生物制氢过程中微生物耐酸响应的生物化学机制研究
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摘要
生物质厌氧发酵产氢过程积累的大量有机酸,会对厌氧微生物产生抑制作用,进而制约生物产氢过程的持续进行。本课题采用经丁酸胁迫的污泥进行厌氧发酵产氢实验,以研究经过酸胁迫后的污泥产氢产酸性能能否提高,并考察经酸胁迫后的生物产氢代谢系统中的脱氢酶、谷氨酸脱羧酶(Glutamate decarboxylase, GAD)等酶活性的变化,以期阐明厌氧颗粒污泥中微生物菌群的耐酸应答机制;同时,对厌氧污泥胞外聚合物(Extracellular polymeric substances, EPS)组成进行研究,以对比不同丁酸胁迫浓度对污泥EPS组分含量的影响;最后采用DGGE技术分析酸胁迫对污泥内微生物DNA含量的影响,从而为更深入研究厌氧污泥酸响应的生物化学机制打下一定基础。
主要内容与结果如下:
1、首先,对污泥厌氧产氢产酸过程中的控制因素进行研究,主要从底物浓度、物料比、pH值调节、添加金属元素等产氢的初步条件进行讨论。研究表明,当葡萄糖初始浓度为0.1 mol/L,物料比为1.5:1,初始pH值设定为5.5,且在发酵过程中调节并保持pH值在5.3-5.7间,镍、铁离子浓度分别为0.04 mg/L、7.5 g/L时,产氢量可达到最大值2240 mL,总有机酸产量为104.08 mmol/L,其中乙酸与丁酸含量分别占29%、60%,属于典型的丁酸型发酵。
2、其次,本研究对污泥预先进行酸胁迫,然后对其厌氧发酵过程进行研究。研究表明,当丁酸胁迫浓度为6 g/L时,厌氧发酵产氢体系中丁酸、乙酸以及氢气产量最高,分别达到107.1 mmol/L,46.3 mmol/L和3690 mL/mol glucose,与对照组相比分别提高了65%、54%、110%。
3、同时,本研究还采用葡萄糖、谷氨酸钠、正丁酸对厌氧污泥进行预处理,以研究不同环境因子对厌氧微生物菌群基于谷氨酸脱羧酶体系的耐酸应答机制的影响。结果表明:经谷氨酸钠处理后污泥GAD活性有所提高,γ-氨基丁酸(GABA)含量较对照组提高55%;而经丁酸处理的组别GAD活性最高,GABA浓度达13.9μmol/(g·TS·h),相对于对照组提高了60%;葡萄糖处理组的污泥GAD活性与对照组相比降低了9%。进而,本研究采用了不同浓度丁酸对厌氧颗粒污泥预处理并进行厌氧发酵实验,结果表明,当丁酸浓度为6 g/L时,污泥谷氨酸脱羧酶和脱氢酶活性最高,分别达到14.7μmol/(g·TS·h),6982.12μgTF/(g·TS·h),相对于对照组分别提高了77%、95%,因此后续实验中均采用6 g/L作为最适宜的酸胁迫浓度。
4、最后,本研究还对厌氧产氢过程中污泥EPS的变化进行了讨论。结果表明:经过丁酸胁迫的厌氧污泥中EPS含量有明显的提高。与对照组相比,松散型多糖和蛋白质以及紧密型多糖和蛋白质含量分别提高了147%,34.8%,35%,21.6%。同时,本研究还采用DGGE技术分析丁酸处理对污泥DNA表达的影响。研究表明,经过6 g/L丁酸胁迫的污泥DNA含量较空白组提高11%,其后随着酸浓度的提高,污泥内DNA的含量呈现降低趋势;此外,经6 g/L丁酸胁迫的污泥电泳条带亮度高、带型整齐,Clostridium sp.和Bacillus sp.微生物成为污泥内的优势种,说明该组产氢微生物活性高于对照组,且产氢性能也较好。而12 g/L丁酸胁迫组的污泥产氢微生物数量大幅降低,产氢性能也远低于对照组,说明过高浓度的丁酸胁迫会对污泥内微生物活性造成伤害,进而导致微生物产氢性能降低。
Along with the anaerobic digestion of biomass, large amounts of organic acids would be accumulated, which resulted in an inhibitory effect on anaerobic microbiology, and the biohydrogen process. In this study, the anaerobic sludge with butyate stress was used to enhance the biohydrogen process, and to explore the glutamate decarboxylase (GAD) represented microbial acid tolerance response (ATR) within the anaerobic sludge. Moreover, effect of different concentration of acid stress on extracellular polymeric substances (EPS) component of the anaerobic sludge was also investigated. Finally, further investigation of the DNA components through DGGE analysis during the anaerobic digestion process was also conducted. The main conclusions of this paper are as followed:
1. Firstly, optimization of the fermentation conditions were investigated. It was found that the accumulation of biohydrogen and organic acids reached 2240 mL and 104.08 mmol/L when substrate concentration was 0.1 mol/L, pH ranged 5.3-5.7, ratio of glucose and inoculating sludge at 1.5:1, and the concentration of Fe2+ and Ni2+ is 0.04 mg/L and 7.5 g/L.
