两段式厌氧工艺产甲烷发酵特性及微生物生态调控机制研究
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摘要
随着世界经济的迅猛发展，石化能源日趋枯竭，环境污染日益加重。亟待开发以高效利用各种废弃物能源化、资源化的新技术。目前，利用有机废弃物厌氧消化产甲烷技术已得到部分地区应用和推广，并取得了一定的经济效益和生态效益。然而，实际上普遍应用的厌氧发酵技术仍依靠传统经验实施，缺乏对厌氧消化微生物过程深入的机理研究。因此造成厌氧消化装置和运行工艺简单，系统运行效率低及稳定性差等。尤其在一些高寒地区，受发酵温度制约，更加难以应用和推广。此外，厌氧消化过程产生的大量残液处理不当还会造成对环境的二次污染，而作为生物有机肥应用在加工及施灌等技术方面还不成熟。
本文围绕沼气应用中的实际问题及待深入研究的理论问题，构建一套两段式厌氧发酵系统，以北方农村典型的废弃物牛粪和水稻秸秆为产甲烷基质，开展了以利用单一底物牛粪的产甲烷特性研究，以及利用水稻秸秆和糖浆废水混合底物共发酵产甲烷特性的研究。同时，为探索低温条件对厌氧消化微生物作用的抑制机理，采取两种研究技术路线，即从自然生境筛选低温复合产甲烷菌群的传统培养技术，结合两段发酵产甲烷相降温驯化适低温产甲烷菌群的技术途径，探求低温条件下的产甲烷效能及微生物特性。此外，在沼液资源化利用方面，提出了以厌氧消化产甲烷残液作为生产生物絮凝剂的廉价替代底物，证实并获得较理想絮凝率和絮凝剂产量的可行性。论文取得研究结果如下。
发现了结合MFC技术构建的复合产酸系统，可显著促进牛粪降解、产酸速率及有利于末端液相有机挥发酸的定向转化。以单一底物牛粪8%TS稳定运行时，末端液相发酵产物以乙酸为主，占挥发酸总量的78.7%，并显著抑制丙酸浓度积累。生物膜表面优势细菌种群为Acinetobacter sp.、Pseudomonas sp.和Flavobacterium sp.。利用复合产酸系统运行40d的酸化物料作为产甲烷相进料基质，以HRT为40d，OLR为2.5kg VSaddm-3d~(-1)运行时，产甲烷相的沼气容积产量可达1.21±0.047m~3m-3reactord~(-1)，甲烷产量为252.6±8.4L kg~(-1)VSfed。产甲烷相优势细菌类群为Lutaonella sp.、Sedimentibacter sp.、Proteiniphilum sp.、和Clostridium sp.；优势产甲烷菌为Methanosaeta sp.、 Methanosarcina sp.和Methanobacterium formicicum。
利用水稻秸秆和糖浆废水混合底物共发酵考察两段发酵系统产甲烷特性，结果发现，8%TS的CSTR产酸系统运行至30d时，pH迅速降为6.42，乙酸浓度为3210mg/L，占挥发酸总量72.2%。产酸相优势细菌为Firmicutes，其次是Bacteroidetes和Proteobacteria，占微生物总数的90%以上。以产酸相运行30d酸化物料为产甲烷相底物基质，HRT为40d，OLR为3.0kg VSaddm~(-3)d~(-1)和3.5kg VSaddm~(-3)d~(-1)运行时，沼气容积产量分别达1.04±0.025m~3m_(reactor)~(-3)d~(-1)和1.12±0.046m~3m_(reactor)~(-3)d~(-1)。产甲烷相的优势细菌属γ-Proteobacteria和Clostridia；优势产甲烷菌为Methanosaeta sp.。
为研究低温对厌氧发酵产甲烷效能的影响，从自然生境筛选了两组低温产甲烷复合菌群，经6次转代富集培养周期累积沼产气量稳定，分别为180ml和195ml。两组低温菌群均在18°C时产气效率最高，最大沼气产量为0.26m~3m_(reactor)~(-3)d~(-1)；18°C时低温复合产甲烷菌群利用牛粪酸化液最大沼气产量为48mld~(-1)。低温复合产甲烷菌群的优势细菌属Firmicutes和Bacteroidetes，可鉴定的产甲烷菌属Methanomicrobiaceae和Methanobacterium sp.。两段发酵产甲烷相降温产甲烷研究发现，25°C和22°C稳定运行时沼气产量可达511.2±15.5和410.1±10.8ml L~(-1)reactord~(-1)。当温度降至20°C以下时，产气波动大且运行不稳定。产甲烷相在30°C、25°C和22°C稳定运行时，优势细菌类群属Proteobacteria、Firmicutes和Bacteroidetes；20°C时过度为以Proteobacteria为绝对优势种群的群落组成，占微生物群落总数的71.36%。低温运行时Methanosarcina sp.和Methanobacterium sp.始终存在，Methanosarcina sp.属最优势产甲烷菌。寒地海林沼气发酵系统夏、冬季沼气产量分别为0.625和0.35m~3m~(-3)digesterd~(-1)，池内优势细菌为Proteiniphilum sp.、Spirochaeta sp.和Wolinella sp.，均属动物消化系统常见菌群，Methanosaeta sp.和Methanosarcina sp.是优势产甲烷古菌。
利用不同底物厌氧发酵残液制取生物絮凝剂可行性的研究，结果表明，以水稻秸秆高浓度厌氧发酵100d的残液，1:1混合配比传统产絮培养基形成复合培养基，絮凝效率可达92.5%，产量为4.28±0.13g/L。对厌氧发酵系统产酸旺盛期细菌群落分析表明，Firmicutes为绝对优势种群，占微生物总序列的92.51%，Clostridiales目Peptostreptococcaceae科的Sporacetigenium属占微生物总量的46.7%。利用海林沼气池残液、以牛粪为底物两段发酵工艺产甲烷残液，以及水稻秸秆与糖浆废水混合底物两段发酵产甲烷残液，分别以1:1配比混合传统产絮培养基，絮凝率分别可达52.3%、91.3%和92.1%，絮凝剂产量分别为1.75±0.03g/L、4.21±0.05g/L和4.18±0.04g/L。
