秸秆类生物质暗发酵产氢关键参数优化及其机理研究
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摘要
氢能因其具有高效、清洁、无污染的特点被公认为最具发展前景的能源之一。在诸多的制氢方法中，生物制氢因其能够在常温常压下操作而备受关注。与光合制氢相比，暗发酵制氢具有产氢速率高、不依赖光照、设备简单、易操作、能够实现废弃生物质资源化等特点而最具产业化前景。目前，该技术普遍存在产氢率低、发酵底物成本高等问题，成为制约暗发酵制氢产业化的主要技术瓶颈。基于此，本文在探寻天然菌源预处理方法的基础上，从高效生物制氢反应器中分离、筛选得到一株底物利用范围广的高效兼性厌氧菌，对其产氢特性进行了研究，并从生化工程的角度对其中的NADH产氢途径进行调控；之后，从降低运行成本的角度，筛选分离在中温条件下能够直接利用未经过预处理的秸秆类生物质高效发酵产氢的微生物，进行菌种鉴定及相关的产氢特性研究。
具体内容如下：
1．探寻新的菌源预处理方式。
系统考察了微波辐射预处理对混合菌源产氢性能的综合影响。结果显示：采用微波辐射对牛粪堆肥进行预处理，能够有效抑制或杀灭嗜氢菌，而保留产氢菌的活性。以20g/L酸解玉米秸秆为底物，利用微波辐射1.5min预处理的牛粪堆肥为菌源，在Na2CO3添加量800mg/L，Fe添加量400mg/L时，产氢量最大，达到144.3ml/g。与其它菌源预处理方式相比，该方法操作安全简单，能耗低，耗时少，且菌源产氢能力更高。
通过AFM，蛋白浓度测定和PCR-DGGE技术研究了微波辐射作用机理在于：微波辐射能够对菌源中微生物细胞壁造成不同程度的损伤和破坏，进而影响微生物细胞的通透性；此外，微波还能对微生物的基因组DNA造成不同程度的损伤，由此导致对微波辐射敏感的嗜氢菌的失活或杀灭；而主要的产氢菌形成的芽孢具有极强的耐辐射和耐热能力，短时的微波辐射不会对其造成损伤，产氢菌的活性得以保持。
2．从生物制氢反应器中分离得到一株高效兼性厌氧产氢菌。首先对其进行形态学特征及生理生化特性研究，结合菌株16S rDNA序列分析，确定该菌株的种属分类。对菌株产氢特性进行研究：菌株Bacillus sp. FS2011能利用多种碳、氮源产氢。以葡萄糖为底物时，最大产氢量为2.26mol/mol。发酵液的初始pH和培养温度对菌株产氢活性有影响，初始pH6.98、35℃为该菌种发酵产氢较为适宜的条件。在单因素实验的基础上，采用Plackett-Burman试验设计法及响应面分析法对菌株Bacillus sp. FS2011发酵产氢条件进行优化。首先采用Plackett-Burman试验设计法筛选出影响菌株产氢的主要因素：葡萄糖浓度、牛肉膏浓度和磷酸缓冲液浓度。在此基础上，利用最陡爬坡路径大致确定最大响应区域，最后利用Box-Behnken试验设计及响应面法进行回归分析。结果表明，三个主要因素的浓度变化显著影响氢气产量。通过求解回归方程，确定最优产氢条件：当葡萄糖浓度13.52g/L，牛肉膏浓度1.95g/L，磷酸缓冲液浓度19.36mmol/L时，氢气产量达到理论最大值3079.0ml/L，实际产氢量达到3081.2ml/L。
3．对Bacillus sp. FS2011中NADH产氢途径进行强化。首先，在培养基中使用与葡萄糖结构类似，氧化还原状态不同的底物作为碳源进行发酵产氢试验，确定FS2011胞内存在NADH产氢途径。
采用丙酮酸脱氢酶系E1抑制剂对菌源产氢过程进行干涉，以期能够充分发挥NADH产氢途径的作用。首先考察了抑制剂浓度对兼性厌氧纯菌和混合菌源发酵产氢量的影响。之后，考察了特定浓度抑制剂的添加对兼性厌氧纯菌和混合菌源产氢性能的综合影响。结果发现，添加适量的抑制剂能够有效提高发酵体系的氢气产量，同时降低体系中乙酸和丁酸等副产物的生成，降低了后续发酵废水的处理难度。
4．从利用生玉米秸秆驯化的生物制氢反应器中分离得到一株能够在中温条件下，不经过底物预处理，高效利用生玉米秸秆发酵产氢的细菌。根据其形态学特征及生理生化特性，并结合菌株16S rDNA序列分析，确定菌株FS3的种属分类。
菌株Clostridium sp. FS3能利用多种碳源产氢。以生玉米秸秆和酸解秸秆为底物时，产氢量分别为73.1和75.3ml/g-cornstalk，初步判断该菌株能够直接利用秸秆中的半纤维素产氢。在单因素实验的基础上，采用响应面分析法对Clostridium sp. FS3发酵产氢培养基进行优化。利用Box-Behnken试验设计及响应面分析法进行回归分析。结果表明，碳酸氢铵、磷酸二氢钾和营养液的浓度变化能够显著影响氢气产量，通过求解回归方程得到如下结论：当生玉米秸秆浓度20g/L，碳酸氢铵浓度1.76g/L，磷酸二氢钾浓度0.91g/L，营养液的添加量为10.4ml/L时，氢气产量达到理论最大值94.1ml/g，实际产氢量达到92.9ml/g。Clostridium sp. FS3作为能够在中温条件下直接利用纤维素类生物质发酵产氢的优良菌种，在生物制氢中有较好的应用前景。
Energy crisis and serious global environment pollution have been caused byexcessive use of fossil fuels. Thus, it is urgent to develop nonpolluting and renewableenergy resource. Hydrogen is deemed as the most promising alternative energy due toits high conversion efficiency, recyclability and nonpolluting nature. In all hydrogenproduction processes, biological hydrogen production stands out because it can beaccomplished at ambient temperature and pressure. In comparison withphotosynthetic fermentation, dark fermentation has many advantages such ashigh-rate of hydrogen production, simple fermentative equipment, independence oflight and bioconversion feasibility from renewable resources. However, the low yieldof hydrogen production and the high cost of the substrate are the main constraints forthe industrialization