牛粪好氧堆肥中微生物多样性及生产应用研究
摘要
近年来,随着畜禽饲养量不断提高,畜禽粪便对环境的污染问题日益凸显。好氧堆肥作为一种集畜禽粪便处理和资源化利用为一体的处理方法,具有生产成本低、污染少、改良土壤等众多优点。本研究从牛粪好氧堆肥菌剂的筛选、堆肥验证、规模化处理设备和参数优化到有机肥料应用并利用16SrRNA方法对微生物多样性分析和鉴定,得出如下主要结论：
(1)从牛粪好氧堆肥过程中温度曲线、升温速度、堆肥高温期持续的天数、堆肥结束后的GI、纤维素、半纤维素降解率来看,筛选出单菌种HB1、HB2、HB3、HB4HB10、LB2、HB14、MB5、MF9、LF11、MC5、Mc9共12种菌为优势单菌种作为本次单菌种试验中筛选出的优势菌株。
(2)采用16SrRNA PCR-DGGE方法从牛粪自然好氧发酵过程中分离出微生物共计417个,其中细菌类微生物177个,放线菌类微生物113个,真菌类微生物127个。细菌类是优势群体,生物多样性指数也是处于2.1以上；放线菌类微生物数量和种类都较少,多样性指数在1.4以下；真菌类微生物在数量和种类上处于中间价态,在降温期密度持续增加,对纤维素分解起到了决定性的作用,,是中后期的优势群体。
(3)将(1)得出的优势单菌种按照复合菌种筛选原则进行组合应用,在牛粪好氧堆肥过程中,接种复合菌种处理1-8均明显高于CK处理,从纤维素类物质分解、温度和养分变化、发芽指数得出,处理2为最佳一蚀木链霉菌(Streptomyces Xylophagus)、嗜冷枯草芽孢杆菌(Psychrobacter sp)、嗜热单胞菌(Thermomonospora)、波茨坦芽孢杆菌(Brevibacillus)、卷枝毛霉菌(Mucor circinelloides)和钋青霉菌(Penicillium polonicum)。
(4)在复合菌种堆肥反应过程中,细菌由15种减少至12种,波茨坦短芽孢杆菌(Brevibacillus borstelensis)消失；放线菌由5种增加到7种,在堆肥开始时成为优势群体：真菌6种增加到10种。复合微生物菌剂确定为：嗜冷枯草芽孢杆菌(Psychrobacter sp)、嗜热单胞菌(Thermomonospora)、蚀木链霉菌(Streptomyces Xylophagus)、卷枝毛霉菌(Mucorcircinelloides)和钋青霉菌(Penicillium polonicum)组成。
(5)基于实验室小规模正交发酵参数,通过正交试验得出规模化生产最佳工艺条件为：含水率为65%,初始C/N为30：1,每天通风30min,有机质初始质量分数为80%,初始温度为30℃,pH值为8.0。
(6)施用有机肥、化肥和不施肥对玉米种植比较得出,施用化肥除双穗率略低施用有机肥外,其他因子均高于施用有机肥和不施肥。从玉米根系来看,施用化肥的根茎大于施用有机肥和不施肥。在根系投影面积等数据方面,施用化肥处理均高于施用有机肥和不施肥；从根系直径分布直方图看出,施用有机肥处理在小于4mm直径范围的根系数量明显高于不施肥和施用化肥处理。
(7)不同的施肥处理条件下,不同的土壤层呈现出不同的生物多样性指数和种类。不施肥和施用有机肥处理从表层至深层具有较高的多样性指数,化肥则相反。团聚体的含量随着粒级的增加呈现先增加后下降的趋势,2-0.25mm粒级团聚体为优势粒级。HS(腐殖物质)各组分以HM(胡敏素)的含量最高。随粒级的增加HS各组分含量没有明显变化。长期施用有机肥使2-0.25mm粒级团聚体中HA(胡敏酸)含量、HM含量、PQ值(某粒级HA占可提取物的百分数)和HS各组分的富集率均增加。
In recent years, with the continuous improvement of the livestock and poultry, livestock and poultry manure pollution of the environment has become increasingly prominent. As a approach of livestock manure treatment and resource use, aerobic composting has many advantages like low production costs, less pollution, soil improvement, and so on. Screening of this study, aerobic compost inocula from cow dung, compost validation, large-scale processing equipment and parameter optimization, the application of organic fertilizers and 16SrRNA method was used for microbial diversity analysis and identification, the main conclusions are:
(1) From the temperature curve, heating rate, days of compost high temperature, the GI after compost, cellulose, hemicellulose degradation rate of cow dung aerobic composting process, we filtered out the single-strain HBi、HB2、HB3、HB4、HB10、LB2、HB14、MB5、MF9、LF11、MC5、MC9. All the 12 kinds of dominant single strain were screened in the single-strain tests.
(2) 16SrRNA PCR-DGGE method was used to isolated 417 microorganisms from cow dung natural aerobic fermentation process. There are 177 bacteria microorganisms1113 actinomycetes microbial and 127 fungal microorganisms. Bacteria is the dominant groups, biodiversity index is more than 2.1; The number and types of actinomycetes microbial are less, and the diversity index is less than 1.4; The number and types of fungal microorganisms are in the central parity states, the density continues to increase in the cooling stage, and it played a decisive role in the cellulose decomposition. It's the advantaged groups in the late.