The depletion of fossil fuel sources and environmental pollution caused by thedevelopment of world economy. It is urgent to seek novel resource utilizationtechnique of using organic wastes to produce renewable energy. Recently, anaerobicdigestion is widely applied to convert biomass waste into methane worldwide, and ithas received considerable economic and environmental benefits. However,application of anaerobic digestion technique mainly depended on traditionalexperience, and lacked of deep mechanism research on microorganisms. Therefore,the anaerobic digester operated at low efficiency and instability due to simpleconfiguration and process tequenology. It was hard for industrial application in lowtemperature regions. Furthermore, biogas slurry was disposed inappropriately couldcaused secondary pollution to environment. Disposal of biogas slurry to producebioorganic fertilizer was inhibited by technique of granulation and irrigation.
Thus, based on practical problems and detailed studies during the anaerobicdigestion process, this study designed a two-stage anaerobic digestion system formethane production from cattle dung and co-digestion of rice straw and molasseswastewater. The research discussed the effect of psychrophilic conditions onmethane production and microorganisms via two technique route. The psychrophilicmethanogens were cultivated from natural habits, and domesticated frommethanogenic phase of two-stage anaerobic digesion. Futhermore, the feasibility ofusing of biogas slurry as substrate to produce bioflocculant was discussed. The mainresults obtained by this study were as follow.
The acidification-phase using MFC technique could promote degradation andVFA production of cattle dung obviously, and which was beneficial to directionalconversation of VFA. The main VFAs found was acetate which occupied78%oftotal VFAs operated at8%TS, and the propionate concentration was inhibited.Microbial comminity of biofilm analysis revealed that bacteria were dominated byAcinetobacter sp., Pseudomonas sp., and Flavobacterium sp.. The acidified cattledung from40-d operation of acidification-phase were used for IC reactor. Biogasproduction reached1.21±0.047m~3m_(reactor)~(-3)d~(-1), and methane production was252.6±8.4L kg~(-1)VSfedwhich operated at an OLR of2.5kg VSaddm~(-3)d~(-1)and a HRTof40d. The dominant bacteria were Lutaonella sp., Sedimentibacter sp.,Proteiniphilum sp, and Clostridium sp., and archaea dominated by Methanosaeta sp.,Methanosarcina sp. and Methanobacterium formicicum during methanogenesis.