of dark fermentative hydrogen production. In this dissertation,the effect of microwave irradiation pretreatment of compost on bio-hydrogenproduction from corn stalk was systematically investigated. The characteristics of anisolated high-efficient hydrogen-producing bacterium and another strain which coulddirectly convert raw cornstalk to hydrogen were analyzed. In addition, biochemicalengineering was applied to manipulate “NADH pathway” in Bacillus sp. FS2011.Main contents and results are follows:
1. The effect of microwave irradiation pretreatment of compost on bio-hydrogenproduction from corn stalk was systematically investigated. The microwavepretreatment of compost was an alternative strategy to suppress the activity ofhydrogen-consuming bacteria and harvest high yield hydrogen-producingsporeforming anaerobes in fermentation producing hydrogen process for its safe andsimple operation, low energy consumption, short time-consuming and higherhydrogen yield. The maximum hydrogen yield of144.3ml/g was obtained from cornstock by the irradiated cow dung compost at fixed substrate concentration of20g/L,Na2CO3dosage of800mg/L and Fe dosage of400mg/L. The diversity and symbiosisrelations of the mixed bacteria in the pretreated compost had been observed, thedamage of microbial cell was further confirmed by AFM, determination of protein content and PCR-DGGE analysis.
2. A new fermentative hydrogen-producing strain FS2011was isolated from aneffluent of bio-hydrogen production reactor, and identified as Bacillusamyloliquefaciens on the basis of16S rDNA gene sequence. The strain could utilizevarious carbon and nitrogen sources to produce hydrogen in a broad range of initialpH (5.29-7.38). Single factor experimental designs and RSM were applied tooptimize culture conditions for hydrogen production from glucose. The optimalparameters for the maximal Ps(3079.0ml/L)were: glucose13.52g/L, beef extract1.95g/L, initial pH6.98, phosphate buffer19.36mmol/L, and35℃, indicatingFS2011was a high-efficiency hydrogen-producing bacterium.
3.“NADH pathway” was determined in Bacillus sp. FS2011by studying theeffect of the redox state of substrates on hydrogen production. The efficient methodsfor extraction and detection of NAD+and NADH in pure and mixed cultures wereestablished. Thereafter, the inhibitor of pyruvate dehydrogenase multienzymecomplex (PDHc) E1:4-((1-((4-amino-2-methylpyrimidin-5-yl) methyl)-5-iodo-1H-1,2,3-triazol-4-yl)methoxy)benzonitrilewas added into the medium to control thehydrogen metabolism of FS2011and mixed culture. And the effect of the inhibitor ofPDHc on hydrogen production by pure and mixed cultures was systematically studied.Results revealed that the appropriate amount of PDC inhibitor could efficientlyenhance hydrogen production and decrease the production of soluble metabolicbyproducts such as butyrate, acetate and so on by both the facultative anaerobeBacillus sp. FS2011and mix culture in batch cultivation.
