生物质高效水解及发酵产氢的机理研究
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摘要
随着环保要求的日益严格和化石能源的日益短缺，氢能作为清洁高效的可再生能源受到人们的普遍重视。本文针对农业废弃生物质秸秆以及世界十大恶性杂草之一的水葫芦进行了厌氧发酵产氢实验研究。
首先针对稻草秸秆，在比较酶水解和酸水解优缺点的基础上，确定了纤维素酶水解的绿色工艺。研究了预处理方法、反应时间、水解温度及pH值等因素对稻草秸秆纤维素酶水解的还原糖转化率的影响规律，确定了稻草秸秆微生物发酵产氢前采用纤维素酶水解时，pH为4.8以及反应温度为50℃的较佳工况点。
稻草秸秆的水解效果是发酵产氢的关键点之一，本文提出了生物质超临界水高效降解以及微生物发酵产氢，采用稻草秸秆近临界水解在280℃和20Mpa等优化条件下获得了较高的还原糖转化率。并分别讨论了物料浓度、水解温度、水解压力以及水解反应时间等参数对稻草秸秆近临界水降解还原糖转化率的影响。采用色质联机分析技术对液体产物中糖以外的副产物进行了深入分析，发现产物中主要成分为一些含甲基、乙基、甲氧基、羟基等官能团的酮类、醛类、醇类和酸类极性化合物。采用一级均相反应模型模拟了试验数据，得到了与实验数据吻合较好的动力学反应曲线。
在纤维素酶水解的基础上，分别讨论不同的预处理方式、不同来源的混合菌群以及不同的菌群接种方式对稻草秸秆发酵产氢的影响。由于秸秆水解产物中包含多种还原糖，其中五碳糖木糖的发酵液相产物中丙酸含量偏高，乙酸、丁酸含量较低，导致NADH含量较低，因此其发酵产氢能力低于六碳糖。这也是不同发酵底料发酵产氢存在差异的原因之一。针对不同糖源，讨论了产气肠杆菌以及混合菌群发酵产氢特性。由于混菌间的协同作用，因此混菌的发酵能力优于纯菌。而混菌发酵产氢也会因为菌群来源以及接种方式的不同而有所差异。在上述研究的基础上，讨论了温度、pH值以及稻草秸秆粒径等影响因子对发酵产氢的影响。并通过修正的Gompertz方程拟合发酵产氢动力学曲线。经过NaOH预处理后的稻草单位最大总产氢量可达到91.5mL／g，最高产氢速率为1.52mL／h·g。
在稻草秸秆发酵产氢研究的基础上，采用厌氧活性污泥为接种物，以水葫芦作为发酵底料进行产氢实验研究。针对水葫芦不同部位对于水域内金属离子吸附能力的差异性，考察了水葫芦不同部位的发酵产氢能力，并在此基础上通过讨论不同金属Fe~(2+)、Mg~(2+)、Cu~(2+)、Ni~(2+)、和Zn~(2+)离子及其浓度对发酵产氢的影响，提出过量的金属离子对于微生物发酵产氢存在抑制作用，从而导致了水葫芦不同部位发酵产氢能力的差异。在实验室三角烧瓶的实验基础上，采用5L容积的发酵罐进行了水葫芦发酵产氢的中小规模实验研究。
最后合成GenBank中登录的阴沟肠杆菌(Enterobacter cloacae)铁氢酶序列，通过质粒将氢酶植入大肠杆菌Escherichia coli BL21中，实现了氢酶在Escherichia coli BL21中的表达，并研究重组后的E.coli BL21的产氢特性。基因改造后的E.coli BL21最大氢气产气量提升了0.5mol／mol葡萄糖。
Hydrogen, as a clean energy that can replace conventional fossil fuels, is attracting worldwide attentions. In this paper, a detail research of hydrogen production by anaerobic fermentation from straw and hyacinth was carried out.
First of all, rice straw was degraded by commercial cellulase. Effect of various process parameters, such as pretreatment methods, reaction time, and hydrolysis temperature and pH values was examined with respect to saccharification rate to determine the optimum of hydrolysis parameters.
Bio-Hydrogen production by combination of Supercritical Water hydrolysis and anaerobic fermentation was proposed. Limited by the experimental apparatus, rice straw was degraded in Near Critical Water. Saccharification rate of rice straw reached to 50.41 % at 280°C and 20Mpa. The sugar components of the liquid products were analyzed by HPLC with the KS-802 sugars column. Other small molecule organic products were detected by GC-MS analysis, as showed that there were some polarity components with methyl, ethyl, methylene and hydroxyl, etc. The experimental data was simulated by heterogeneous first-order reactions model and the experimental date was coincided with the model.
Experiments were carried out to show the effects of pretreatment methods, different mixed bacteria and different inoculation methods on fermentative hydrogen production by rice straw. And hydrogen production by Enterobacter aerogenes and mixed bacteria from different sugars was also investigated. The influence of the main parameters such as particle size, the ratio of substrate to cellulase, pretreatment method and pH value on the straw fermentation was investigated Gompertz equation was used to simulated the dynamics curve of hydrogen production.
Hydrogen from the fermentation of hyacinth by digested sludge is demonstrated. The leaf and root of the hyacinth showed different hydrogen production potential because of the diverse metals content in different parts of the hyacinth. And a further study was carried out to investigate effects of different metal ion and its concentration on hydrogen production by Enterobacter aerogenes from glucose. Finally, hydrogen production by hyacinth was carried out in a fermenter with a volume of 5L.
