厌氧发酵产酸微生物种群生态及互营关系研究
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摘要
产酸微生物是有机物厌氧发酵过程中一类重要的微生物，其产生的挥发性有机酸（VFAs）是主要的中间产物之一，也是有机化工行业重要生产原料。而且，产酸过程与有机物降解和沼气的产生都有密切联系，产酸微生物与水解微生物、产甲烷微生物，甚至产酸微生物之间均有复杂的互营关系。近年来，高通量测序技术的发展突飞猛进，正成为微生物生态研究者们经常使用的生物技术。与传统测序方法通过对DNA片断中碱基的特异性切割不同，高通量测序技术能一次并行，对几十万到几百万条DNA分子进行序列测定。在产酸反应器中，为研究产酸微生物种群生态，添加产甲烷抑制剂是一种常用的技术方法，它能够阻止产甲烷菌利用乙酸的同时，不影响产酸微生物的生长。
本论文利用稳定性同位素标记和高通量测序等环境微生物学技术，研究了产酸微生物在厌氧发酵过程中所起的作用、明确了各产酸菌的种群结构以及它们之间的互营关系、阐明了不同功能菌群在生产VFAs中各自的贡献、并通过菌群结构与数量的调节，增强互营产酸以达到乙酸高产的目的。在此基础上，找出该工艺的最优操作条件，并建立动力学模型，为污泥厌氧发酵过程的研究丰富了理论与应用基础。主要的研究结果如下：
1葡萄糖厌氧降解中产酸微生物种群生态
单糖降解是有机物厌氧发酵中重要步骤，然而，对于BES抑制产甲烷情况下，厌氧污泥对葡萄糖降解起关键性作用的微生物研究则较少。利用DNA-SIP、克隆文库及高通量测序技术，考察污泥厌氧产酸与产甲烷两种模式下，微生物群落的演替情况后发现：未抑制产甲烷的厌氧发酵中占优势的是梭状芽孢杆菌（Clostridia），其丰度占到总细菌分类的51.88%，而芽孢杆菌（Bacilli）则在抑制产甲烷的厌氧发酵中占绝对优势，丰度占50%；未抑制产甲烷培养中Peptococcaceae、Syntrophomonadaceae以及Syntrophobacterale这三个菌属的丰度明显大于抑制产甲烷培养，说明产氢产乙酸菌与产甲烷菌的互营关系在厌氧产甲烷体系中发挥着重要作用。
2产氢产乙酸菌与同型产乙酸菌的互营关系
在污泥厌氧产酸体系中，通过加入丁酸盐强化产氢产乙酸过程可以显著提高同型产乙酸反应的活性。同型产乙酸反应的吉布斯自由能降至-15KJ/mol左右，同型产乙酸菌数量明显大于其它反应器；同型产乙酸菌的存在可“拉动”产氢产乙酸过程，但这种互营关系是有限的。在H2分压为3.2KPa，4g/L的丁酸和6g/L的乙酸体系中，产氢产乙酸和同型产乙酸的协同作用停止；在反应吉布斯自由能达到临界值时，产氢产乙酸菌与同型产乙酸菌可利用协同作用产生乙酸。在人为强化产氢产乙酸反应的情况下，协同作用中产氢产乙酸菌与同型产乙酸菌产生乙酸的比例为7:3。
3富集同型产乙酸菌对产酸微生物生态的影响
在厌氧污泥中选择性培养同型产乙酸，通过检测培养过程中有机酸、顶空气体以及关键酶活来鉴定同型产乙酸菌的富集情况。对富集前后的污泥进行高通量测序后发现，富集前的污泥多样性极大，其中厚壁菌门(Firmicutes)和变形菌门(Proteobacteria)分别占到32.3%和42.9%。而富集后的污泥则集中在梭状芽孢杆菌属(Clostridium)，占总细菌的41.3%。另外富集前污泥中含有少量古菌(47OTU)，富集后污泥则未检测到古菌存在。
4强化同型产乙酸工艺条件优化
设计单因素影响实验，研究种泥浓度、底物浓度和pH值等条件对富集同型产乙菌的污泥在厌氧发酵过程中有机酸分布及乙酸累积的影响，结果表明，碱性条件有利于产乙酸菌的生长，但影响产氢产乙酸菌，尤其是丁酸降解菌的反应。产氢产乙酸菌和同型产乙酸菌的互营作用在6天后广泛存在，而且不同pH条件下，丙酸降解菌和丁酸降解菌的反应情况不竟相同；底物浓度过大，产氢产乙酸与同型产乙酸的互营机制受到抑制。
5强化同型产乙酸工艺的动力学模型
在ADM1模型基础上，构建强化同型产乙酸工艺的动力学模型，通过简化后的动力学模型与实验数据的模拟验证表明，拟合结果较为理想，可作为强化同型产乙酸工艺的设计依据。
Acidogenic fermentation is a critical step in the process of anaerobicfermentation of organic matter. Volatile organic acids (VAFAs) is not only one of themain intermediate product, but also an important raw material for the organicchemical industry. Prductions of VAFAs is closely contact with organic matterdegradation and biogas generation. Hydrolysis microorganisms&acidogenicmicroorganisms, methane-producing microorganisms&VFAs-producingmicroorganisms have complex mutual camps relations. In recent years,high-throughput sequencing technology has been used by a growing number ofmicro-organisms workers and has been a very promising biotechnology. Unliketraditional sequencing methods with nucleotide specificity of the DNA fragments,high-throughput sequencing technology can parallel sequence to hundreds ofthousands to millions of DNA molecules. In VFAs-producing reactor, VFAs-producing microorganism population ecology was study. The methanogenicinhibitor addtion which does not affect the acid-producing microorganisms, volatilefatty acids and other intermediate metabolites is a common technique method.