4. A new hydrogen-producing strain Clostridium sp. FS3was successfullyisolated and identified, which could directly convert raw cornstalk to hydrogenwithout substrate pretreatment in moderate temperature. The strain could utilizevarious carbon sources to produce hydrogen. According to the results of RSMexperiment,20g/L raw corn stalk,1.76g/L NH4HCO3,0.91g/L KH2PO4and10.4ml/L nutrient solution were established to be the most favorable for hydrogenproduction. The maximal hydrogen yield of92.9ml/g-corn stalk was observed underthe optimal conditions. Clostridium sp. FS3was considered an ideal bacterium forhydrogen production from raw cellulosic biomass.
具体内容如下：
1．探寻新的菌源预处理方式。
系统考察了微波辐射预处理对混合菌源产氢性能的综合影响。结果显示：采用微波辐射对牛粪堆肥进行预处理，能够有效抑制或杀灭嗜氢菌，而保留产氢菌的活性。以20g/L酸解玉米秸秆为底物，利用微波辐射1.5min预处理的牛粪堆肥为菌源，在Na2CO3添加量800mg/L，Fe添加量400mg/L时，产氢量最大，达到144.3ml/g。与其它菌源预处理方式相比，该方法操作安全简单，能耗低，耗时少，且菌源产氢能力更高。
通过AFM，蛋白浓度测定和PCR-DGGE技术研究了微波辐射作用机理在于：微波辐射能够对菌源中微生物细胞壁造成不同程度的损伤和破坏，进而影响微生物细胞的通透性；此外，微波还能对微生物的基因组DNA造成不同程度的损伤，由此导致对微波辐射敏感的嗜氢菌的失活或杀灭；而主要的产氢菌形成的芽孢具有极强的耐辐射和耐热能力，短时的微波辐射不会对其造成损伤，产氢菌的活性得以保持。
2．从生物制氢反应器中分离得到一株高效兼性厌氧产氢菌。首先对其进行形态学特征及生理生化特性研究，结合菌株16S rDNA序列分析，确定该菌株的种属分类。对菌株产氢特性进行研究：菌株Bacillus sp. FS2011能利用多种碳、氮源产氢。以葡萄糖为底物时，最大产氢量为2.26mol/mol。发酵液的初始pH和培养温度对菌株产氢活性有影响，初始pH6.98、35℃为该菌种发酵产氢较为适宜的条件。在单因素实验的基础上，采用Plackett-Burman试验设计法及响应面分析法对菌株Bacillus sp. FS2011发酵产氢条件进行优化。首先采用Plackett-Burman试验设计法筛选出影响菌株产氢的主要因素：葡萄糖浓度、牛肉膏浓度和磷酸缓冲液浓度。在此基础上，利用最陡爬坡路径大致确定最大响应区域，最后利用Box-Behnken试验设计及响应面法进行回归分析。结果表明，三个主要因素的浓度变化显著影响氢气产量。通过求解回归方程，确定最优产氢条件：当葡萄糖浓度13.52g/L，牛肉膏浓度1.95g/L，磷酸缓冲液浓度19.36mmol/L时，氢气产量达到理论最大值3079.0ml/L，实际产氢量达到3081.2ml/L。
3．对Bacillus sp. FS2011中NADH产氢途径进行强化。首先，在培养基中使用与葡萄糖结构类似，氧化还原状态不同的底物作为碳源进行发酵产氢试验，确定FS2011胞内存在NADH产氢途径。
采用丙酮酸脱氢酶系E1抑制剂对菌源产氢过程进行干涉，以期能够充分发挥NADH产氢途径的作用。首先考察了抑制剂浓度对兼性厌氧纯菌和混合菌源发酵产氢量的影响。之后，考察了特定浓度抑制剂的添加对兼性厌氧纯菌和混合菌源产氢性能的综合影响。结果发现，添加适量的抑制剂能够有效提高发酵体系的氢气产量，同时降低体系中乙酸和丁酸等副产物的生成，降低了后续发酵废水的处理难度。
4．从利用生玉米秸秆驯化的生物制氢反应器中分离得到一株能够在中温条件下，不经过底物预处理，高效利用生玉米秸秆发酵产氢的细菌。根据其形态学特征及生理生化特性，并结合菌株16S rDNA序列分析，确定菌株FS3的种属分类。
菌株Clostridium sp. FS3能利用多种碳源产氢。以生玉米秸秆和酸解秸秆为底物时，产氢量分别为73.1和75.3ml/g-cornstalk，初步判断该菌株能够直接利用秸秆中的半纤维素产氢。在单因素实验的基础上，采用响应面分析法对Clostridium sp. FS3发酵产氢培养基进行优化。利用Box-Behnken试验设计及响应面分析法进行回归分析。结果表明，碳酸氢铵、磷酸二氢钾和营养液的浓度变化能够显著影响氢气产量，通过求解回归方程得到如下结论：当生玉米秸秆浓度20g/L，碳酸氢铵浓度1.76g/L，磷酸二氢钾浓度0.91g/L，营养液的添加量为10.4ml/L时，氢气产量达到理论最大值94.1ml/g，实际产氢量达到92.9ml/g。Clostridium sp. FS3作为能够在中温条件下直接利用纤维素类生物质发酵产氢的优良菌种，在生物制氢中有较好的应用前景。
Energy crisis and serious global environment pollution have been caused byexcessive use of fossil fuels. Thus, it is urgent to develop nonpolluting and renewableenergy resource. Hydrogen is deemed as the most promising alternative energy due toits high conversion efficiency, recyclability and nonpolluting nature. In all hydrogenproduction processes, biological hydrogen production stands out because it can beaccomplished at ambient temperature and pressure. In comparison withphotosynthetic fermentation, dark fermentation has many advantages such ashigh-rate of hydrogen production, simple fermentative equipment, independence oflight and bioconversion feasibility from renewable resources. However, the low yieldof hydrogen production and the high cost of the substrate are the main constraints forthe industrialization of dark fermentative hydrogen production. In this dissertation,the effect of microwave irradiation pretreatment of compost on bio-hydrogenproduction from corn stalk was systematically investigated. The characteristics of anisolated high-efficient hydrogen-producing bacterium and another strain which coulddirectly convert raw cornstalk to hydrogen were analyzed. In addition, biochemicalengineering was applied to manipulate “NADH pathway” in Bacillus sp. FS2011.Main contents and results are follows:
