利用PCR-SSCP技术分析猪粪堆肥微生物群落动态
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摘要
本试验以猪粪和稻草为原料进行室内堆肥，设置2个试验组分别为堆体1(被动通风静态垛)和堆体2(强制通风静态垛)，在为期30d的堆程中，均测定了2个堆体上、中、下层的一系列理化指标如温度、CO_2、PH、含水率、总有机碳(TOC)、全氮(TKN)和C／N随时间的变化，也用分子生物学方法(PCR-SSCP)检测了每堆体三层的细菌和真菌群落数随时间的变化，并调用SAS6.12软件中的T检验程序和相关分析程序处理数据，旨在研究堆肥微生物群落结构动态，其中涉及到同一堆体不同堆肥阶段微生物群落结构比较；不同堆体或不同堆肥工艺微生物群落结构比较；微生物群落结构演替与堆肥理化性质变化的关系；真菌群落动态和细菌群落动态的比较。试验结果如下：
不论是从堆料的物理性状还是化学指标来看，堆体0～30d都快速经历了完整的常规堆肥一次发酵过程。且微生物分子生物学检测(PCR—SSCP)表明这个过程中细菌和真菌构成的微生物群落发生了很大的变化。
堆体内细菌类群数在0～100d的堆肥期中总体呈振荡下降，梯级递减的趋势。而2个堆体的真菌群落动态表明：第9d真菌群落已有繁荣迹象，但15～24d处于最繁荣的阶段。
将细菌与真菌群落动态进行比较可知：两堆体各层的细菌变化与真菌变化主要表现为较弱的负相关(r＜0，p＞0.05)；堆体各层条带数均是真菌平均数大于细菌平均数：但全过程中，真菌类群数的变异要大于细菌；不管是真菌还是细菌，强制通风堆每个点的类群平均数明显大于被动通风堆(真菌为3.128个对2.641个；细菌为2.436个对1.54个)。
将细菌和真菌的类群变化结合起来，得出：在14d以前，细菌类群明显占据优势，14d以后真菌类群数超过细菌，并一直处于优势状态，尤其是在15～24d内十分突出，类群数远远高于细菌。
将理化指标平均数与细菌和真菌的相关性进行分析可知：细菌类群数与温度和CO_2释放量呈显著正相关(r＞0，p＜0.05)，与OC和C／N呈极显著正相关(r＞0，p＜0.01)，而与KN呈显著负相关((r＜0，p＜0.05)；真菌类群数只与pH和含水率呈显著正相关(r＞0，p＜0.05)。
细菌类群数量变化与堆体的温度和C／N变化趋势比较一致，在第一次发酵(0～30d阶段)中，18d以前是细菌最活跃的阶段，之后细菌成为群落中的劣势微生物类群。由于堆肥30d以前堆体温度主要在44～71℃，在SSCP图谱上的条带可能是好氧高温细菌占主导。
利用取样日的温度、C／N、KN和真菌类群数的标准化数据进行比较作图，可以看出：真菌最适宜的温度可能是42.5～50℃；C／N变得不适宜于细菌生长似乎也间接有利于真菌的繁殖；而KN的相对含量过高可能对真菌的繁殖有抑制作用，从而协同温度造成了真菌类群数下降。
把细菌和真菌的类群数数据除以相应的平均数，得到无量纲标准化数据可知，第15d是一个明显的分界线，前期细菌的优势地位在15d后被真菌取而代之，从整个微生物群落的角度看，第27d以前群落是兴旺的，以后则趋向衰弱。
In this study, swine manure and rice straw were used to be compostingmaterials indoor. Two trial groups were set up, which were called staticwindrow of passive aeration (Pile One) and static windrow of forced aeration(Pile Two), respectively. Physical and Chemical parameters, such astemperature, carbon dioxide, pH, moisture content, total organic carbon,total kjeldahl nitrogen, the ratio of carbon to nitrogen, were determined inthirty days. The test samples were taken from both piles at three points ofdifferent depth every 3d, In addition, bacteria and fungi populations weredetected with the biological technique of Single Strand ConformationPolymorphism. The procedures of t-test and correlation analysis in thesoftware of SAS 6.12 were applied to analyze statistically the data obtainedin the experiment in order to understand the development of the microbialstructures within and between piles in different composting phases, therelationship between macroscopic parameters and the succession Of microbialcommunities, and comparing the community of fungi with that of bacterial.
The variance curve of physical and chemical parameters indicated that thetwo piles hadexperienced an integrity course of fermentation in compostingprophase, while the results of PCR-SSCP detection manifested that themicrobial populations, composed of bacteria and fungi, changed greatly.