Fe-hydrogenase coded gene from Enterobacter cloacae was express by vector in non-hydrogen producing Escherichia coli BL21. And fermentative hydrogen production was carried out in batch experiment using recombinant E.coli.
首先针对稻草秸秆，在比较酶水解和酸水解优缺点的基础上，确定了纤维素酶水解的绿色工艺。研究了预处理方法、反应时间、水解温度及pH值等因素对稻草秸秆纤维素酶水解的还原糖转化率的影响规律，确定了稻草秸秆微生物发酵产氢前采用纤维素酶水解时，pH为4.8以及反应温度为50℃的较佳工况点。
稻草秸秆的水解效果是发酵产氢的关键点之一，本文提出了生物质超临界水高效降解以及微生物发酵产氢，采用稻草秸秆近临界水解在280℃和20Mpa等优化条件下获得了较高的还原糖转化率。并分别讨论了物料浓度、水解温度、水解压力以及水解反应时间等参数对稻草秸秆近临界水降解还原糖转化率的影响。采用色质联机分析技术对液体产物中糖以外的副产物进行了深入分析，发现产物中主要成分为一些含甲基、乙基、甲氧基、羟基等官能团的酮类、醛类、醇类和酸类极性化合物。采用一级均相反应模型模拟了试验数据，得到了与实验数据吻合较好的动力学反应曲线。
在纤维素酶水解的基础上，分别讨论不同的预处理方式、不同来源的混合菌群以及不同的菌群接种方式对稻草秸秆发酵产氢的影响。由于秸秆水解产物中包含多种还原糖，其中五碳糖木糖的发酵液相产物中丙酸含量偏高，乙酸、丁酸含量较低，导致NADH含量较低，因此其发酵产氢能力低于六碳糖。这也是不同发酵底料发酵产氢存在差异的原因之一。针对不同糖源，讨论了产气肠杆菌以及混合菌群发酵产氢特性。由于混菌间的协同作用，因此混菌的发酵能力优于纯菌。而混菌发酵产氢也会因为菌群来源以及接种方式的不同而有所差异。在上述研究的基础上，讨论了温度、pH值以及稻草秸秆粒径等影响因子对发酵产氢的影响。并通过修正的Gompertz方程拟合发酵产氢动力学曲线。经过NaOH预处理后的稻草单位最大总产氢量可达到91.5mL／g，最高产氢速率为1.52mL／h·g。
在稻草秸秆发酵产氢研究的基础上，采用厌氧活性污泥为接种物，以水葫芦作为发酵底料进行产氢实验研究。针对水葫芦不同部位对于水域内金属离子吸附能力的差异性，考察了水葫芦不同部位的发酵产氢能力，并在此基础上通过讨论不同金属Fe~(2+)、Mg~(2+)、Cu~(2+)、Ni~(2+)、和Zn~(2+)离子及其浓度对发酵产氢的影响，提出过量的金属离子对于微生物发酵产氢存在抑制作用，从而导致了水葫芦不同部位发酵产氢能力的差异。在实验室三角烧瓶的实验基础上，采用5L容积的发酵罐进行了水葫芦发酵产氢的中小规模实验研究。
最后合成GenBank中登录的阴沟肠杆菌(Enterobacter cloacae)铁氢酶序列，通过质粒将氢酶植入大肠杆菌Escherichia coli BL21中，实现了氢酶在Escherichia coli BL21中的表达，并研究重组后的E.coli BL21的产氢特性。基因改造后的E.coli BL21最大氢气产气量提升了0.5mol／mol葡萄糖。
Hydrogen, as a clean energy that can replace conventional fossil fuels, is attracting worldwide attentions. In this paper, a detail research of hydrogen production by anaerobic fermentation from straw and hyacinth was carried out.
First of all, rice straw was degraded by commercial cellulase. Effect of various process parameters, such as pretreatment methods, reaction time, and hydrolysis temperature and pH values was examined with respect to saccharification rate to determine the optimum of hydrolysis parameters.
Bio-Hydrogen production by combination of Supercritical Water hydrolysis and anaerobic fermentation was proposed. Limited by the experimental apparatus, rice straw was degraded in Near Critical Water. Saccharification rate of rice straw reached to 50.41 % at 280°C and 20Mpa. The sugar components of the liquid products were analyzed by HPLC with the KS-802 sugars column. Other small molecule organic products were detected by GC-MS analysis, as showed that there were some polarity components with methyl, ethyl, methylene and hydroxyl, etc. The experimental data was simulated by heterogeneous first-order reactions model and the experimental date was coincided with the model.
Experiments were carried out to show the effects of pretreatment methods, different mixed bacteria and different inoculation methods on fermentative hydrogen production by rice straw. And hydrogen production by Enterobacter aerogenes and mixed bacteria from different sugars was also investigated. The influence of the main parameters such as particle size, the ratio of substrate to cellulase, pretreatment method and pH value on the straw fermentation was investigated Gompertz equation was used to simulated the dynamics curve of hydrogen production.
Hydrogen from the fermentation of hyacinth by digested sludge is demonstrated. The leaf and root of the hyacinth showed different hydrogen production potential because of the diverse metals content in different parts of the hyacinth. And a further study was carried out to investigate effects of different metal ion and its concentration on hydrogen production by Enterobacter aerogenes from glucose. Finally, hydrogen production by hyacinth was carried out in a fermenter with a volume of 5L.
Fe-hydrogenase coded gene from Enterobacter cloacae was express by vector in non-hydrogen producing Escherichia coli BL21. And fermentative hydrogen production was carried out in batch experiment using recombinant E.coli.
引文
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