1. The effect of microwave irradiation pretreatment of compost on bio-hydrogenproduction from corn stalk was systematically investigated. The microwavepretreatment of compost was an alternative strategy to suppress the activity ofhydrogen-consuming bacteria and harvest high yield hydrogen-producingsporeforming anaerobes in fermentation producing hydrogen process for its safe andsimple operation, low energy consumption, short time-consuming and higherhydrogen yield. The maximum hydrogen yield of144.3ml/g was obtained from cornstock by the irradiated cow dung compost at fixed substrate concentration of20g/L,Na2CO3dosage of800mg/L and Fe dosage of400mg/L. The diversity and symbiosisrelations of the mixed bacteria in the pretreated compost had been observed, thedamage of microbial cell was further confirmed by AFM, determination of protein content and PCR-DGGE analysis.
2. A new fermentative hydrogen-producing strain FS2011was isolated from aneffluent of bio-hydrogen production reactor, and identified as Bacillusamyloliquefaciens on the basis of16S rDNA gene sequence. The strain could utilizevarious carbon and nitrogen sources to produce hydrogen in a broad range of initialpH (5.29-7.38). Single factor experimental designs and RSM were applied tooptimize culture conditions for hydrogen production from glucose. The optimalparameters for the maximal Ps(3079.0ml/L)were: glucose13.52g/L, beef extract1.95g/L, initial pH6.98, phosphate buffer19.36mmol/L, and35℃, indicatingFS2011was a high-efficiency hydrogen-producing bacterium.
3.“NADH pathway” was determined in Bacillus sp. FS2011by studying theeffect of the redox state of substrates on hydrogen production. The efficient methodsfor extraction and detection of NAD+and NADH in pure and mixed cultures wereestablished. Thereafter, the inhibitor of pyruvate dehydrogenase multienzymecomplex (PDHc) E1:4-((1-((4-amino-2-methylpyrimidin-5-yl) methyl)-5-iodo-1H-1,2,3-triazol-4-yl)methoxy)benzonitrilewas added into the medium to control thehydrogen metabolism of FS2011and mixed culture. And the effect of the inhibitor ofPDHc on hydrogen production by pure and mixed cultures was systematically studied.Results revealed that the appropriate amount of PDC inhibitor could efficientlyenhance hydrogen production and decrease the production of soluble metabolicbyproducts such as butyrate, acetate and so on by both the facultative anaerobeBacillus sp. FS2011and mix culture in batch cultivation.
4. A new hydrogen-producing strain Clostridium sp. FS3was successfullyisolated and identified, which could directly convert raw cornstalk to hydrogenwithout substrate pretreatment in moderate temperature. The strain could utilizevarious carbon sources to produce hydrogen. According to the results of RSMexperiment,20g/L raw corn stalk,1.76g/L NH4HCO3,0.91g/L KH2PO4and10.4ml/L nutrient solution were established to be the most favorable for hydrogenproduction. The maximal hydrogen yield of92.9ml/g-corn stalk was observed underthe optimal conditions. Clostridium sp. FS3was considered an ideal bacterium forhydrogen production from raw cellulosic biomass.
引文
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