For our study, VFAs-producing microorganisms from the population andcommunity levels in the anaerobic fermentation was used by environment molecularmicrobial ecology technology, such as high-throughput sequencing, the populationstructure of the VFAs-producing bacteria was determined, the contribution ofvarious flora acetic acid was clarified, the yield of VFAs production was enhancedby flora structure and regulation. On these basis the optimal operating conditions ofthe process was identified, and a dynamic model was constructed. This study shouldenrich the theory and application of the anaerobic fermentation process.. The mainfindings are as follows:
1Glucose-degrading microorganisms in activated sludge under methanogenicconditions
Monosaccharide degradation is an important step in the anaerobic fermentationof organic matter, the case for BES inhibition of methanogenesis, anaerobic sludgeglucose degradation plays a critical role in microbial less. The modes of anaerobicacid production and the production of methane was studied by DNA-SIP, clonelibrary and high-throughput sequencing technology, The results showed thatClostridia was dominant in the inhibition of methane-producing anaerobic fermentation, whose abundance accounted for51.88%of the total bacteria andBacilli was dominant in the inhibition of methanogenic anaerobic fermentation,whose abundance accounted for50%; The abundance of Peptococcaceae,Syntrophomonadaceae and Syntrophobacterale genus in the methanogenic culture issignificantly larger than the culture of inhibit methanogenis. The trophic linkbetween hydrogen-producing acetogenic bacteria and methanogenic bacteria p lay animportant role in anaerobic methane-producing system.
2Trophic link between syntrophic acetogens and homoacetogens
In the anaerobic system, strengthen acetogenic reaction. by adding formate cansignificantly improve the activity of the hydrogen-producing process. Isotypeproducing acetic acid of the Gibbs free energy is reduced to-15KJ/mol, the sametype of acetogenic bacteria count was significantly greater than the other reactor; thepresence of acetic acid bacteria of the same type of production to "pull" thehydrogen production process, but this trophic link is limited. In the system of H2pressure was3.2KPa, butyric acid was4g/L and acetic acid was6g/L, the Gibbsfree energy reaches a critical value, the trophic link between syntrophic acetogensand homoacetogens was stopped; The acetate can be producted by the syntrophicacetogens/homoacetogens synergism and the proportion of acetate producted bysyntrophic acetogens and homoacetogens is7:3
3Acidogenic microbial ecology in the enrichment of
Homoacetogens was enriched by selective culture. At the same time, organicacids, the headspace gas and the activities of key enzymes was detected in theculture process. Homoacetogens was identified by compared the high-throughputsequencing at the befor and after enrichment and Firmicutes and Proteobacteriaaccounted for32.3%and42.9%respectively. Clostridium of the enriched sludgerepresent41.3%of the total bacteria. Archaea in original sludge was found (47OTU)and does not detected in enrichment sludge.
4Conditions optimization to the system of homoacetogens enrichment
The single-factor experiments were conducted to study the effects of substrateconcentrations, sludge concentration and pH value conditions in the enrichment ofhomoacetogens. The distribution of organic acid and the accumulation of acetic acidin the anaerobic fermentation process was studied. The results showed that thealkaline conditions conducive to the production of acetic acid, but hydrogenproduction bacteria, especially butyric acid degrading bacteria was inhibited.Production of hydrogen production bacteria and homoacetogens was widely present at just6days, and propionic and butyric acid degrading bacteria reaction was notactually the same at different pH conditions; The concentration of substrate was sohigh that the trophic link between syntrophic acetogens and homoacetogens issuppressed.
