兔粪堆肥控制参数研究及其微生物群落变化的PCR-DGGE分析
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摘要
本试验采用主动通风方式,以兔粪和菌渣为原料进行室内高温堆肥试验。设置了2个试验组和2个纯兔粪对照组,在为期51d的堆肥过程中,研究了堆体温度、含水率、pH、总有机碳、总氮、总磷、总钾、种子发芽指数、细菌群落总数和大肠杆菌菌群数的变化规律及其影响因素。利用PCR-DGGE分子生物学方法研究了在堆肥高温发酵过程中微生物群落结构动态及其变化规律,并分析了微生物群落结构演替与理化指标之间的关系,从而进一步了解兔粪高温发酵的作用机制。旨在为兔粪高温堆肥过程中的辅料选择、配方确定以及控制各种影响因素等提供科学依据,从而探索一种兔粪无害化、减量化和资源化利用的高效实用的堆肥方法,为规模化兔场兔粪堆肥处理提供技术储备。试验结果表明：
1、兔粪高温堆肥生产中,利用兔粪和菌渣按2.6：1混合,初始水分控制在55%-60%,进行堆肥能正常发酵,且能达到无害化处理的目的,在生产中可以此为参考对兔粪进行堆肥生产有机肥料。
2、在整个堆制过程中,兔粪菌渣混合堆肥和纯兔粪堆肥的TOC相对含量和C／N持续下降,TN相对含量则持续升高,TP、TK的绝对含量基本不变,这与其他畜禽粪便堆肥规律相似。细菌总数整体呈下降趋势,纯兔粪组下降趋势不明显；大肠杆菌在堆肥高温期大部分死亡,在腐熟阶段少量苏醒繁殖。
3、整个堆肥过程中,试验组和对照组的pH分别为8.70和8.73；C／N分别为22.53和17.86；随着堆肥化的进行抑制种子发芽的物质被慢慢消除,在37天以后种子发芽指数全部达到0.8以上,以上各指标均基本符合国家堆肥腐熟的标准。
4、利用PCR-DGGE技术对堆肥微生物的群落结构进行了研究,发现堆肥过程中微生物群落结构的演替比较清楚。DGGE指纹图谱显示堆肥细菌的变化规律：“升高-降低-平稳”,在高温阶段有比较丰富的优势细菌群落。
5、在整个堆制期间,DGGE指纹图谱多样性与传统培养细菌结果并不十分吻合；对照组的细菌多样性指数平均为1.82,略高于试验组1.74,表明,对照组中细菌群落数多于试验组,但其优势菌群不如试验组明显；堆体中微生物主要来源于兔粪中的土著微生物,所以二者的DGGE条带相似度在50%以上；兔粪高温堆肥中细菌多样性与堆体温度变化趋势相似而与C／N比变化趋相反。
6、试验中对照组除pH值变化与试验组差异较大外,其它参数变化均与试验组趋于一致。50℃以上的高温期能持续9天,大肠杆菌和GI值也符合堆肥腐熟的要求,TN、TP水平略高于试验组。说明生产中如调节好初始水分,采用被动通风堆肥方式,即可以利用兔粪不加辅料进行堆肥,这样可以使兔粪堆肥更加简单,成本更低。
The mushroom residue were used as the auxiliary organic materials in the rabbit manure composting experiment, and in which aeration windrow composting process was adopted. Two trial groups(mushroom residue-rabbit manure) and two control group (pure rabbit manure), were set up respectively,composting process of about 51 days. The temperature of piles, moisture content, pH,total organic carbon content, total kjeldahl nitrogen content,C/N,total phosphorus content,total potassium content,seed germination index(GI), general bacterial population and then E.coli population of were studied.Use of the biological technique of Denaturing Gradien Gel Electrophoresis understand the development and variation of the microbial structures during the composting,the relationship between the physical characteristic,chemical parameters and the succession of microbial communities, further studies the mechanism of thermophilic fermentation during rabbit manure composting.The experiment in order to understand the physical characteristic and chemical parameters influencing composting and the auxiliary organic materials selecting provide scientific basis for rabbit manure composting. Explored rabbit manures resources,minimization,using the sound of effective channel and provide theoretical basis for rabbit manure composting treatment on large rabbit farms. The following results have been obtained:
1. Rabbit manure and mushroom residue cooperation ratio were 2.6:1, initial moisture content of piles was maintained to 55%-60% during the composting, compost pile temperature arrived at the normal fermentation temperature in composting process athe results taken as reference in the actual production.
