基于分子生物学技术的堆肥微生物群落、功能与应用研究
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摘要
堆肥法可以实现有机城生活垃圾的减量化、无害化和资源化处理,是一种很有应用前景垃圾处理技术,但目前堆肥法周期过长、产品质量较差,其在国内的应用较少,因此,提高堆肥效率和产品质量是促进其在生活垃圾处理应用中的关键。微生物是堆肥系统中最主要的生物因素,全面、准确地掌握堆肥中微生物的群落结构、种群动态、功能活性可为堆肥工艺的优化调整与技术革新提供最重要的微生物学依据。现代分子生物学技术可以在非培养的条件下研究环境微生物,应用聚合酶链式反应(PCR)、变性梯度凝胶电泳(DGGE)、实时荧光定量PCR (qPCR)及DNA测序等现代分子生物学技术及时、快速、全面地获取堆肥微生物的群落组成和种群动态等信息对优化、改进现有堆肥工艺技术,提高堆肥效率与产品质量具有重要意义。
本论文以分子生物学技术为主要研究手段,结合使用传统分离培养技术,全面、深入地研究了堆肥系统中的微生物群落,并以获得的微生物学信息进行了堆肥工艺改进、技术革新方面的有益探索。
采用传统堆肥法对以人工分选到的厨余垃圾进行堆肥,证明厨余垃圾是良好的堆肥材料,微生物在堆肥高温期降解的有机质量占堆肥过程中有机质降解总量的60%以上,堆肥高温期微生物对有机质的降解和堆肥的腐熟贡献最大；以PCR、 DGGE、DNA测序等对堆肥高温期样品中的微生物进行研究,16/18S rDNA序列分析结果表明微生物群落在堆肥高温期存在较明显的动态演替,其中优势细菌为芽孢杆菌,优势放线菌为链霉菌和诺卡氏菌,优势真菌为曲霉。
基于传统堆肥过程高温期的堆肥材料降解速度快、堆体内微生物群落多样性较高的研究结果,提出了一种通过人为加热方式使堆肥全过程均在50℃高温下完成的全程高温堆肥法,并以4种采用不同温度控制方式的全程高温堆肥法与传统堆肥法进行比较研究,结果表明直接将堆体温度快速升高至50℃以上但不控制最高温度的全程高温堆肥法可以获得最快的腐熟速度,其堆肥周期为14天,而传统堆肥法的周期为28天。在比较研究的基础上总结出一种较佳的全程高温堆肥法：在堆肥初期采取外加热源的方式使堆体温度快速升高到50℃以上并使其一直维持在50℃以上、保持堆体约60%的含水量、以0.25L/(min·kg)的供气量保持堆体好氧的环境并每天翻堆。
结合使用PCR、DGGE及DNA测序、qPCR等分子生物学技术和传统分离培养技术对比研究了传统堆肥法和全程高温堆肥法中的微生物群落,结果表明全程高温堆肥法降低了堆体中细菌群落的多样性,优势细菌以嗜热的芽孢杆菌为主,增加了细菌尤其是芽孢杆菌的数量,但过高的堆体温度会使细菌数量减少；全程高温堆肥法提高了堆体中放线菌群落的多样性,增加了放线菌的数量,优势放线菌包括链霉菌、微球菌、无枝酸菌和其他一些嗜热放线菌；全程高温堆肥法会强烈抑制真菌的生长。
以高温期堆肥和土壤等的悬浮液为接种剂、以稻草和木屑混合物为培养基在55℃水浴中驯化培养30天,以羧甲基纤维素钠平板培养基对其分离培养、以纤维素刚果红培养基对嗜热菌的纤维素降解能力进行筛选,获得了15株纤维素高效降解菌。生理生化、形态学和16S rDNA序列分析结果显示其中13株与芽孢杆菌有97%以上的相似性,1株与波茨坦短芽孢杆菌有97%以上的相似性,1株与类芽孢杆菌有80%的相似性,可能是某未知菌种。将单个菌株的培养液以5mL/kg堆肥的接种量添加至堆体PA中,结果显示该堆体的高温期比不接种的堆体PN提早1天进入高温期,高温期的时间也延长了2天；堆体PA中的TOC含量比未接种的堆体PN中的降得更快,但氮元素的损失更小；以C/N值对其堆肥周期进行评价,结果显示堆体PA和PN分别在第21天和27天达到腐熟,添加接种剂可以提前6天实现堆肥的腐熟；以纤维素和半纤维素的降解为评价指标探讨了接种剂对堆肥的影响,结果表明堆肥的高温期是降解纤维素和半纤维素主要阶段,添加接种剂促进了纤维素和半纤维素的降解；以有机质消耗动力学方程评价接种剂对堆肥的影响,结果显示接种剂的添加使有机质的降解速度更快,降解度更高,因而接种剂可以加速堆肥的腐熟并获得更稳定的产品。
Composting is a promising technology which can simultaneously achieve the reduction, reclamation and safe treatment of organic municipal solid waste (MSW). The most important thing for the promotion of composting in MSW treatment is to improve the composting efficiency and the quality of compost since the long composting cycle and the low quality of compost have restricted the application of composting in China. Microorganism is the major biological factor in a composting system. An understanding of the microbial community structure, the population dynamics and the functions of microbes will provide us important microbiological knowledge for the improvement of composting. With the help of modern molecular biology techniques, researchers can study microorganisms without culturing them, and by using molecular biology techniques, such as polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), real-time fluorescence quantitative PCR (qPCR) and DNA sequencing, researchers can obtain the microbial community structure and the population dynamics promptly, which is very important for the the improvement of composting.
In this paper, molecular biology techniques, combinated with culture method, was applied for investigating the microbial community in compost system, and the information about the microbial community was used as basic kwonledge for the improvement of composting technology.