2. Secondly, anaerobic sludge with acid stress was adopted for hydrogen production. It was found that the production of butyric acid, acetic acid and hydrogen reached 107.1 mmol/L, 46.2 mmol/L and 3690 mL/mol glucose when the concentration of butyric acid reached 6.0 g/L which were 65%, 54% and 110% of the control, respectively.
3. Thirdly, three different stimulators as glucose, glutamine and butyrate were used to induce the the glutamate-dependent system based microbial ATR. It was found that GAD activity decreased 9% compared with the control in the case of glucose. While the GAD activity reached 13.9μmol/(g·TS·h) when butyrate was adopted, which increased by 55% compared with the control. Following, concentration of adopted for further acid stress was 6.0 g/L, as the activity of GAD and dehydrogenase activity reached 14.7μmol/(g·TS·h) and 6982.12μgTF/(g·TS·h), which increased by 77% and 95% compared with the control, respectively.
4. Finally, it indicated that the content of EPS, and loosely bound protein, loosely bound polysaccharide, tightly bound protein and tightly bound polysaccharide content were 147%, 34.8%, 35%, 21.6% higher than that of the control, respectively, with acid stress on anaerobic sludge. On the other hand, total amount of DNA was found to be increased by 11% compared with the control with appropriate acid stress. Moreover, denaturing gradient gel electrophoresis (DGGE) investigation indicated that the amplification of the DNA with 6 g/L of acid stress was intensified obviously in comparison with the control and 12 g/L stress groups. Clostridium sp. and Bacillus sp. are dominant kinds of microbials in the sludge under acid stress was 6.0 g/L.
主要内容与结果如下:
1、首先,对污泥厌氧产氢产酸过程中的控制因素进行研究,主要从底物浓度、物料比、pH值调节、添加金属元素等产氢的初步条件进行讨论。研究表明,当葡萄糖初始浓度为0.1 mol/L,物料比为1.5:1,初始pH值设定为5.5,且在发酵过程中调节并保持pH值在5.3-5.7间,镍、铁离子浓度分别为0.04 mg/L、7.5 g/L时,产氢量可达到最大值2240 mL,总有机酸产量为104.08 mmol/L,其中乙酸与丁酸含量分别占29%、60%,属于典型的丁酸型发酵。
2、其次,本研究对污泥预先进行酸胁迫,然后对其厌氧发酵过程进行研究。研究表明,当丁酸胁迫浓度为6 g/L时,厌氧发酵产氢体系中丁酸、乙酸以及氢气产量最高,分别达到107.1 mmol/L,46.3 mmol/L和3690 mL/mol glucose,与对照组相比分别提高了65%、54%、110%。
3、同时,本研究还采用葡萄糖、谷氨酸钠、正丁酸对厌氧污泥进行预处理,以研究不同环境因子对厌氧微生物菌群基于谷氨酸脱羧酶体系的耐酸应答机制的影响。结果表明:经谷氨酸钠处理后污泥GAD活性有所提高,γ-氨基丁酸(GABA)含量较对照组提高55%;而经丁酸处理的组别GAD活性最高,GABA浓度达13.9μmol/(g·TS·h),相对于对照组提高了60%;葡萄糖处理组的污泥GAD活性与对照组相比降低了9%。进而,本研究采用了不同浓度丁酸对厌氧颗粒污泥预处理并进行厌氧发酵实验,结果表明,当丁酸浓度为6 g/L时,污泥谷氨酸脱羧酶和脱氢酶活性最高,分别达到14.7μmol/(g·TS·h),6982.12μgTF/(g·TS·h),相对于对照组分别提高了77%、95%,因此后续实验中均采用6 g/L作为最适宜的酸胁迫浓度。
4、最后,本研究还对厌氧产氢过程中污泥EPS的变化进行了讨论。结果表明:经过丁酸胁迫的厌氧污泥中EPS含量有明显的提高。与对照组相比,松散型多糖和蛋白质以及紧密型多糖和蛋白质含量分别提高了147%,34.8%,35%,21.6%。同时,本研究还采用DGGE技术分析丁酸处理对污泥DNA表达的影响。研究表明,经过6 g/L丁酸胁迫的污泥DNA含量较空白组提高11%,其后随着酸浓度的提高,污泥内DNA的含量呈现降低趋势;此外,经6 g/L丁酸胁迫的污泥电泳条带亮度高、带型整齐,Clostridium sp.和Bacillus sp.微生物成为污泥内的优势种,说明该组产氢微生物活性高于对照组,且产氢性能也较好。而12 g/L丁酸胁迫组的污泥产氢微生物数量大幅降低,产氢性能也远低于对照组,说明过高浓度的丁酸胁迫会对污泥内微生物活性造成伤害,进而导致微生物产氢性能降低。