2. Test groups and control groups total organic canbon and C:N ratio were showed downtrend, total kjeldahl nitrogen relative content was increased, total phosphorus and total potassium was maintained to unchangeableness throughout the experiment,similar trend was observed with respect to other domestic animal manure. Test groups the decrease general bacterial population in the process while the decrease was not obvious in control groups.the E.coli were mass mortality in the high temperature period,bacterial reproduction in the cooling-maturity period.
3. Total organic carbon,pH,C:N ratio and seed germination index can applied to maturity evaluation of composting productions well.Test groups and control groups,pH were 8.70 and 8.73,C:N ratio were 22.53 and 17.86,the seed germination index above to 0.8, all parameters which indicate the compost was complete mature.
4. PCR-DGGE was used to analyzed the structure of the microbial communities. The result showed that the successional of microbial communities of the compost was obviously during the composting. Denaturing gradien gel electrophoresis patterns showed that:the composting process in the quantities of becateria change in ghe ternd was "higher-lower-constancy",in the high temperature period the quantity of microbial was more.
5. In the whole composting process, quantity of microbial became more and more,this result accrod with the results by conventional culture. The average shannon-weaver index of bacteria in control groups were higher than test groups (1.82 versus 1.72 to bacteria diversity),showed that bacterial communities in the control group more than in the test groups, but the dominant bacteria were significantly better than test groups. The control groups and the test groups similarity coefficient were above 50% due to the piles microbial from rabbit manure indigenous microbial The bacterial number change tendency was similar to the trend of temperature and opposite tend with the C:N ratio.
6. In addition,the change of pH in control group and test groups quite different, other parameters were in line with test group. Control group duration of 50℃high temperature period 9 days, E.coli and GI is also consistent with the requirements of compost maturity, total kjeldahl nitrogen and total phosphorus slightly higher than the test group.The results also showed that the raw rabbit manure could be directly used in composting after moisture content was adjusted to a proper range, and the process was not only simple but also low cost.
1、兔粪高温堆肥生产中,利用兔粪和菌渣按2.6：1混合,初始水分控制在55%-60%,进行堆肥能正常发酵,且能达到无害化处理的目的,在生产中可以此为参考对兔粪进行堆肥生产有机肥料。
2、在整个堆制过程中,兔粪菌渣混合堆肥和纯兔粪堆肥的TOC相对含量和C／N持续下降,TN相对含量则持续升高,TP、TK的绝对含量基本不变,这与其他畜禽粪便堆肥规律相似。细菌总数整体呈下降趋势,纯兔粪组下降趋势不明显；大肠杆菌在堆肥高温期大部分死亡,在腐熟阶段少量苏醒繁殖。
3、整个堆肥过程中,试验组和对照组的pH分别为8.70和8.73；C／N分别为22.53和17.86；随着堆肥化的进行抑制种子发芽的物质被慢慢消除,在37天以后种子发芽指数全部达到0.8以上,以上各指标均基本符合国家堆肥腐熟的标准。
4、利用PCR-DGGE技术对堆肥微生物的群落结构进行了研究,发现堆肥过程中微生物群落结构的演替比较清楚。DGGE指纹图谱显示堆肥细菌的变化规律：“升高-降低-平稳”,在高温阶段有比较丰富的优势细菌群落。
5、在整个堆制期间,DGGE指纹图谱多样性与传统培养细菌结果并不十分吻合；对照组的细菌多样性指数平均为1.82,略高于试验组1.74,表明,对照组中细菌群落数多于试验组,但其优势菌群不如试验组明显；堆体中微生物主要来源于兔粪中的土著微生物,所以二者的DGGE条带相似度在50%以上；兔粪高温堆肥中细菌多样性与堆体温度变化趋势相似而与C／N比变化趋相反。
6、试验中对照组除pH值变化与试验组差异较大外,其它参数变化均与试验组趋于一致。50℃以上的高温期能持续9天,大肠杆菌和GI值也符合堆肥腐熟的要求,TN、TP水平略高于试验组。说明生产中如调节好初始水分,采用被动通风堆肥方式,即可以利用兔粪不加辅料进行堆肥,这样可以使兔粪堆肥更加简单,成本更低。