Methane production from co-digestion of rice straw and molasses wastewaterwas studied. The ph of acidification-phase decreased dramatically to6.42, and concentration of acetate was3210mg/l which occupied72.2%of total VFAsoperated at8%TS after30days of operation. The dominant bacteria wereFirmicutes, Bacteroidetes and Proteobacteria. Biogas production reached1.12±0.046m~3m_(reactor)~(-3)d~(-1), which operated at a HRT of40d and an OLR of3.5kgVSaddm~(-3)d~(-1). The dominant bacteria were γ-Proteobacteria and Clostridia. Thedominant archaea were Methanosaeta sp..
Two group of psychrophilic methanogens were collected from nature habitat.Cumulative biogas production maintained stability after6generations cultivation,and biogas production reached180ml and195ml. The optimum biogas productionwas0.26m~3m~3reactord~(-1)operated at18°C. The dominant bacteria of psychrophilicmicrooganisms were Firmicutes and Bacteroidetes. Archaea could be identified wereMethanomicrobiaceae and Methanobacterium sp.. The effect of decreasingtemperature gradually on biogas production of IC reactor was studied. The biogasproduction achieved511.2±15.5and410.1±10.8ml L~(-1)reactord~(-1)while operating at25°C and22°C, respectively. The biogas production was fluctuant whiletemperature decreased to20°C. The dominant bacteria were Proteobacteria,Firmicutes and Bacteroidetes while operating at a temperature of30°C,25°C and22°C during a stable process. The most predominant bacteria were Proteobacteria at20°C, accounted for71.36%of total populations. Methanosarcina sp. dominatedduring low temperature operation process. The biogas production of Hailin biogasdigester in cold region reached0.625and0.35m~3m_(reactor)~(-3)d~(-1)in winter and summerof operation. The domiant bacteria belonged to Proteiniphilum sp.、Spirochaeta sp.and Wolinella sp., which were found in animal digestive system. Methanosaeta sp.and Methanosarcina sp. were the dominant archaea.
Fermentation liquor from different substrates were used to producebioflocculants. The maximum flocculation activity,92.45%was achieved when100-day fermentation liquor of rice straw during a dry fermentation process andconventional bioflocculants medium were mixed at an equal ratio. The bioflocculantyield was4.28±0.13g/L. Notably, Sporacetigenium sp.(46.7%) was the dominantmicrobial population during VFA accumulation time. Use of biogas slurry toproduce bioflocullant, which collected from Hailin digester, two-stage anaerobicdigestion of cattle dung and co-digestion of rice straw and molasses wastewater. Theflocculation activity were52.3%,91.3%and92.1%, while fermentation liquor andconventional bioflocculants medium were mixed at an equal ratio. The bioflocculantyield were1.75±0.03g/L,4.21±0.05g/L and4.18±0.04g/L, respectively.
本文围绕沼气应用中的实际问题及待深入研究的理论问题，构建一套两段式厌氧发酵系统，以北方农村典型的废弃物牛粪和水稻秸秆为产甲烷基质，开展了以利用单一底物牛粪的产甲烷特性研究，以及利用水稻秸秆和糖浆废水混合底物共发酵产甲烷特性的研究。同时，为探索低温条件对厌氧消化微生物作用的抑制机理，采取两种研究技术路线，即从自然生境筛选低温复合产甲烷菌群的传统培养技术，结合两段发酵产甲烷相降温驯化适低温产甲烷菌群的技术途径，探求低温条件下的产甲烷效能及微生物特性。此外，在沼液资源化利用方面，提出了以厌氧消化产甲烷残液作为生产生物絮凝剂的廉价替代底物，证实并获得较理想絮凝率和絮凝剂产量的可行性。论文取得研究结果如下。