Traditional composting method was used for composting kitchen discards that manually separated from MSW, and composting was proved to be good compost materials. Among the degraded organic matters during the active composting phases, more than60%of them were degraded in the high-temperature phase, which indicated that the high-temperature phase is the most important phsase for the degradation of organic matters. PCR, DGGE and DNA sequencing were applied for investigating the microbial community of samples from the high-temperature phase, and the analysis results of16/18S rDNA sequence showed a relatively abvious dynamics of the microbial community. The studies also showed that, in the samples from the high-temperature phase, the dominant bacteria, the dominant actinomysetes and the dominnat fungi are Bacillus spp., Streptomyces spp. and Nocardia spp., and Aspergillus spp., respectively.
Based on the study results above, the paper proposed a new method of continuously thermophilic composting (CTC), which composted the materials under high-temperature (>50℃) during the whole composting process. Four runs with different operation manner were compared with a traditional composting run. The run which directly heated the materials to50℃without any other temperature control showed the fastest speed of degradation and matured in14days, while the composting cycle of traditional run was28days. On the basis of research results above, the paper suggested an effective CTC method as follows:heat the materials up to50℃in the initial phase of compossting, maintain the pile temperature at least at50℃, keep a moisture content of60%, supply a gas flow of0.25L/(min-kg) and turn the pile daily.
With the help of PCR, DGGE and DNA sequencing, qPCR and traditional culture techniques, the paper investigated the microbial community in the traditional composting process and the CTC process. The study showed that the CTC methods decreased the diversity of bacterial community and increased the numbers of bacteria, and the major dominant bacteria are Bacillus spp.. The study also showd that the CTC methods increased the diversity of actinomycetes and the numbers of actinomycetes, and the dominant actinomycetes are Streptomyces spp., Mocromonospora spp., Amycolatopsis spp. and some other thermophilic actinomycetes. In addition, the CTC methods might have suppressed the growth of fungi.
In the study, suspension of soil and compost samples from high-temperature phase was used as source inouculant and the suspension was mixed with rice straw and chips, which were used as a medium. The mixture was cultured in a water bath at55℃for30days. A medium with sodium salt of carboxymethyl cellulose was used for separation of cellulolytic thermophiles from the culture mixture. Fifteen strains were selected from the cultured thermophiles after determine their degradation ability on a medium with cellulose and Congo red. Among the15strains,13strains were97%identical to Bacillus spp., one strain was97%identical to Brevibacillus borstelensis, and one strain was80%identical to Paenibacillus sp., which was determined by the biochemical or physiological identidication, the morphology characterization and the analysis of16S rDNA sequence. Each cultured strain with a volume of5mL/(kg compost) was inoculated into pile PA, with a negative control PN. The results showed a faster decrease of total organic carbon and a less loss of nitrogen in pile PA. The results also showed that pile PA matured six days earlier than pile PN, which suggested that a inoculation have spped up the mature process. The inoculant also had speed up the degradation of cellulose and hemicellulose.