The mushroom residue were used as the auxiliary organic materials in the rabbit manure composting experiment, and in which aeration windrow composting process was adopted. Two trial groups(mushroom residue-rabbit manure) and two control group (pure rabbit manure), were set up respectively,composting process of about 51 days. The temperature of piles, moisture content, pH,total organic carbon content, total kjeldahl nitrogen content,C/N,total phosphorus content,total potassium content,seed germination index(GI), general bacterial population and then E.coli population of were studied.Use of the biological technique of Denaturing Gradien Gel Electrophoresis understand the development and variation of the microbial structures during the composting,the relationship between the physical characteristic,chemical parameters and the succession of microbial communities, further studies the mechanism of thermophilic fermentation during rabbit manure composting.The experiment in order to understand the physical characteristic and chemical parameters influencing composting and the auxiliary organic materials selecting provide scientific basis for rabbit manure composting. Explored rabbit manures resources,minimization,using the sound of effective channel and provide theoretical basis for rabbit manure composting treatment on large rabbit farms. The following results have been obtained:
1. Rabbit manure and mushroom residue cooperation ratio were 2.6:1, initial moisture content of piles was maintained to 55%-60% during the composting, compost pile temperature arrived at the normal fermentation temperature in composting process athe results taken as reference in the actual production.
2. Test groups and control groups total organic canbon and C:N ratio were showed downtrend, total kjeldahl nitrogen relative content was increased, total phosphorus and total potassium was maintained to unchangeableness throughout the experiment,similar trend was observed with respect to other domestic animal manure. Test groups the decrease general bacterial population in the process while the decrease was not obvious in control groups.the E.coli were mass mortality in the high temperature period,bacterial reproduction in the cooling-maturity period.
3. Total organic carbon,pH,C:N ratio and seed germination index can applied to maturity evaluation of composting productions well.Test groups and control groups,pH were 8.70 and 8.73,C:N ratio were 22.53 and 17.86,the seed germination index above to 0.8, all parameters which indicate the compost was complete mature.
4. PCR-DGGE was used to analyzed the structure of the microbial communities. The result showed that the successional of microbial communities of the compost was obviously during the composting. Denaturing gradien gel electrophoresis patterns showed that:the composting process in the quantities of becateria change in ghe ternd was "higher-lower-constancy",in the high temperature period the quantity of microbial was more.
5. In the whole composting process, quantity of microbial became more and more,this result accrod with the results by conventional culture. The average shannon-weaver index of bacteria in control groups were higher than test groups (1.82 versus 1.72 to bacteria diversity),showed that bacterial communities in the control group more than in the test groups, but the dominant bacteria were significantly better than test groups. The control groups and the test groups similarity coefficient were above 50% due to the piles microbial from rabbit manure indigenous microbial The bacterial number change tendency was similar to the trend of temperature and opposite tend with the C:N ratio.
6. In addition,the change of pH in control group and test groups quite different, other parameters were in line with test group. Control group duration of 50℃high temperature period 9 days, E.coli and GI is also consistent with the requirements of compost maturity, total kjeldahl nitrogen and total phosphorus slightly higher than the test group.The results also showed that the raw rabbit manure could be directly used in composting after moisture content was adjusted to a proper range, and the process was not only simple but also low cost.
引文
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