猪场废弃物堆肥中芽孢杆菌属和梭菌属细菌的分子生态学研究
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摘要
规模化养猪业的迅速发展一方面提高了人民生活水平,另一方面,养猪场粪便的大量集中排放给环境造成了巨大压力。目前,寻找具有良好环境效应的畜禽场废弃物无害化、资源化技术成为我国畜禽规模化养殖业面临的一项重要课题。堆肥技术通常用于有机固体废弃物的资源化利用,成功的堆肥可以实现无害化、减量化和资源化的处理目标。因此,堆肥被认为是养猪业与环境协调发展的有效途径之一,对堆肥过程中微生物多样性的探讨是近几年来的研究热点。
本课题组在前期的研究中发现,芽孢杆菌属和梭菌属是猪场废弃物堆肥过程中的优势菌群,本研究将PCR-DGGE技术与传统分离方法相结合,进一步在种和属的水平上对芽孢杆菌属和梭菌属在堆肥过程中的动态变化、种群结构和多样性差异进行了研究；利用FISH技术对其进行了原位空间分布研究及快速定量分析；采用生理生化鉴定与16S rRNA序列分析技术对分离培养的细菌进行了特异性菌种鉴定。具体结果如下：
1.堆肥试验
利用强制通风静态仓堆肥系统进行了堆肥试验,以猪粪为主料,木屑为调理剂,物料的初始碳氮比(C/N)调整为20%,初始含水率为60%。堆体温度的变化过程经历了典型的升温期、高温期、降温期三个阶段,堆体中层温度在5天时达到了60℃,且在50℃以上的时间持续了30天,高于55℃上持续了25天。整个堆肥过程历时42d。堆制结束时,堆体体积减少了42.68%,大肠杆菌值和蛔虫卵杀灭率分别为0.069和100%,达到了《粪便无害化卫生标准(GB7959-87)》的规定。本次堆肥试验取得成功,为后续各项研究工作奠定了基础。
2.核酸的提取
参照Zhou等(1996)的方法,对猪粪堆肥样品中DNA提取方法进行了改良,从堆肥过程中升温期、高温期、降温期上、中、下共9个样品中提取了基因组DNA,并用试剂盒对其进行了纯化。琼脂糖凝胶电泳检测DNA片段大小均在2kbp以上。使用DNA/RNA浓度测定仪,测得纯化后的DNA浓度为370n/μl,其A260/A280的比值为1.766。结果表明,采用改良后的该方法提取、并用试剂盒纯化后获得的核酸结果较好,能满足后面分子生物学研究的需要。
3.芽孢杆菌属细菌的分离及鉴定
(1)采用传统平板稀释法从猪粪堆肥过程中不同时期、不同高度层堆肥样品中分离得到了540株芽孢杆菌,利用HinfⅠ, HaeⅢ和Msp Ⅰ三种限制性内切酶对这些芽孢杆菌进行了RFLP分型,得到8种不同的芽孢杆菌,辅以生理生化鉴定试验,并依据16S rRNA序列分析对这些菌株进行了鉴定,初步得出猪场废弃物堆肥过程中可培养的芽孢杆菌属细菌的多样性信息。它们分别是Bacillus subtilis, Bacillus megaterium, Bacillus anthracis, Bacillus thuringiensis, Bacillus cereus, Bacillus pycnus, Bacillus oleronius和Bacillus circulans。
(2)获得了8株不同芽孢杆菌的16S rDNA序列,并提交到了GenBank数据库,获得了登录号：JF833086-JF833093。
4.可培养的芽孢杆菌的时空分布
RFLP分析结果显示猪粪堆肥过程中,可培养的芽孢杆菌属细菌存在着丰富的多样性和明显的时空分布特性。Bacillus subtilis是整个堆肥过程中的优势菌；升温期和降温期样品中优势芽孢杆菌还分别包括Bacillus anthracis, Bacillus cereus和Bacillus circulans。升温期和降温期中层样品中的可培养的芽孢杆菌属细菌的多样性相对更丰富,包括5种不同的芽孢杆菌。多样性最低的是升温期下层和高温期上层和中层样品。
5. PCR-DGGE分析
(1) PCR-DGGE分析结果显示,猪粪堆肥过程中不同时期、不同高度层堆肥样品中有着丰富的芽孢杆菌属和梭菌属细菌多样性,PCR-DGGE图谱中优势性条带都达到了9条以上,其中高温期样品中芽孢杆菌属细菌的多样性相对更丰富；同时存在明显的芽孢杆菌属种群结构变化,而梭菌属种群结构变化不明显,这些都表明猪粪堆肥过程中存在宝贵的芽孢杆菌属和梭菌属细菌资源。
(2)根据指纹图谱聚类分析和Cs值分析结果显示,处于同一堆肥阶段的三个不同高度层样品之间的芽孢杆菌属种群的相似性最高,说明处于同一阶段的各高度层样品的芽孢杆菌属细菌种群结构最接近；9个堆肥样品彼此间梭菌属种群的相似性都很高,说明各样品之间梭菌属细菌种群结构很接近。9个堆肥样品之间的两属细菌的Cs值都没有明显的变化规律,说明堆肥过程中芽孢杆菌属和梭菌属细菌的演替规律复杂,更替过程更复杂,需要我们进行更深入的探讨。
(3)对芽孢杆菌属和梭菌属DGGE图谱中优势条带进行了回收和测序,结果发现,堆肥过程中大部分都是未能培养的芽孢杆菌,并得到了利用纯培养方法未能分离到的芽孢杆菌属细菌信息,它们包括：Bacillus cereus, Bacillus marisflavi, Bacillus koreensis和Bacillus fordii,而Bacillus cereus也是猪粪堆肥过程中的优势菌,存在于整个堆肥过程中。堆肥过程中都是未能培养的梭茵,未检测到可培养的梭菌。说明PCR-DGGE在分子生态学方面具有更大的优越性,可以获得更多的微生物信息。