本论文以分子生物学技术为主要研究手段,结合使用传统分离培养技术,全面、深入地研究了堆肥系统中的微生物群落,并以获得的微生物学信息进行了堆肥工艺改进、技术革新方面的有益探索。
采用传统堆肥法对以人工分选到的厨余垃圾进行堆肥,证明厨余垃圾是良好的堆肥材料,微生物在堆肥高温期降解的有机质量占堆肥过程中有机质降解总量的60%以上,堆肥高温期微生物对有机质的降解和堆肥的腐熟贡献最大；以PCR、 DGGE、DNA测序等对堆肥高温期样品中的微生物进行研究,16/18S rDNA序列分析结果表明微生物群落在堆肥高温期存在较明显的动态演替,其中优势细菌为芽孢杆菌,优势放线菌为链霉菌和诺卡氏菌,优势真菌为曲霉。
基于传统堆肥过程高温期的堆肥材料降解速度快、堆体内微生物群落多样性较高的研究结果,提出了一种通过人为加热方式使堆肥全过程均在50℃高温下完成的全程高温堆肥法,并以4种采用不同温度控制方式的全程高温堆肥法与传统堆肥法进行比较研究,结果表明直接将堆体温度快速升高至50℃以上但不控制最高温度的全程高温堆肥法可以获得最快的腐熟速度,其堆肥周期为14天,而传统堆肥法的周期为28天。在比较研究的基础上总结出一种较佳的全程高温堆肥法：在堆肥初期采取外加热源的方式使堆体温度快速升高到50℃以上并使其一直维持在50℃以上、保持堆体约60%的含水量、以0.25L/(min·kg)的供气量保持堆体好氧的环境并每天翻堆。
结合使用PCR、DGGE及DNA测序、qPCR等分子生物学技术和传统分离培养技术对比研究了传统堆肥法和全程高温堆肥法中的微生物群落,结果表明全程高温堆肥法降低了堆体中细菌群落的多样性,优势细菌以嗜热的芽孢杆菌为主,增加了细菌尤其是芽孢杆菌的数量,但过高的堆体温度会使细菌数量减少；全程高温堆肥法提高了堆体中放线菌群落的多样性,增加了放线菌的数量,优势放线菌包括链霉菌、微球菌、无枝酸菌和其他一些嗜热放线菌；全程高温堆肥法会强烈抑制真菌的生长。
以高温期堆肥和土壤等的悬浮液为接种剂、以稻草和木屑混合物为培养基在55℃水浴中驯化培养30天,以羧甲基纤维素钠平板培养基对其分离培养、以纤维素刚果红培养基对嗜热菌的纤维素降解能力进行筛选,获得了15株纤维素高效降解菌。生理生化、形态学和16S rDNA序列分析结果显示其中13株与芽孢杆菌有97%以上的相似性,1株与波茨坦短芽孢杆菌有97%以上的相似性,1株与类芽孢杆菌有80%的相似性,可能是某未知菌种。将单个菌株的培养液以5mL/kg堆肥的接种量添加至堆体PA中,结果显示该堆体的高温期比不接种的堆体PN提早1天进入高温期,高温期的时间也延长了2天；堆体PA中的TOC含量比未接种的堆体PN中的降得更快,但氮元素的损失更小；以C/N值对其堆肥周期进行评价,结果显示堆体PA和PN分别在第21天和27天达到腐熟,添加接种剂可以提前6天实现堆肥的腐熟；以纤维素和半纤维素的降解为评价指标探讨了接种剂对堆肥的影响,结果表明堆肥的高温期是降解纤维素和半纤维素主要阶段,添加接种剂促进了纤维素和半纤维素的降解；以有机质消耗动力学方程评价接种剂对堆肥的影响,结果显示接种剂的添加使有机质的降解速度更快,降解度更高,因而接种剂可以加速堆肥的腐熟并获得更稳定的产品。
Composting is a promising technology which can simultaneously achieve the reduction, reclamation and safe treatment of organic municipal solid waste (MSW). The most important thing for the promotion of composting in MSW treatment is to improve the composting efficiency and the quality of compost since the long composting cycle and the low quality of compost have restricted the application of composting in China. Microorganism is the major biological factor in a composting system. An understanding of the microbial community structure, the population dynamics and the functions of microbes will provide us important microbiological knowledge for the improvement of composting. With the help of modern molecular biology techniques, researchers can study microorganisms without culturing them, and by using molecular biology techniques, such as polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), real-time fluorescence quantitative PCR (qPCR) and DNA sequencing, researchers can obtain the microbial community structure and the population dynamics promptly, which is very important for the the improvement of composting.
