城市生活垃圾好氧堆肥实验及嗜热微生物群落研究
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摘要
好氧堆肥是依靠自然界广泛分布的细菌、真菌、放线菌等微生物,在分解有机质的过程中产生热量使得堆体温度升高,促进堆体中各类物质分解的过程,是自然界微生物降解过程的强化,微生物是堆肥处理系统中的主有功能生物,而堆肥的高温期更是堆肥过程中有机质等物质迅速降解的阶段,因此充分了解该阶段中有机质等的降解情况和微生物
变化情况以及两者之间的关系,能为改进堆肥处理工艺、提高堆肥处理效率提供理论依据。本实验采用传统的培养方法对高温期微生物群落进行计数,同时利用扩增链式反应-变性梯度凝胶电泳技术(PCR-DGGE)对微生物进行了研究。
本实验分别采用园林垃圾和餐厨垃圾作为堆肥原料,进行了20d好氧堆肥,堆体经历了中温阶段、高温阶段和降温阶段三个过程,堆体高温期(≥50℃)持续了10d和8d,堆肥后期pH值基本维持在7.5～8.0之间,保持了一个微碱性环境。堆肥前后整个过程中含水量减少率分别为38.5%和40.5%,有机质减少率分别为46.3%和44.6%,而高温期有机质减少率分别占总减少量占58.3%和48.0%,高温阶段堆肥有机质的降解力度较其他阶段要大。堆体最终C/N比均接近20,基本满足堆肥稳定时C/N比的变化要求,综合各个指标推断,两个堆体在对置20d的时候基本达到稳定。堆体中纤维素降解量分别达到45.0%和41.0%,半纤维素降解量分别达到33.9%和32.3%,而两堆体木质素降解量仅达到27.0%左右;两堆体高温期纤维素的降解量分别占到了总降解量的49..0%和48.0%,半纤维素的降解分别占总降解量的68.0%和54.0%,木质素的降解分别占总降解量的65.0%和52.0%,这些数据说明在堆肥过程中,纤维素和半纤维素降解幅度较大,木质素降解幅度较小,但总体来说高温期仍是纤维素、半纤维素和木质素高速降解的一个阶段。
使用传统的培养方法和PCR-DGGE技术对好氧堆肥高温期嗜热细菌、嗜热真菌和嗜热放线菌群落结构进行了研究。分别对两堆体高温期样品进行稀释平板混菌培养,细菌总数、真菌总数和放线菌总数分别呈“升高-降低”、“降低-升高”和“升高-降低-升高”的趋势,这与高温期有机质降解率呈“下降-上升-下降-上升”的变化规律有着密切的关系。同时提取高温期样品总DNA,分别使用细菌引物对(GC-341F/907R)、真菌引物对(GC-NS7/NS8)和放线菌引物对(F243/GC-R513)从总DNA中成功扩增得到目标产物,长度分别为630bp、340bp和270bp,再对目标产物进行DGGE分离。DGGE图谱显示,两堆体高温期嗜热细菌较真菌、放线菌种类要多,但优势菌种没有真菌明显;嗜热真菌优势菌明显,但整体菌群种类不多;嗜热放线菌优势菌不及同时期嗜热真菌明显,但菌群种类比嗜热细菌少、比嗜热真菌多,这与传统培养方法获得的结果一致。DGGE图谱聚类分析结果表明,堆肥高温期对于嗜热细菌也分成两个阶段,但是没有找到统一且确切的分段规律;而高温期嗜热真菌以升温时56℃为界,嗜热放线菌以58℃为界,分成两个变化阶段,每阶段内部聚类关系较近,阶段间关系较远;低于分界温度,部分菌种不能适应高温环境而逐渐消失,高于分界温度,能适应56℃或58℃以上温度环境的菌种迅速生长.温度对高温期各类嗜热微生物种群具有明显的筛选作用。
通过将DGGE图中条带切胶测序,获得与条带序列同源性序列,再建立系统发育树可知,得到的与芽孢杆菌属亲缘关系较近的嗜热细菌,与青霉菌、曲霉菌等有较近亲缘关系的嗜热真菌,与链酶菌和诺卡氏菌等有较近亲缘关系的嗜热放线菌,都是与降解纤维素、半纤维素和木质素有关的微生物,这些微生物是堆肥高温期有机质降解的主要作用微生物。
Aerobic composting is the natural process of the strengthening of microbial degradation and relies on the widely distributed nature of the bacteria,fungi,actinomycetes. Microorganisms are key organisms in the composting systems. Moreover,the thermophilic phases is a key stage with a quick degradation of organic materials. Studying the changes of microbial communities and the degradation of the organic materials in composting system is of great importance for technique improvement and efficiency enhancement. In this paper , a culture - dependent approach and molecular thechnologies polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) were chosen to study the succession profiles of thermophilic microorganisms communities during the thermophilic composting phases.