Along with the anaerobic digestion of biomass, large amounts of organic acids would be accumulated, which resulted in an inhibitory effect on anaerobic microbiology, and the biohydrogen process. In this study, the anaerobic sludge with butyate stress was used to enhance the biohydrogen process, and to explore the glutamate decarboxylase (GAD) represented microbial acid tolerance response (ATR) within the anaerobic sludge. Moreover, effect of different concentration of acid stress on extracellular polymeric substances (EPS) component of the anaerobic sludge was also investigated. Finally, further investigation of the DNA components through DGGE analysis during the anaerobic digestion process was also conducted. The main conclusions of this paper are as followed:
1. Firstly, optimization of the fermentation conditions were investigated. It was found that the accumulation of biohydrogen and organic acids reached 2240 mL and 104.08 mmol/L when substrate concentration was 0.1 mol/L, pH ranged 5.3-5.7, ratio of glucose and inoculating sludge at 1.5:1, and the concentration of Fe2+ and Ni2+ is 0.04 mg/L and 7.5 g/L.
2. Secondly, anaerobic sludge with acid stress was adopted for hydrogen production. It was found that the production of butyric acid, acetic acid and hydrogen reached 107.1 mmol/L, 46.2 mmol/L and 3690 mL/mol glucose when the concentration of butyric acid reached 6.0 g/L which were 65%, 54% and 110% of the control, respectively.
3. Thirdly, three different stimulators as glucose, glutamine and butyrate were used to induce the the glutamate-dependent system based microbial ATR. It was found that GAD activity decreased 9% compared with the control in the case of glucose. While the GAD activity reached 13.9μmol/(g·TS·h) when butyrate was adopted, which increased by 55% compared with the control. Following, concentration of adopted for further acid stress was 6.0 g/L, as the activity of GAD and dehydrogenase activity reached 14.7μmol/(g·TS·h) and 6982.12μgTF/(g·TS·h), which increased by 77% and 95% compared with the control, respectively.
4. Finally, it indicated that the content of EPS, and loosely bound protein, loosely bound polysaccharide, tightly bound protein and tightly bound polysaccharide content were 147%, 34.8%, 35%, 21.6% higher than that of the control, respectively, with acid stress on anaerobic sludge. On the other hand, total amount of DNA was found to be increased by 11% compared with the control with appropriate acid stress. Moreover, denaturing gradient gel electrophoresis (DGGE) investigation indicated that the amplification of the DNA with 6 g/L of acid stress was intensified obviously in comparison with the control and 12 g/L stress groups. Clostridium sp. and Bacillus sp. are dominant kinds of microbials in the sludge under acid stress was 6.0 g/L.
引文
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2杨占春.餐厨垃圾生物法制高浓度氢气的研究[D]:[硕士学位论文].南京:南京工业大学生物化工专业, 2006
3张全国,李刚,荆艳艳.生物制氢技术现状及其研究进展[C].见:中华人民共和国农业部,亚洲开发银行主编. 2008中国农村生物质能源国际研讨会暨东盟与中日韩生物质能源论坛论文集.北京:中国农业出版社, 2008. 388-394
4朱建良,何世颖.活性污泥降解有机物制氢技术[J].化工纵横, 2003, 17(4): 5-8
5章佩丽.再生植物纤维厌氧发酵生物制氢试验研究[D]:[硕士学位论文].广州:华南理工大学环境科学与工程学院, 2011
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