In this paper, molecular biology techniques, combinated with culture method, was applied for investigating the microbial community in compost system, and the information about the microbial community was used as basic kwonledge for the improvement of composting technology.
Traditional composting method was used for composting kitchen discards that manually separated from MSW, and composting was proved to be good compost materials. Among the degraded organic matters during the active composting phases, more than60%of them were degraded in the high-temperature phase, which indicated that the high-temperature phase is the most important phsase for the degradation of organic matters. PCR, DGGE and DNA sequencing were applied for investigating the microbial community of samples from the high-temperature phase, and the analysis results of16/18S rDNA sequence showed a relatively abvious dynamics of the microbial community. The studies also showed that, in the samples from the high-temperature phase, the dominant bacteria, the dominant actinomysetes and the dominnat fungi are Bacillus spp., Streptomyces spp. and Nocardia spp., and Aspergillus spp., respectively.
Based on the study results above, the paper proposed a new method of continuously thermophilic composting (CTC), which composted the materials under high-temperature (>50℃) during the whole composting process. Four runs with different operation manner were compared with a traditional composting run. The run which directly heated the materials to50℃without any other temperature control showed the fastest speed of degradation and matured in14days, while the composting cycle of traditional run was28days. On the basis of research results above, the paper suggested an effective CTC method as follows:heat the materials up to50℃in the initial phase of compossting, maintain the pile temperature at least at50℃, keep a moisture content of60%, supply a gas flow of0.25L/(min-kg) and turn the pile daily.
With the help of PCR, DGGE and DNA sequencing, qPCR and traditional culture techniques, the paper investigated the microbial community in the traditional composting process and the CTC process. The study showed that the CTC methods decreased the diversity of bacterial community and increased the numbers of bacteria, and the major dominant bacteria are Bacillus spp.. The study also showd that the CTC methods increased the diversity of actinomycetes and the numbers of actinomycetes, and the dominant actinomycetes are Streptomyces spp., Mocromonospora spp., Amycolatopsis spp. and some other thermophilic actinomycetes. In addition, the CTC methods might have suppressed the growth of fungi.
In the study, suspension of soil and compost samples from high-temperature phase was used as source inouculant and the suspension was mixed with rice straw and chips, which were used as a medium. The mixture was cultured in a water bath at55℃for30days. A medium with sodium salt of carboxymethyl cellulose was used for separation of cellulolytic thermophiles from the culture mixture. Fifteen strains were selected from the cultured thermophiles after determine their degradation ability on a medium with cellulose and Congo red. Among the15strains,13strains were97%identical to Bacillus spp., one strain was97%identical to Brevibacillus borstelensis, and one strain was80%identical to Paenibacillus sp., which was determined by the biochemical or physiological identidication, the morphology characterization and the analysis of16S rDNA sequence. Each cultured strain with a volume of5mL/(kg compost) was inoculated into pile PA, with a negative control PN. The results showed a faster decrease of total organic carbon and a less loss of nitrogen in pile PA. The results also showed that pile PA matured six days earlier than pile PN, which suggested that a inoculation have spped up the mature process. The inoculant also had speed up the degradation of cellulose and hemicellulose.
引文
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