In this paper,two aerobic composting were separately processed with garden waste and kitchen trash for 20 days going through mesophilic phase,thermophilic phase and cooling phase. And the thermophilic period of the two composting (≥50℃) lasted 10d and 8d respectively. The final pH value maintained at 7.5～8.0 in a tiny alkalescence environment. The reduce rate of water content and organic materials were 38.5% and 40.5%,46.3% and 44.6%, While in the thermophilic phases the reduce rate of degradation of organic materials taked up 58.3% and 48.0%. The final C/N ratio was close to 20. Considering all the indicators,we inferred that the two composting piles satisfied with the demand of composting stabilization. Besides,the degradation percentage of cellulose,hemicellulose and lignin of the two piles were 45.0% and 41.0%,33.9% and 32.3% and both of 27.0%,respectively. And the degradation rate of the three indicators in thermophilic phases accounting for the total degradation quantity were 49.0% and 48.0%,68.0% and 54.0%,65.0% and 52.0%. All the datas showed that the degradation of cellulose and hemicellulose were bigger than that of lignin.
The mixed bacteria,fungi and actinomycetes in samples during thermophilic phases of two piles were cultured by plate count,and the result was that the total population of bacteria,fungi and actinomycetes showed a wave-like curve,i.e“up-down”,“down-up”and“up-down- up”,which showed close relationship with the degradation of organic materials with a change of“down-up-down-up”. And the total microbial DNA was extracted from samples by method of Protein K-CTAB. PCR amplifications were performed with primers GC-341F and 907R targeting the V3 region of bacteria 16 S rRNA,GC-NS7 and NS8 targrting the V8-V9 region of fungal 18S rRNA and primers F243 and GC-R513 targeting the V3 region of 16S rRNA genes of actinomycetes. And the PCR products were separated by DGGE for a segment of 630bp,340bp and 270bp,respectively. The results of culture-dependent approach and DGGE profiles showed that the population of the thermophilic bacteria were more than that of fungi and actinomycetes during thermophilic phase in the two piles,while the predominant kinds of the themophilic actinomycetes were less than that of fungi and bacteria. A total of 28 DGGE single bands were isolated from the profiles by cloning. The obtained sequences of these clones showed similarity to some known organisms in a range of 91% to 100%. The clones identified as Bacillus spp.,Aspergillus spp., Penicillium spp., Stretomyces spp. and Nocardioides spp. are characterized as cellulose and lignin-degrading bacteria,fungi and actinomycetes. The bacteria,fungi and actinomycetes composition for different composting materials processes similarly. This study would be beneficial for definituding the thermophilic microorganisms of thermophilic phases in aerobic composting. The clustering analysis indicated that themophilic fungi and actinomycetes in high temperature stages of composting can be divided into two phases,the dividing lines were temperature of 56℃and 58℃,when heating up,respectively. But there was no exact rules for the changes of thermophilic bacteria. The clustering relation in phrases were close to each other,while between phrases was distant. Temperature played a great role in sieving thermophilic fungi and actinomycetes of high temperature stage in composting.
变化情况以及两者之间的关系,能为改进堆肥处理工艺、提高堆肥处理效率提供理论依据。本实验采用传统的培养方法对高温期微生物群落进行计数,同时利用扩增链式反应-变性梯度凝胶电泳技术(PCR-DGGE)对微生物进行了研究。
本实验分别采用园林垃圾和餐厨垃圾作为堆肥原料,进行了20d好氧堆肥,堆体经历了中温阶段、高温阶段和降温阶段三个过程,堆体高温期(≥50℃)持续了10d和8d,堆肥后期pH值基本维持在7.5～8.0之间,保持了一个微碱性环境。堆肥前后整个过程中含水量减少率分别为38.5%和40.5%,有机质减少率分别为46.3%和44.6%,而高温期有机质减少率分别占总减少量占58.3%和48.0%,高温阶段堆肥有机质的降解力度较其他阶段要大。堆体最终C/N比均接近20,基本满足堆肥稳定时C/N比的变化要求,综合各个指标推断,两个堆体在对置20d的时候基本达到稳定。堆体中纤维素降解量分别达到45.0%和41.0%,半纤维素降解量分别达到33.9%和32.3%,而两堆体木质素降解量仅达到27.0%左右;两堆体高温期纤维素的降解量分别占到了总降解量的49..0%和48.0%,半纤维素的降解分别占总降解量的68.0%和54.0%,木质素的降解分别占总降解量的65.0%和52.0%,这些数据说明在堆肥过程中,纤维素和半纤维素降解幅度较大,木质素降解幅度较小,但总体来说高温期仍是纤维素、半纤维素和木质素高速降解的一个阶段。
使用传统的培养方法和PCR-DGGE技术对好氧堆肥高温期嗜热细菌、嗜热真菌和嗜热放线菌群落结构进行了研究。分别对两堆体高温期样品进行稀释平板混菌培养,细菌总数、真菌总数和放线菌总数分别呈“升高-降低”、“降低-升高”和“升高-降低-升高”的趋势,这与高温期有机质降解率呈“下降-上升-下降-上升”的变化规律有着密切的关系。同时提取高温期样品总DNA,分别使用细菌引物对(GC-341F/907R)、真菌引物对(GC-NS7/NS8)和放线菌引物对(F243/GC-R513)从总DNA中成功扩增得到目标产物,长度分别为630bp、340bp和270bp,再对目标产物进行DGGE分离。DGGE图谱显示,两堆体高温期嗜热细菌较真菌、放线菌种类要多,但优势菌种没有真菌明显;嗜热真菌优势菌明显,但整体菌群种类不多;嗜热放线菌优势菌不及同时期嗜热真菌明显,但菌群种类比嗜热细菌少、比嗜热真菌多,这与传统培养方法获得的结果一致。DGGE图谱聚类分析结果表明,堆肥高温期对于嗜热细菌也分成两个阶段,但是没有找到统一且确切的分段规律;而高温期嗜热真菌以升温时56℃为界,嗜热放线菌以58℃为界,分成两个变化阶段,每阶段内部聚类关系较近,阶段间关系较远;低于分界温度,部分菌种不能适应高温环境而逐渐消失,高于分界温度,能适应56℃或58℃以上温度环境的菌种迅速生长.温度对高温期各类嗜热微生物种群具有明显的筛选作用。