The number of bacterial groups descended vibrationally in the 0～100dcomposting process. Community dynamics of fungi in two piles proved that fungipopulation had already flourished in the 9d, whose prosperous stage was the15～24d.
Comparing the community of bacterial and fungi, it can be realized that,the correlation analysis of community succession between bacterial and fungimainly reflected weakly negative correlation (r＜0,p＞0.05),that more fungispecies OTUs were amplified by PCR-SSCP than bacteria species in this researchdue to the length of fungi primer being shorter than bacteria primer, that the variance of fungi species was significantly greater than that of bacteriaspecies, that the average numbers of bacteria and fungi in the pile of forcedaeration were higher than those in passive aeration pile (2.436 versus 1.54to bacteria species and 3.128 versus 2.641 to fungi species).
Integrating community variation of bacterial and fungi, the results weregained that, the bacterial community predominated obviously before the 15thday. However, the number of fungi groups surpassed that of bacterial groupsand had the advantage all the time, especially in the period of 15～24d, whichkept far away from that of bacterial groups.
The number of bacterial groups was significantly positive correlativewith the change of the temperature and carbon dioxide (r＞0,p＜0. 05), and hadextremely significantly positive correlation with the change of total organiccarbon and the ratio of carbon to nitrogen(r＞0,p＜0.01),but had significantlynegative correlation with the change of total kjeldahl nitrogen. While thenumber of fungi groups was significant positive correlative with the changeof pH and moisture content.
The bacterial number change tendency was similar to the trend oftemperature and the ratio of carbon to nitrogen changing. In 0～30d, the activestage of bacteria was before the eighteenth day, however, bacterial communitybecame inferior microbial species after that phase. Because the piletemperature was mainly in the scope of forty-four celsius degree toseventy-one celsius degree during thirty composting days, aerobic andthermophilic bacteria amplified by PCR-Single Strand ConformationPolymorphism may be the prevailing microbial population.
Through the curve of the standard data derived from the fungi communitynumber and the macroscopic parameters such as temperature, the ratio of carbonto nitrogen and total kjeldahl nitrogen, it was observed that, the optimaltemperature for fungi may be 42.5～50 celsius degree. And the ratio of carbonbecame unsuitable for bacteria to grow, which may benefit the growth of fungi.The relative amount of total kjeldahl nitrogen became more and more high, which may inhibit the propagating of fungi in coordination with temperature.
Standard data transformed from the ratio of the number of bacterial andfungi groups to their corresponding average number, indicated that, thebacterial community was predominated before the 15th day while the fungicommunity was prosperous afterwards. In conclusion, the compostingmicroorganism community was thrifty before the 27th day, but ran downafterward.
不论是从堆料的物理性状还是化学指标来看，堆体0～30d都快速经历了完整的常规堆肥一次发酵过程。且微生物分子生物学检测(PCR—SSCP)表明这个过程中细菌和真菌构成的微生物群落发生了很大的变化。
堆体内细菌类群数在0～100d的堆肥期中总体呈振荡下降，梯级递减的趋势。而2个堆体的真菌群落动态表明：第9d真菌群落已有繁荣迹象，但15～24d处于最繁荣的阶段。
将细菌与真菌群落动态进行比较可知：两堆体各层的细菌变化与真菌变化主要表现为较弱的负相关(r＜0，p＞0.05)；堆体各层条带数均是真菌平均数大于细菌平均数：但全过程中，真菌类群数的变异要大于细菌；不管是真菌还是细菌，强制通风堆每个点的类群平均数明显大于被动通风堆(真菌为3.128个对2.641个；细菌为2.436个对1.54个)。
将细菌和真菌的类群变化结合起来，得出：在14d以前，细菌类群明显占据优势，14d以后真菌类群数超过细菌，并一直处于优势状态，尤其是在15～24d内十分突出，类群数远远高于细菌。
将理化指标平均数与细菌和真菌的相关性进行分析可知：细菌类群数与温度和CO_2释放量呈显著正相关(r＞0，p＜0.05)，与OC和C／N呈极显著正相关(r＞0，p＜0.01)，而与KN呈显著负相关((r＜0，p＜0.05)；真菌类群数只与pH和含水率呈显著正相关(r＞0，p＜0.05)。
细菌类群数量变化与堆体的温度和C／N变化趋势比较一致，在第一次发酵(0～30d阶段)中，18d以前是细菌最活跃的阶段，之后细菌成为群落中的劣势微生物类群。由于堆肥30d以前堆体温度主要在44～71℃，在SSCP图谱上的条带可能是好氧高温细菌占主导。
利用取样日的温度、C／N、KN和真菌类群数的标准化数据进行比较作图，可以看出：真菌最适宜的温度可能是42.5～50℃；C／N变得不适宜于细菌生长似乎也间接有利于真菌的繁殖；而KN的相对含量过高可能对真菌的繁殖有抑制作用，从而协同温度造成了真菌类群数下降。
把细菌和真菌的类群数数据除以相应的平均数，得到无量纲标准化数据可知，第15d是一个明显的分界线，前期细菌的优势地位在15d后被真菌取而代之，从整个微生物群落的角度看，第27d以前群落是兴旺的，以后则趋向衰弱。