(3) The advantage single strain derived from (1) was combinated according to the composite strain screening principles and was used in the process of cattle manure aerobic composting. Composite strain treatment 1-8 were significantly higher than the CK treatment. From the decomposition of cellulosic materials, changes in temperature and nutrients, germination index,we obtained:treatment 2 is the best-xylophagous Streptomyces (Streptomyces Xylophagus), addicted to cold Bacillus subtilis (Psychrobacter sp.), Thermophilic Aeromonas (Thermomonospora), Potsdam Bacillus bacilli (Brevibacillus), Volume sticks Mucor (Mucor circinelloides) and polonium Penicillium (Penicillium. polonicum).
(4) In the compost reaction process of the composite strain, the kinds of bacteria reduced from 15 to 12, and the Potsdam short Bacillus (Brevibacillus borstelensis)was disappear; The kinds of actinomycetes increased from 5 to 7 and become the dominant groups at the beginning of compost; The kinds of Fungi increased from 6 to 10. Complex microbial are:psychrophilic Bacillus subtilis {Psychrobacter sp.), Thermophilic Aeromonas (Thermomonospora) the xylophagous Streptomyces {Streptomyces Xylophagus), volume branch mucormycosis (Mucorcircinelloides), and polonium Penicillium (Penicillium polonicum) composition.
(5) Based on the parameters of the laboratory-scale orthogonal fermentation, the optimal conditions for large-scale production by orthogonal test are:water content was 65%, the initial C/N was 30:1, daily ventilation 30min, the initial mass fraction of organic matter was 80%, the initial temperature was 30℃, pH value 8.0.
(6) The effect on maize under different treatment (organic manure, chemical fertilizer and no fertilization) were compared:the two-spike rate of treatment under chemical fertilizer was slightly lower than the treatment under organic fertilizer, but the other factors were higher than both of the treatment under organic manure and on fertilization. Maize rhizome of treatment chemical fertilizer was larger than the other two treatments. The number of roots less than 4mm diameter range of treatment organic fertilizer was significantly higher than the other two treatments.
(7) Different soil layers showing different biological diversity index and species under different fertilization treatments. Treatments fertilization and organic fertilizer has a high diversity index from the surface to the deep, on the contrary of fertilizers. Content of aggregates first increased and then decreased with the increase of particle size,2-0.25 mm size aggregates was the dominant grain size. Content of HM was the highest in each components of HS. The content of each component in HS did not change significantly with the increase of particle size. Long-term application of organic fertilizer made of 2-0.25mm aggregates in HA content, HM content, PQ value and HS component enrichment rate increased.
(1)从牛粪好氧堆肥过程中温度曲线、升温速度、堆肥高温期持续的天数、堆肥结束后的GI、纤维素、半纤维素降解率来看,筛选出单菌种HB1、HB2、HB3、HB4HB10、LB2、HB14、MB5、MF9、LF11、MC5、Mc9共12种菌为优势单菌种作为本次单菌种试验中筛选出的优势菌株。
(2)采用16SrRNA PCR-DGGE方法从牛粪自然好氧发酵过程中分离出微生物共计417个,其中细菌类微生物177个,放线菌类微生物113个,真菌类微生物127个。细菌类是优势群体,生物多样性指数也是处于2.1以上；放线菌类微生物数量和种类都较少,多样性指数在1.4以下；真菌类微生物在数量和种类上处于中间价态,在降温期密度持续增加,对纤维素分解起到了决定性的作用,,是中后期的优势群体。
(3)将(1)得出的优势单菌种按照复合菌种筛选原则进行组合应用,在牛粪好氧堆肥过程中,接种复合菌种处理1-8均明显高于CK处理,从纤维素类物质分解、温度和养分变化、发芽指数得出,处理2为最佳一蚀木链霉菌(Streptomyces Xylophagus)、嗜冷枯草芽孢杆菌(Psychrobacter sp)、嗜热单胞菌(Thermomonospora)、波茨坦芽孢杆菌(Brevibacillus)、卷枝毛霉菌(Mucor circinelloides)和钋青霉菌(Penicillium polonicum)。
(4)在复合菌种堆肥反应过程中,细菌由15种减少至12种,波茨坦短芽孢杆菌(Brevibacillus borstelensis)消失；放线菌由5种增加到7种,在堆肥开始时成为优势群体：真菌6种增加到10种。复合微生物菌剂确定为：嗜冷枯草芽孢杆菌(Psychrobacter sp)、嗜热单胞菌(Thermomonospora)、蚀木链霉菌(Streptomyces Xylophagus)、卷枝毛霉菌(Mucorcircinelloides)和钋青霉菌(Penicillium polonicum)组成。
(5)基于实验室小规模正交发酵参数,通过正交试验得出规模化生产最佳工艺条件为：含水率为65%,初始C/N为30：1,每天通风30min,有机质初始质量分数为80%,初始温度为30℃,pH值为8.0。
(6)施用有机肥、化肥和不施肥对玉米种植比较得出,施用化肥除双穗率略低施用有机肥外,其他因子均高于施用有机肥和不施肥。从玉米根系来看,施用化肥的根茎大于施用有机肥和不施肥。在根系投影面积等数据方面,施用化肥处理均高于施用有机肥和不施肥；从根系直径分布直方图看出,施用有机肥处理在小于4mm直径范围的根系数量明显高于不施肥和施用化肥处理。
(7)不同的施肥处理条件下,不同的土壤层呈现出不同的生物多样性指数和种类。不施肥和施用有机肥处理从表层至深层具有较高的多样性指数,化肥则相反。团聚体的含量随着粒级的增加呈现先增加后下降的趋势,2-0.25mm粒级团聚体为优势粒级。HS(腐殖物质)各组分以HM(胡敏素)的含量最高。随粒级的增加HS各组分含量没有明显变化。长期施用有机肥使2-0.25mm粒级团聚体中HA(胡敏酸)含量、HM含量、PQ值(某粒级HA占可提取物的百分数)和HS各组分的富集率均增加。