5The kinetic model of the system of homoacetogens enrichment
The kinetic model of homoacetogens enrichment based on ADM1model wasbuilded and the kinetic model was simulated and verified to experimental data. Theresult showed that the this model can be used as the basis to design the process ofhomoacetogens enrichment.
本论文利用稳定性同位素标记和高通量测序等环境微生物学技术，研究了产酸微生物在厌氧发酵过程中所起的作用、明确了各产酸菌的种群结构以及它们之间的互营关系、阐明了不同功能菌群在生产VFAs中各自的贡献、并通过菌群结构与数量的调节，增强互营产酸以达到乙酸高产的目的。在此基础上，找出该工艺的最优操作条件，并建立动力学模型，为污泥厌氧发酵过程的研究丰富了理论与应用基础。主要的研究结果如下：
1葡萄糖厌氧降解中产酸微生物种群生态
单糖降解是有机物厌氧发酵中重要步骤，然而，对于BES抑制产甲烷情况下，厌氧污泥对葡萄糖降解起关键性作用的微生物研究则较少。利用DNA-SIP、克隆文库及高通量测序技术，考察污泥厌氧产酸与产甲烷两种模式下，微生物群落的演替情况后发现：未抑制产甲烷的厌氧发酵中占优势的是梭状芽孢杆菌（Clostridia），其丰度占到总细菌分类的51.88%，而芽孢杆菌（Bacilli）则在抑制产甲烷的厌氧发酵中占绝对优势，丰度占50%；未抑制产甲烷培养中Peptococcaceae、Syntrophomonadaceae以及Syntrophobacterale这三个菌属的丰度明显大于抑制产甲烷培养，说明产氢产乙酸菌与产甲烷菌的互营关系在厌氧产甲烷体系中发挥着重要作用。
2产氢产乙酸菌与同型产乙酸菌的互营关系
在污泥厌氧产酸体系中，通过加入丁酸盐强化产氢产乙酸过程可以显著提高同型产乙酸反应的活性。同型产乙酸反应的吉布斯自由能降至-15KJ/mol左右，同型产乙酸菌数量明显大于其它反应器；同型产乙酸菌的存在可“拉动”产氢产乙酸过程，但这种互营关系是有限的。在H2分压为3.2KPa，4g/L的丁酸和6g/L的乙酸体系中，产氢产乙酸和同型产乙酸的协同作用停止；在反应吉布斯自由能达到临界值时，产氢产乙酸菌与同型产乙酸菌可利用协同作用产生乙酸。在人为强化产氢产乙酸反应的情况下，协同作用中产氢产乙酸菌与同型产乙酸菌产生乙酸的比例为7:3。
3富集同型产乙酸菌对产酸微生物生态的影响
在厌氧污泥中选择性培养同型产乙酸，通过检测培养过程中有机酸、顶空气体以及关键酶活来鉴定同型产乙酸菌的富集情况。对富集前后的污泥进行高通量测序后发现，富集前的污泥多样性极大，其中厚壁菌门(Firmicutes)和变形菌门(Proteobacteria)分别占到32.3%和42.9%。而富集后的污泥则集中在梭状芽孢杆菌属(Clostridium)，占总细菌的41.3%。另外富集前污泥中含有少量古菌(47OTU)，富集后污泥则未检测到古菌存在。
4强化同型产乙酸工艺条件优化
设计单因素影响实验，研究种泥浓度、底物浓度和pH值等条件对富集同型产乙菌的污泥在厌氧发酵过程中有机酸分布及乙酸累积的影响，结果表明，碱性条件有利于产乙酸菌的生长，但影响产氢产乙酸菌，尤其是丁酸降解菌的反应。产氢产乙酸菌和同型产乙酸菌的互营作用在6天后广泛存在，而且不同pH条件下，丙酸降解菌和丁酸降解菌的反应情况不竟相同；底物浓度过大，产氢产乙酸与同型产乙酸的互营机制受到抑制。
5强化同型产乙酸工艺的动力学模型
在ADM1模型基础上，构建强化同型产乙酸工艺的动力学模型，通过简化后的动力学模型与实验数据的模拟验证表明，拟合结果较为理想，可作为强化同型产乙酸工艺的设计依据。
Acidogenic fermentation is a critical step in the process of anaerobicfermentation of organic matter. Volatile organic acids (VAFAs) is not only one of themain intermediate product, but also an important raw material for the organicchemical industry. Prductions of VAFAs is closely contact with organic matterdegradation and biogas generation. Hydrolysis microorganisms&acidogenicmicroorganisms, methane-producing microorganisms&VFAs-producingmicroorganisms have complex mutual camps relations. In recent years,high-throughput sequencing technology has been used by a growing number ofmicro-organisms workers and has been a very promising biotechnology. Unliketraditional sequencing methods with nucleotide specificity of the DNA fragments,high-throughput sequencing technology can parallel sequence to hundreds ofthousands to millions of DNA molecules. In VFAs-producing reactor, VFAs-producing microorganism population ecology was study. The methanogenicinhibitor addtion which does not affect the acid-producing microorganisms, volatilefatty acids and other intermediate metabolites is a common technique method.