通过将DGGE图中条带切胶测序,获得与条带序列同源性序列,再建立系统发育树可知,得到的与芽孢杆菌属亲缘关系较近的嗜热细菌,与青霉菌、曲霉菌等有较近亲缘关系的嗜热真菌,与链酶菌和诺卡氏菌等有较近亲缘关系的嗜热放线菌,都是与降解纤维素、半纤维素和木质素有关的微生物,这些微生物是堆肥高温期有机质降解的主要作用微生物。
Aerobic composting is the natural process of the strengthening of microbial degradation and relies on the widely distributed nature of the bacteria,fungi,actinomycetes. Microorganisms are key organisms in the composting systems. Moreover,the thermophilic phases is a key stage with a quick degradation of organic materials. Studying the changes of microbial communities and the degradation of the organic materials in composting system is of great importance for technique improvement and efficiency enhancement. In this paper , a culture - dependent approach and molecular thechnologies polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) were chosen to study the succession profiles of thermophilic microorganisms communities during the thermophilic composting phases.
In this paper,two aerobic composting were separately processed with garden waste and kitchen trash for 20 days going through mesophilic phase,thermophilic phase and cooling phase. And the thermophilic period of the two composting (≥50℃) lasted 10d and 8d respectively. The final pH value maintained at 7.5～8.0 in a tiny alkalescence environment. The reduce rate of water content and organic materials were 38.5% and 40.5%,46.3% and 44.6%, While in the thermophilic phases the reduce rate of degradation of organic materials taked up 58.3% and 48.0%. The final C/N ratio was close to 20. Considering all the indicators,we inferred that the two composting piles satisfied with the demand of composting stabilization. Besides,the degradation percentage of cellulose,hemicellulose and lignin of the two piles were 45.0% and 41.0%,33.9% and 32.3% and both of 27.0%,respectively. And the degradation rate of the three indicators in thermophilic phases accounting for the total degradation quantity were 49.0% and 48.0%,68.0% and 54.0%,65.0% and 52.0%. All the datas showed that the degradation of cellulose and hemicellulose were bigger than that of lignin.
The mixed bacteria,fungi and actinomycetes in samples during thermophilic phases of two piles were cultured by plate count,and the result was that the total population of bacteria,fungi and actinomycetes showed a wave-like curve,i.e“up-down”,“down-up”and“up-down- up”,which showed close relationship with the degradation of organic materials with a change of“down-up-down-up”. And the total microbial DNA was extracted from samples by method of Protein K-CTAB. PCR amplifications were performed with primers GC-341F and 907R targeting the V3 region of bacteria 16 S rRNA,GC-NS7 and NS8 targrting the V8-V9 region of fungal 18S rRNA and primers F243 and GC-R513 targeting the V3 region of 16S rRNA genes of actinomycetes. And the PCR products were separated by DGGE for a segment of 630bp,340bp and 270bp,respectively. The results of culture-dependent approach and DGGE profiles showed that the population of the thermophilic bacteria were more than that of fungi and actinomycetes during thermophilic phase in the two piles,while the predominant kinds of the themophilic actinomycetes were less than that of fungi and bacteria. A total of 28 DGGE single bands were isolated from the profiles by cloning. The obtained sequences of these clones showed similarity to some known organisms in a range of 91% to 100%. The clones identified as Bacillus spp.,Aspergillus spp., Penicillium spp., Stretomyces spp. and Nocardioides spp. are characterized as cellulose and lignin-degrading bacteria,fungi and actinomycetes. The bacteria,fungi and actinomycetes composition for different composting materials processes similarly. This study would be beneficial for definituding the thermophilic microorganisms of thermophilic phases in aerobic composting. The clustering analysis indicated that themophilic fungi and actinomycetes in high temperature stages of composting can be divided into two phases,the dividing lines were temperature of 56℃and 58℃,when heating up,respectively. But there was no exact rules for the changes of thermophilic bacteria. The clustering relation in phrases were close to each other,while between phrases was distant. Temperature played a great role in sieving thermophilic fungi and actinomycetes of high temperature stage in composting.
引文
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