发现了结合MFC技术构建的复合产酸系统，可显著促进牛粪降解、产酸速率及有利于末端液相有机挥发酸的定向转化。以单一底物牛粪8%TS稳定运行时，末端液相发酵产物以乙酸为主，占挥发酸总量的78.7%，并显著抑制丙酸浓度积累。生物膜表面优势细菌种群为Acinetobacter sp.、Pseudomonas sp.和Flavobacterium sp.。利用复合产酸系统运行40d的酸化物料作为产甲烷相进料基质，以HRT为40d，OLR为2.5kg VSaddm-3d~(-1)运行时，产甲烷相的沼气容积产量可达1.21±0.047m~3m-3reactord~(-1)，甲烷产量为252.6±8.4L kg~(-1)VSfed。产甲烷相优势细菌类群为Lutaonella sp.、Sedimentibacter sp.、Proteiniphilum sp.、和Clostridium sp.；优势产甲烷菌为Methanosaeta sp.、 Methanosarcina sp.和Methanobacterium formicicum。
利用水稻秸秆和糖浆废水混合底物共发酵考察两段发酵系统产甲烷特性，结果发现，8%TS的CSTR产酸系统运行至30d时，pH迅速降为6.42，乙酸浓度为3210mg/L，占挥发酸总量72.2%。产酸相优势细菌为Firmicutes，其次是Bacteroidetes和Proteobacteria，占微生物总数的90%以上。以产酸相运行30d酸化物料为产甲烷相底物基质，HRT为40d，OLR为3.0kg VSaddm~(-3)d~(-1)和3.5kg VSaddm~(-3)d~(-1)运行时，沼气容积产量分别达1.04±0.025m~3m_(reactor)~(-3)d~(-1)和1.12±0.046m~3m_(reactor)~(-3)d~(-1)。产甲烷相的优势细菌属γ-Proteobacteria和Clostridia；优势产甲烷菌为Methanosaeta sp.。
为研究低温对厌氧发酵产甲烷效能的影响，从自然生境筛选了两组低温产甲烷复合菌群，经6次转代富集培养周期累积沼产气量稳定，分别为180ml和195ml。两组低温菌群均在18°C时产气效率最高，最大沼气产量为0.26m~3m_(reactor)~(-3)d~(-1)；18°C时低温复合产甲烷菌群利用牛粪酸化液最大沼气产量为48mld~(-1)。低温复合产甲烷菌群的优势细菌属Firmicutes和Bacteroidetes，可鉴定的产甲烷菌属Methanomicrobiaceae和Methanobacterium sp.。两段发酵产甲烷相降温产甲烷研究发现，25°C和22°C稳定运行时沼气产量可达511.2±15.5和410.1±10.8ml L~(-1)reactord~(-1)。当温度降至20°C以下时，产气波动大且运行不稳定。产甲烷相在30°C、25°C和22°C稳定运行时，优势细菌类群属Proteobacteria、Firmicutes和Bacteroidetes；20°C时过度为以Proteobacteria为绝对优势种群的群落组成，占微生物群落总数的71.36%。低温运行时Methanosarcina sp.和Methanobacterium sp.始终存在，Methanosarcina sp.属最优势产甲烷菌。寒地海林沼气发酵系统夏、冬季沼气产量分别为0.625和0.35m~3m~(-3)digesterd~(-1)，池内优势细菌为Proteiniphilum sp.、Spirochaeta sp.和Wolinella sp.，均属动物消化系统常见菌群，Methanosaeta sp.和Methanosarcina sp.是优势产甲烷古菌。
利用不同底物厌氧发酵残液制取生物絮凝剂可行性的研究，结果表明，以水稻秸秆高浓度厌氧发酵100d的残液，1:1混合配比传统产絮培养基形成复合培养基，絮凝效率可达92.5%，产量为4.28±0.13g/L。对厌氧发酵系统产酸旺盛期细菌群落分析表明，Firmicutes为绝对优势种群，占微生物总序列的92.51%，Clostridiales目Peptostreptococcaceae科的Sporacetigenium属占微生物总量的46.7%。利用海林沼气池残液、以牛粪为底物两段发酵工艺产甲烷残液，以及水稻秸秆与糖浆废水混合底物两段发酵产甲烷残液，分别以1:1配比混合传统产絮培养基，絮凝率分别可达52.3%、91.3%和92.1%，絮凝剂产量分别为1.75±0.03g/L、4.21±0.05g/L和4.18±0.04g/L。
The depletion of fossil fuel sources and environmental pollution caused by thedevelopment of world economy. It is urgent to seek novel resource utilizationtechnique of using organic wastes to produce renewable energy. Recently, anaerobicdigestion is widely applied to convert biomass waste into methane worldwide, and ithas received considerable economic and environmental benefits. However,application of anaerobic digestion technique mainly depended on traditionalexperience, and lacked of deep mechanism research on microorganisms. Therefore,the anaerobic digester operated at low efficiency and instability due to simpleconfiguration and process tequenology. It was hard for industrial application in lowtemperature regions. Furthermore, biogas slurry was disposed inappropriately couldcaused secondary pollution to environment. Disposal of biogas slurry to producebioorganic fertilizer was inhibited by technique of granulation and irrigation.