In recent years, with the continuous improvement of the livestock and poultry, livestock and poultry manure pollution of the environment has become increasingly prominent. As a approach of livestock manure treatment and resource use, aerobic composting has many advantages like low production costs, less pollution, soil improvement, and so on. Screening of this study, aerobic compost inocula from cow dung, compost validation, large-scale processing equipment and parameter optimization, the application of organic fertilizers and 16SrRNA method was used for microbial diversity analysis and identification, the main conclusions are:
(1) From the temperature curve, heating rate, days of compost high temperature, the GI after compost, cellulose, hemicellulose degradation rate of cow dung aerobic composting process, we filtered out the single-strain HBi、HB2、HB3、HB4、HB10、LB2、HB14、MB5、MF9、LF11、MC5、MC9. All the 12 kinds of dominant single strain were screened in the single-strain tests.
(2) 16SrRNA PCR-DGGE method was used to isolated 417 microorganisms from cow dung natural aerobic fermentation process. There are 177 bacteria microorganisms1113 actinomycetes microbial and 127 fungal microorganisms. Bacteria is the dominant groups, biodiversity index is more than 2.1; The number and types of actinomycetes microbial are less, and the diversity index is less than 1.4; The number and types of fungal microorganisms are in the central parity states, the density continues to increase in the cooling stage, and it played a decisive role in the cellulose decomposition. It's the advantaged groups in the late.
(3) The advantage single strain derived from (1) was combinated according to the composite strain screening principles and was used in the process of cattle manure aerobic composting. Composite strain treatment 1-8 were significantly higher than the CK treatment. From the decomposition of cellulosic materials, changes in temperature and nutrients, germination index,we obtained:treatment 2 is the best-xylophagous Streptomyces (Streptomyces Xylophagus), addicted to cold Bacillus subtilis (Psychrobacter sp.), Thermophilic Aeromonas (Thermomonospora), Potsdam Bacillus bacilli (Brevibacillus), Volume sticks Mucor (Mucor circinelloides) and polonium Penicillium (Penicillium. polonicum).
(4) In the compost reaction process of the composite strain, the kinds of bacteria reduced from 15 to 12, and the Potsdam short Bacillus (Brevibacillus borstelensis)was disappear; The kinds of actinomycetes increased from 5 to 7 and become the dominant groups at the beginning of compost; The kinds of Fungi increased from 6 to 10. Complex microbial are:psychrophilic Bacillus subtilis {Psychrobacter sp.), Thermophilic Aeromonas (Thermomonospora) the xylophagous Streptomyces {Streptomyces Xylophagus), volume branch mucormycosis (Mucorcircinelloides), and polonium Penicillium (Penicillium polonicum) composition.
(5) Based on the parameters of the laboratory-scale orthogonal fermentation, the optimal conditions for large-scale production by orthogonal test are:water content was 65%, the initial C/N was 30:1, daily ventilation 30min, the initial mass fraction of organic matter was 80%, the initial temperature was 30℃, pH value 8.0.
(6) The effect on maize under different treatment (organic manure, chemical fertilizer and no fertilization) were compared:the two-spike rate of treatment under chemical fertilizer was slightly lower than the treatment under organic fertilizer, but the other factors were higher than both of the treatment under organic manure and on fertilization. Maize rhizome of treatment chemical fertilizer was larger than the other two treatments. The number of roots less than 4mm diameter range of treatment organic fertilizer was significantly higher than the other two treatments.
(7) Different soil layers showing different biological diversity index and species under different fertilization treatments. Treatments fertilization and organic fertilizer has a high diversity index from the surface to the deep, on the contrary of fertilizers. Content of aggregates first increased and then decreased with the increase of particle size,2-0.25 mm size aggregates was the dominant grain size. Content of HM was the highest in each components of HS. The content of each component in HS did not change significantly with the increase of particle size. Long-term application of organic fertilizer made of 2-0.25mm aggregates in HA content, HM content, PQ value and HS component enrichment rate increased.
引文
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