通过紫外分光光度计的测定表明:溶壁酶法提取固态发酵中微生物总DNA产量最高,超声波法次之,液氮研磨+CTAB法最差。所以综合以上各结果,溶壁酶法最优。
采用筛选到的最佳提取DNA方法,从农业废物垃圾好氧堆肥样品中提取出堆肥混合菌群的总DNA,粗提DNA经过纯化后,采用GC-341F/907R引物进行PCR扩增16SrDNA基因段,用DGGE进行分离和鉴定PCR扩增产物,从而得出农业废物垃圾堆肥过程中微生物种群多样性的信息。DGGE图谱显示,堆肥初期有较多的微生物多样性,随着堆肥温度的升高,一些中温期的微生物被淘汰,高温期微生物多样性减少,同时噬热菌出现,并成为优势菌群。高温期过后,微生物多样性进一步锐减。从而表明堆肥的温度是影响堆体中微生物生长的重要因素,不同的温度,堆体具有适应这个温度的不同的优势菌种。同时,对几种特征堆肥参数进行了测定,结果显示,经过39d的堆肥,堆肥产物基本达到要求。而通过对木质纤维素降解以及各酶活(Lac、LiP、MnP)的测定,可以发现在微生物种群数增加阶段,纤维素、半纤维素降解速度和酶活量都有增加。而在微生物数递减阶段,木质纤维素的降解速度有所缓慢且酶活量有下降趋势。从而看出,微生物的种群数和木质纤维素的降解有一定的相关关系。从以上研究表明,将溶壁酶法用于研究堆肥过程中菌落的多样性、演替情况以及群落与生物降解关系是可行的。此研究对于堆肥菌剂的开发提供了很有意义的方法借鉴,有助于解决在菌剂开发中选择菌株缺乏依据的问题,还可以为其他环境体系的菌落研究提供参考。
Composting takes the advantage of natural microorganisms, under the manual control condition, it is a process which will transform biodegradable organic to fertilizer. However, the microbes can be cultured, only 1% to 10% of the microbes from natural habitats, so it can’t well reflect the microbial diversity in the natural environment of the original state. With the development of molecular biology and phylogenetic analysis, based on 16SrDNA gene molecular biology methods are gradually being used to analyze complex ecosystems. In this paper, composting used polymerase chain reaction - denaturing gradient gel electrophoresis (PCR-DGGE) technology to study the relationship between colonies and lignocellulose degradation during the composting process.
Lyticase lysis, ultrasonic lysis and grinding lysis in liquid nitrogen with CTAB as genomic DNA extraction methods for bacteria and fungi were studied to analyze the diversity and succession of the microbes during solid-state fermentation. Quantity and purity of DNA were measured by ultraviolet spectrophotometer. PCR amplification was carried out with a eubacterial 16S rDNA targeted primer pair (341F and 907R) and a fungi 18S rDNA targeted primer pair (NU-SSU-0817 and NU-SSU-1196). The bacterial and fungal diversity during solid-state fermentation was analyzed by denaturing gradient gel electrophoresis (DGGE). The results showed that the rude and purified DNA fragments extracted during solid-state fermentation have a length of about 23 kb. The length of DNA fragments after bacterial and fungal PCR amplification is about 586 bp and 422 bp, respectively. DNA band profiles by DGGE analysis for either bacterial or fungal PCR products testify the similar microbial diversity for three DNA extract methods. The results by the ultraviolet spectrophotometer showed that lyticase lysis yielded the highest quantity of DNA, ultrasonic lysis followed and grinding lysis in liquid nitrogen with CTAB produced the lowest quantity of DNA. Therefor, Lyticase lysis was best.
According to the screening method of DNA extraction, we choosed lyticase lysis by which the total DNA of the organic solid waste composting microorganisms was extracted. After purification, the total DNA was amplified with PCR using the GC-341F/907R primers to obtain the 16S rDNA genes. The PCR amplified products were separated and identified by DGGE,which could show the information of organic solid waste composting microorganisms. It showed that there was more microbial diversities in the initial stage of composting. Some mesothermal microorganisms were eliminated with the rising of the temperature, and the microbial diversities of high temperature stage decreased. The thermophilic bacterias emergence became to the preponderant microorganisms. After the high temperature stage, the diversity of microorganisms still dereased. The temperature of the composting was an important factor effecting the microorganisms of the pile. And the pile might have different preponderant microorganisms in different temperature. At the same time, several characteristics of composting parameters were measured. After 39 days compost, the results showed that the product of compost met the basic requirements. Measurement of the lignocellulose degradation as well as the enzyme activity of Lac, Lip, Mnp showed that when microbial populations increased, cellulose, hemicellulose degradation rate and volume of activity also increased. When the number of microbial decreased, the rate of lignocellulose degradation and enzyme activity would slow. So we could see that the number of microbial populations and lignocellulose degradation have a certain relationship.
In conclusion, Lyticase lysis which was used to study diversity and succession of the microbes and the relationship between communities and biodegradation was feasible. These methods would be helpful for solving the problem of subjectivity about strains selecting, they could provide useful information for the researching of the environment systematic communities.
引文
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