In this study, swine manure and rice straw were used to be compostingmaterials indoor. Two trial groups were set up, which were called staticwindrow of passive aeration (Pile One) and static windrow of forced aeration(Pile Two), respectively. Physical and Chemical parameters, such astemperature, carbon dioxide, pH, moisture content, total organic carbon,total kjeldahl nitrogen, the ratio of carbon to nitrogen, were determined inthirty days. The test samples were taken from both piles at three points ofdifferent depth every 3d, In addition, bacteria and fungi populations weredetected with the biological technique of Single Strand ConformationPolymorphism. The procedures of t-test and correlation analysis in thesoftware of SAS 6.12 were applied to analyze statistically the data obtainedin the experiment in order to understand the development of the microbialstructures within and between piles in different composting phases, therelationship between macroscopic parameters and the succession Of microbialcommunities, and comparing the community of fungi with that of bacterial.
The variance curve of physical and chemical parameters indicated that thetwo piles hadexperienced an integrity course of fermentation in compostingprophase, while the results of PCR-SSCP detection manifested that themicrobial populations, composed of bacteria and fungi, changed greatly.
The number of bacterial groups descended vibrationally in the 0～100dcomposting process. Community dynamics of fungi in two piles proved that fungipopulation had already flourished in the 9d, whose prosperous stage was the15～24d.
Comparing the community of bacterial and fungi, it can be realized that,the correlation analysis of community succession between bacterial and fungimainly reflected weakly negative correlation (r＜0,p＞0.05),that more fungispecies OTUs were amplified by PCR-SSCP than bacteria species in this researchdue to the length of fungi primer being shorter than bacteria primer, that the variance of fungi species was significantly greater than that of bacteriaspecies, that the average numbers of bacteria and fungi in the pile of forcedaeration were higher than those in passive aeration pile (2.436 versus 1.54to bacteria species and 3.128 versus 2.641 to fungi species).
Integrating community variation of bacterial and fungi, the results weregained that, the bacterial community predominated obviously before the 15thday. However, the number of fungi groups surpassed that of bacterial groupsand had the advantage all the time, especially in the period of 15～24d, whichkept far away from that of bacterial groups.
The number of bacterial groups was significantly positive correlativewith the change of the temperature and carbon dioxide (r＞0,p＜0. 05), and hadextremely significantly positive correlation with the change of total organiccarbon and the ratio of carbon to nitrogen(r＞0,p＜0.01),but had significantlynegative correlation with the change of total kjeldahl nitrogen. While thenumber of fungi groups was significant positive correlative with the changeof pH and moisture content.
The bacterial number change tendency was similar to the trend oftemperature and the ratio of carbon to nitrogen changing. In 0～30d, the activestage of bacteria was before the eighteenth day, however, bacterial communitybecame inferior microbial species after that phase. Because the piletemperature was mainly in the scope of forty-four celsius degree toseventy-one celsius degree during thirty composting days, aerobic andthermophilic bacteria amplified by PCR-Single Strand ConformationPolymorphism may be the prevailing microbial population.
Through the curve of the standard data derived from the fungi communitynumber and the macroscopic parameters such as temperature, the ratio of carbonto nitrogen and total kjeldahl nitrogen, it was observed that, the optimaltemperature for fungi may be 42.5～50 celsius degree. And the ratio of carbonbecame unsuitable for bacteria to grow, which may benefit the growth of fungi.The relative amount of total kjeldahl nitrogen became more and more high, which may inhibit the propagating of fungi in coordination with temperature.
Standard data transformed from the ratio of the number of bacterial andfungi groups to their corresponding average number, indicated that, thebacterial community was predominated before the 15th day while the fungicommunity was prosperous afterwards. In conclusion, the compostingmicroorganism community was thrifty before the 27th day, but ran downafterward.
引文
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