6. FISH分析
(1)对芽孢杆菌属特异性探针LGC353b和梭菌属特异性探针Chis150的杂交条件进行了优化。探针LGC353b和Chis150的最佳杂交温度都是46℃；最佳杂交时间分别为4h和5.5h；杂交液中最佳甲酰胺浓度分别为20%和35%。
(2) FISH分析结果显示,猪粪堆肥过程中不同时期、不同高度层堆肥样品中总细菌、芽孢杆菌属和梭菌属细菌数目有着明显的动态变化和的时空分布特征。三者的FISH检测数量级都达到了106个/克(湿重)的数量级。总细菌、芽孢杆菌属、梭菌属的时间分布特征是细菌总数随着温度的升高而增加,随着温度的下降而减少；空间分布特征是处于同一堆肥阶段的三个不同高度层中,中层样品中芽孢杆菌属细菌数目和其占总细菌数的百分比是最低的；梭菌属的空间分布特征不明显。
The rapid development of large-scale pig industry has improved people's living standard, on the other hand, concentrated feces exhaust resulted in remendous pressure on the environment. Now, looking for livestock and poulty harmless and recycling technique having good environmental effect has become an important issue faced large-scale livestock and poultry breeding in China. Composting technology is typically used for organic solid waste recycling. Successful composting can achieve harmless reduction and recycling processing. So, composting was considered to be one of the effective way of pig industry and the environment coordinated development and the study of microbial diversity in the process of composting is a hot research topic in recent years.
1. Composting experiment
Composting experiment was conducted by using forced ventilation composting system, swine manure was the major component and rice bran was used to as the conditioner for swine composting. The initial carbon nitrogen ratio was adjusted to20, the initial moisture content of the mixture was60%. The composting process went through three classic phases:rapid temperature-rising stage, sustained high-temperature stage, and gradual cooling stage. The temperature of the middle layer of pile achieved60℃within5d, and kept above50℃for30days and55℃for25days. The composting process lasted for42days. When the composting process finished, the volume of the composting pile decreased by42.68%. The E. coli and killing rate of ascarid eggs were100%and0.069, which met the sanitary standards of the non-hazardous treatment of night soil (GB7959-87). The composting experiment had been successful, which laid a good foundation for the later studies.