Thus, based on practical problems and detailed studies during the anaerobicdigestion process, this study designed a two-stage anaerobic digestion system formethane production from cattle dung and co-digestion of rice straw and molasseswastewater. The research discussed the effect of psychrophilic conditions onmethane production and microorganisms via two technique route. The psychrophilicmethanogens were cultivated from natural habits, and domesticated frommethanogenic phase of two-stage anaerobic digesion. Futhermore, the feasibility ofusing of biogas slurry as substrate to produce bioflocculant was discussed. The mainresults obtained by this study were as follow.
The acidification-phase using MFC technique could promote degradation andVFA production of cattle dung obviously, and which was beneficial to directionalconversation of VFA. The main VFAs found was acetate which occupied78%oftotal VFAs operated at8%TS, and the propionate concentration was inhibited.Microbial comminity of biofilm analysis revealed that bacteria were dominated byAcinetobacter sp., Pseudomonas sp., and Flavobacterium sp.. The acidified cattledung from40-d operation of acidification-phase were used for IC reactor. Biogasproduction reached1.21±0.047m~3m_(reactor)~(-3)d~(-1), and methane production was252.6±8.4L kg~(-1)VSfedwhich operated at an OLR of2.5kg VSaddm~(-3)d~(-1)and a HRTof40d. The dominant bacteria were Lutaonella sp., Sedimentibacter sp.,Proteiniphilum sp, and Clostridium sp., and archaea dominated by Methanosaeta sp.,Methanosarcina sp. and Methanobacterium formicicum during methanogenesis.
Methane production from co-digestion of rice straw and molasses wastewaterwas studied. The ph of acidification-phase decreased dramatically to6.42, and concentration of acetate was3210mg/l which occupied72.2%of total VFAsoperated at8%TS after30days of operation. The dominant bacteria wereFirmicutes, Bacteroidetes and Proteobacteria. Biogas production reached1.12±0.046m~3m_(reactor)~(-3)d~(-1), which operated at a HRT of40d and an OLR of3.5kgVSaddm~(-3)d~(-1). The dominant bacteria were γ-Proteobacteria and Clostridia. Thedominant archaea were Methanosaeta sp..
Two group of psychrophilic methanogens were collected from nature habitat.Cumulative biogas production maintained stability after6generations cultivation,and biogas production reached180ml and195ml. The optimum biogas productionwas0.26m~3m~3reactord~(-1)operated at18°C. The dominant bacteria of psychrophilicmicrooganisms were Firmicutes and Bacteroidetes. Archaea could be identified wereMethanomicrobiaceae and Methanobacterium sp.. The effect of decreasingtemperature gradually on biogas production of IC reactor was studied. The biogasproduction achieved511.2±15.5and410.1±10.8ml L~(-1)reactord~(-1)while operating at25°C and22°C, respectively. The biogas production was fluctuant whiletemperature decreased to20°C. The dominant bacteria were Proteobacteria,Firmicutes and Bacteroidetes while operating at a temperature of30°C,25°C and22°C during a stable process. The most predominant bacteria were Proteobacteria at20°C, accounted for71.36%of total populations. Methanosarcina sp. dominatedduring low temperature operation process. The biogas production of Hailin biogasdigester in cold region reached0.625and0.35m~3m_(reactor)~(-3)d~(-1)in winter and summerof operation. The domiant bacteria belonged to Proteiniphilum sp.、Spirochaeta sp.and Wolinella sp., which were found in animal digestive system. Methanosaeta sp.and Methanosarcina sp. were the dominant archaea.
Fermentation liquor from different substrates were used to producebioflocculants. The maximum flocculation activity,92.45%was achieved when100-day fermentation liquor of rice straw during a dry fermentation process andconventional bioflocculants medium were mixed at an equal ratio. The bioflocculantyield was4.28±0.13g/L. Notably, Sporacetigenium sp.(46.7%) was the dominantmicrobial population during VFA accumulation time. Use of biogas slurry toproduce bioflocullant, which collected from Hailin digester, two-stage anaerobicdigestion of cattle dung and co-digestion of rice straw and molasses wastewater. Theflocculation activity were52.3%,91.3%and92.1%, while fermentation liquor andconventional bioflocculants medium were mixed at an equal ratio. The bioflocculantyield were1.75±0.03g/L,4.21±0.05g/L and4.18±0.04g/L, respectively.
引文
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