For our study, VFAs-producing microorganisms from the population andcommunity levels in the anaerobic fermentation was used by environment molecularmicrobial ecology technology, such as high-throughput sequencing, the populationstructure of the VFAs-producing bacteria was determined, the contribution ofvarious flora acetic acid was clarified, the yield of VFAs production was enhancedby flora structure and regulation. On these basis the optimal operating conditions ofthe process was identified, and a dynamic model was constructed. This study shouldenrich the theory and application of the anaerobic fermentation process.. The mainfindings are as follows:
1Glucose-degrading microorganisms in activated sludge under methanogenicconditions
Monosaccharide degradation is an important step in the anaerobic fermentationof organic matter, the case for BES inhibition of methanogenesis, anaerobic sludgeglucose degradation plays a critical role in microbial less. The modes of anaerobicacid production and the production of methane was studied by DNA-SIP, clonelibrary and high-throughput sequencing technology, The results showed thatClostridia was dominant in the inhibition of methane-producing anaerobic fermentation, whose abundance accounted for51.88%of the total bacteria andBacilli was dominant in the inhibition of methanogenic anaerobic fermentation,whose abundance accounted for50%; The abundance of Peptococcaceae,Syntrophomonadaceae and Syntrophobacterale genus in the methanogenic culture issignificantly larger than the culture of inhibit methanogenis. The trophic linkbetween hydrogen-producing acetogenic bacteria and methanogenic bacteria p lay animportant role in anaerobic methane-producing system.
2Trophic link between syntrophic acetogens and homoacetogens
In the anaerobic system, strengthen acetogenic reaction. by adding formate cansignificantly improve the activity of the hydrogen-producing process. Isotypeproducing acetic acid of the Gibbs free energy is reduced to-15KJ/mol, the sametype of acetogenic bacteria count was significantly greater than the other reactor; thepresence of acetic acid bacteria of the same type of production to "pull" thehydrogen production process, but this trophic link is limited. In the system of H2pressure was3.2KPa, butyric acid was4g/L and acetic acid was6g/L, the Gibbsfree energy reaches a critical value, the trophic link between syntrophic acetogensand homoacetogens was stopped; The acetate can be producted by the syntrophicacetogens/homoacetogens synergism and the proportion of acetate producted bysyntrophic acetogens and homoacetogens is7:3
3Acidogenic microbial ecology in the enrichment of
Homoacetogens was enriched by selective culture. At the same time, organicacids, the headspace gas and the activities of key enzymes was detected in theculture process. Homoacetogens was identified by compared the high-throughputsequencing at the befor and after enrichment and Firmicutes and Proteobacteriaaccounted for32.3%and42.9%respectively. Clostridium of the enriched sludgerepresent41.3%of the total bacteria. Archaea in original sludge was found (47OTU)and does not detected in enrichment sludge.
4Conditions optimization to the system of homoacetogens enrichment
The single-factor experiments were conducted to study the effects of substrateconcentrations, sludge concentration and pH value conditions in the enrichment ofhomoacetogens. The distribution of organic acid and the accumulation of acetic acidin the anaerobic fermentation process was studied. The results showed that thealkaline conditions conducive to the production of acetic acid, but hydrogenproduction bacteria, especially butyric acid degrading bacteria was inhibited.Production of hydrogen production bacteria and homoacetogens was widely present at just6days, and propionic and butyric acid degrading bacteria reaction was notactually the same at different pH conditions; The concentration of substrate was sohigh that the trophic link between syntrophic acetogens and homoacetogens issuppressed.
5The kinetic model of the system of homoacetogens enrichment
The kinetic model of homoacetogens enrichment based on ADM1model wasbuilded and the kinetic model was simulated and verified to experimental data. Theresult showed that the this model can be used as the basis to design the process ofhomoacetogens enrichment.
引文
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