2. Nucleic acid extraction
A method for obtaining DNA from swine manure composting was modified, based on the procedures described by Zhou (1996), and the genomic DNA were obtained from9samples of rapid temperature-rising stage, sustained high-temperature stage, and gradual cooling stage in the composting process, and was purified with Commercial kit. The length of the DNA fragment was above2Kbp by detecting with agarose gel eletrophoresis. The concentration of purified DNA was about370ng/uL by using DNA/RNA concentration determinator. The A260/A280ratio was1.766. The results showed that the quantity of nucleinic acid was high by using this method and could meet the need of molecular biology research.
3. Separation and identification
(1) A total of540Bacillus were obtained from the samples of different phrases and layers in swine manure composting using traditional plate dilution method. An RFLP analysis was conducted for these isolates which were then divided eight different patterns. Basing on physiology and biochemistry tests and16S rDNA sequence analysis, the diversity of cultured Bacillus in swine manure composting was obtained. There are Bacillus subtilis, Bacillus megaterium, Bacillus anthracis, Bacillus thuringiensis, Bacillus cereus, Bacillus pycnus, Bacillus oleronius and Bacillus circulans.
(2)16S rDNA sequences of eight different Bacillus were obtained, and have been deposited in GenBank database under accession numbers JF833086-JT833093.
4. Temporal-spatial distribution of cultured Bacillus
The results of RFLP analysis showed that the diversity of cultured Bacillus was rich and the characteristic of temporal-spatial distribution was obvious. Bacillus subtilis was the dominant bacteria in the whole composting; the dominant bacteria at rapid temperature-rising stage and gradual cooling stage also included Bacillus anthracis, Bacillus cereus and Bacillus circulans, respectively. The diversity of cultured Bacillus in the samples of middle layer at stages of rapid temperature-rising and gradual cooling was relatively more richer, including5different Bacillus. The diversities of bottom layer at rapid temperature-rising stage and top and middle layers at high temperature stage were the lowest.
5. PCR-DGGE analysis
(1) The results of PCR-DGGE analysis showed that the bacterial diversities of Bacillus and Clostridium in the samples of different stages and different locations in swine manure composting were comparatively rich. The dominant bands in PCR-DGGR maps achieved to over10. The diversity of Bacillus in the sample of high temperature stage was relatively more richer; and the structure of population of Bacillus existed obvious changes, while the change of populative structure of Clostridium was not obvious, which indicated that precious resources of Bacillus and Clostridium in the swine manure composting was very rich.
(2) The results of fingerprinting clustering and Cs value analysis showed that the diversities of Bacillus in the samples of different locations each other at the same stage was the highest. However, the diversities of Clostridium in9samples each other were very high, indicating that the composition of Clostridium among all samples were similar. The change laws of the Cs value of two genus among9samples were not obvious, indicating that the succession of Bacillus and Clostridium in the composting process was complicated, and the succession process was more complicated, further studies are needed.
(3) The dominant bands in the Bacillus and Clostridium maps were retrieved and sequenced, the results indicated that the most Bacillus species were uncultured, some Bacillus which were not found using cultured method were obtained, including Bacillus cereus, Bacillus marisflavi, Bacillus koreensis and Bacillus fordii, and Bacillus cereus was also the dominant bacteria in the whole composting. All Clostridium species were uncultured, and cultured Clostridium were not detected. All of these indicated that PCE-DGGE technology is provided with more advantages in the respect of molecular biology.
6. FISH analysis
(1) The hybridization conditions of the specific probes of Bacillus LGC353b and Clostridium Chis150have been optimized. The optimal hybridization temperature of two probes were46℃, optimal hybridization time were4h and5.5, and the formamide concentration in hybridization buffer were20%and30%, respectively.
(2) The results of FISH analysis showed that the number of total cells, Bacillus and Clostridium were presented obvious dynamic changes and temporal-spatial distribution characteristic. The detected limits of them using FISH method were approximately106cells g"'(wet weight). The characteristic of temporal distribution of total cells, Bacillus and Clostridium was that the number of cells detected would rise with the rising of temperature and vice versa; the spatial distribution of Bacillus bacteria was that the number and the relative level of the Bacillus cells detected in the middle-level of composting were the lowest at each of composting, while that of Clostridium in the compositing was not obvious.
本课题组在前期的研究中发现,芽孢杆菌属和梭菌属是猪场废弃物堆肥过程中的优势菌群,本研究将PCR-DGGE技术与传统分离方法相结合,进一步在种和属的水平上对芽孢杆菌属和梭菌属在堆肥过程中的动态变化、种群结构和多样性差异进行了研究；利用FISH技术对其进行了原位空间分布研究及快速定量分析；采用生理生化鉴定与16S rRNA序列分析技术对分离培养的细菌进行了特异性菌种鉴定。具体结果如下：
1.堆肥试验
利用强制通风静态仓堆肥系统进行了堆肥试验,以猪粪为主料,木屑为调理剂,物料的初始碳氮比(C/N)调整为20%,初始含水率为60%。堆体温度的变化过程经历了典型的升温期、高温期、降温期三个阶段,堆体中层温度在5天时达到了60℃,且在50℃以上的时间持续了30天,高于55℃上持续了25天。整个堆肥过程历时42d。堆制结束时,堆体体积减少了42.68%,大肠杆菌值和蛔虫卵杀灭率分别为0.069和100%,达到了《粪便无害化卫生标准(GB7959-87)》的规定。本次堆肥试验取得成功,为后续各项研究工作奠定了基础。
2.核酸的提取
参照Zhou等(1996)的方法,对猪粪堆肥样品中DNA提取方法进行了改良,从堆肥过程中升温期、高温期、降温期上、中、下共9个样品中提取了基因组DNA,并用试剂盒对其进行了纯化。琼脂糖凝胶电泳检测DNA片段大小均在2kbp以上。使用DNA/RNA浓度测定仪,测得纯化后的DNA浓度为370n/μl,其A260/A280的比值为1.766。结果表明,采用改良后的该方法提取、并用试剂盒纯化后获得的核酸结果较好,能满足后面分子生物学研究的需要。
3.芽孢杆菌属细菌的分离及鉴定
(1)采用传统平板稀释法从猪粪堆肥过程中不同时期、不同高度层堆肥样品中分离得到了540株芽孢杆菌,利用HinfⅠ, HaeⅢ和Msp Ⅰ三种限制性内切酶对这些芽孢杆菌进行了RFLP分型,得到8种不同的芽孢杆菌,辅以生理生化鉴定试验,并依据16S rRNA序列分析对这些菌株进行了鉴定,初步得出猪场废弃物堆肥过程中可培养的芽孢杆菌属细菌的多样性信息。它们分别是Bacillus subtilis, Bacillus megaterium, Bacillus anthracis, Bacillus thuringiensis, Bacillus cereus, Bacillus pycnus, Bacillus oleronius和Bacillus circulans。
(2)获得了8株不同芽孢杆菌的16S rDNA序列,并提交到了GenBank数据库,获得了登录号：JF833086-JF833093。
4.可培养的芽孢杆菌的时空分布
RFLP分析结果显示猪粪堆肥过程中,可培养的芽孢杆菌属细菌存在着丰富的多样性和明显的时空分布特性。Bacillus subtilis是整个堆肥过程中的优势菌；升温期和降温期样品中优势芽孢杆菌还分别包括Bacillus anthracis, Bacillus cereus和Bacillus circulans。升温期和降温期中层样品中的可培养的芽孢杆菌属细菌的多样性相对更丰富,包括5种不同的芽孢杆菌。多样性最低的是升温期下层和高温期上层和中层样品。
5. PCR-DGGE分析
(1) PCR-DGGE分析结果显示,猪粪堆肥过程中不同时期、不同高度层堆肥样品中有着丰富的芽孢杆菌属和梭菌属细菌多样性,PCR-DGGE图谱中优势性条带都达到了9条以上,其中高温期样品中芽孢杆菌属细菌的多样性相对更丰富；同时存在明显的芽孢杆菌属种群结构变化,而梭菌属种群结构变化不明显,这些都表明猪粪堆肥过程中存在宝贵的芽孢杆菌属和梭菌属细菌资源。
(2)根据指纹图谱聚类分析和Cs值分析结果显示,处于同一堆肥阶段的三个不同高度层样品之间的芽孢杆菌属种群的相似性最高,说明处于同一阶段的各高度层样品的芽孢杆菌属细菌种群结构最接近；9个堆肥样品彼此间梭菌属种群的相似性都很高,说明各样品之间梭菌属细菌种群结构很接近。9个堆肥样品之间的两属细菌的Cs值都没有明显的变化规律,说明堆肥过程中芽孢杆菌属和梭菌属细菌的演替规律复杂,更替过程更复杂,需要我们进行更深入的探讨。
(3)对芽孢杆菌属和梭菌属DGGE图谱中优势条带进行了回收和测序,结果发现,堆肥过程中大部分都是未能培养的芽孢杆菌,并得到了利用纯培养方法未能分离到的芽孢杆菌属细菌信息,它们包括：Bacillus cereus, Bacillus marisflavi, Bacillus koreensis和Bacillus fordii,而Bacillus cereus也是猪粪堆肥过程中的优势菌,存在于整个堆肥过程中。堆肥过程中都是未能培养的梭茵,未检测到可培养的梭菌。说明PCR-DGGE在分子生态学方面具有更大的优越性,可以获得更多的微生物信息。
6. FISH分析
(1)对芽孢杆菌属特异性探针LGC353b和梭菌属特异性探针Chis150的杂交条件进行了优化。探针LGC353b和Chis150的最佳杂交温度都是46℃；最佳杂交时间分别为4h和5.5h；杂交液中最佳甲酰胺浓度分别为20%和35%。
(2) FISH分析结果显示,猪粪堆肥过程中不同时期、不同高度层堆肥样品中总细菌、芽孢杆菌属和梭菌属细菌数目有着明显的动态变化和的时空分布特征。三者的FISH检测数量级都达到了106个/克(湿重)的数量级。总细菌、芽孢杆菌属、梭菌属的时间分布特征是细菌总数随着温度的升高而增加,随着温度的下降而减少；空间分布特征是处于同一堆肥阶段的三个不同高度层中,中层样品中芽孢杆菌属细菌数目和其占总细菌数的百分比是最低的；梭菌属的空间分布特征不明显。
The rapid development of large-scale pig industry has improved people's living standard, on the other hand, concentrated feces exhaust resulted in remendous pressure on the environment. Now, looking for livestock and poulty harmless and recycling technique having good environmental effect has become an important issue faced large-scale livestock and poultry breeding in China. Composting technology is typically used for organic solid waste recycling. Successful composting can achieve harmless reduction and recycling processing. So, composting was considered to be one of the effective way of pig industry and the environment coordinated development and the study of microbial diversity in the process of composting is a hot research topic in recent years.
1. Composting experiment
Composting experiment was conducted by using forced ventilation composting system, swine manure was the major component and rice bran was used to as the conditioner for swine composting. The initial carbon nitrogen ratio was adjusted to20, the initial moisture content of the mixture was60%. The composting process went through three classic phases:rapid temperature-rising stage, sustained high-temperature stage, and gradual cooling stage. The temperature of the middle layer of pile achieved60℃within5d, and kept above50℃for30days and55℃for25days. The composting process lasted for42days. When the composting process finished, the volume of the composting pile decreased by42.68%. The E. coli and killing rate of ascarid eggs were100%and0.069, which met the sanitary standards of the non-hazardous treatment of night soil (GB7959-87). The composting experiment had been successful, which laid a good foundation for the later studies.
2. Nucleic acid extraction
A method for obtaining DNA from swine manure composting was modified, based on the procedures described by Zhou (1996), and the genomic DNA were obtained from9samples of rapid temperature-rising stage, sustained high-temperature stage, and gradual cooling stage in the composting process, and was purified with Commercial kit. The length of the DNA fragment was above2Kbp by detecting with agarose gel eletrophoresis. The concentration of purified DNA was about370ng/uL by using DNA/RNA concentration determinator. The A260/A280ratio was1.766. The results showed that the quantity of nucleinic acid was high by using this method and could meet the need of molecular biology research.
3. Separation and identification
(1) A total of540Bacillus were obtained from the samples of different phrases and layers in swine manure composting using traditional plate dilution method. An RFLP analysis was conducted for these isolates which were then divided eight different patterns. Basing on physiology and biochemistry tests and16S rDNA sequence analysis, the diversity of cultured Bacillus in swine manure composting was obtained. There are Bacillus subtilis, Bacillus megaterium, Bacillus anthracis, Bacillus thuringiensis, Bacillus cereus, Bacillus pycnus, Bacillus oleronius and Bacillus circulans.
(2)16S rDNA sequences of eight different Bacillus were obtained, and have been deposited in GenBank database under accession numbers JF833086-JT833093.
4. Temporal-spatial distribution of cultured Bacillus
The results of RFLP analysis showed that the diversity of cultured Bacillus was rich and the characteristic of temporal-spatial distribution was obvious. Bacillus subtilis was the dominant bacteria in the whole composting; the dominant bacteria at rapid temperature-rising stage and gradual cooling stage also included Bacillus anthracis, Bacillus cereus and Bacillus circulans, respectively. The diversity of cultured Bacillus in the samples of middle layer at stages of rapid temperature-rising and gradual cooling was relatively more richer, including5different Bacillus. The diversities of bottom layer at rapid temperature-rising stage and top and middle layers at high temperature stage were the lowest.
5. PCR-DGGE analysis
(1) The results of PCR-DGGE analysis showed that the bacterial diversities of Bacillus and Clostridium in the samples of different stages and different locations in swine manure composting were comparatively rich. The dominant bands in PCR-DGGR maps achieved to over10. The diversity of Bacillus in the sample of high temperature stage was relatively more richer; and the structure of population of Bacillus existed obvious changes, while the change of populative structure of Clostridium was not obvious, which indicated that precious resources of Bacillus and Clostridium in the swine manure composting was very rich.
(2) The results of fingerprinting clustering and Cs value analysis showed that the diversities of Bacillus in the samples of different locations each other at the same stage was the highest. However, the diversities of Clostridium in9samples each other were very high, indicating that the composition of Clostridium among all samples were similar. The change laws of the Cs value of two genus among9samples were not obvious, indicating that the succession of Bacillus and Clostridium in the composting process was complicated, and the succession process was more complicated, further studies are needed.
(3) The dominant bands in the Bacillus and Clostridium maps were retrieved and sequenced, the results indicated that the most Bacillus species were uncultured, some Bacillus which were not found using cultured method were obtained, including Bacillus cereus, Bacillus marisflavi, Bacillus koreensis and Bacillus fordii, and Bacillus cereus was also the dominant bacteria in the whole composting. All Clostridium species were uncultured, and cultured Clostridium were not detected. All of these indicated that PCE-DGGE technology is provided with more advantages in the respect of molecular biology.
6. FISH analysis
(1) The hybridization conditions of the specific probes of Bacillus LGC353b and Clostridium Chis150have been optimized. The optimal hybridization temperature of two probes were46℃, optimal hybridization time were4h and5.5, and the formamide concentration in hybridization buffer were20%and30%, respectively.
(2) The results of FISH analysis showed that the number of total cells, Bacillus and Clostridium were presented obvious dynamic changes and temporal-spatial distribution characteristic. The detected limits of them using FISH method were approximately106cells g"'(wet weight). The characteristic of temporal distribution of total cells, Bacillus and Clostridium was that the number of cells detected would rise with the rising of temperature and vice versa; the spatial distribution of Bacillus bacteria was that the number and the relative level of the Bacillus cells detected in the middle-level of composting were the lowest at each of composting, while that of Clostridium in the compositing was not obvious.
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