【作者】 宗文明；

【导师】 樊美珍； 周志华；

【作者基本信息】 安徽农业大学 ， 微生物学， 2009， 博士

【摘要】 生物制氢技术以其常温、常压、能耗低、环保等特性,已经引起各国科学家的高度重视。在目前备受关注的几种生物制氢方法中,以废弃生物质为底物的发酵法生物制氢,是更接近实用技术的方法。近年来的研究结果表明,暗发酵与光发酵产H₂在底物利用方面具有很好的互补性,暗-光发酵耦联制氢技术不仅可以提高底物向产氢的转化效率,还可以达到发酵液上清直接排放的清洁标准,为生物质向氢能的转化提供了一种理想的清洁生产途径。本论文以进一步提高暗、光发酵法制氢的效率为研究目标,在混合菌群暗发酵过程中微生物组成与产H₂效率关系的解析、高效产H₂菌株的筛选与产H₂条件的优化以及利用自然光的光发酵制氢等方面展开了研究。具体研究内容包括以下五个部分:1.利用不同底物进行暗-光耦联两步法生物制氢的研究。针对寻找合适原料这一开发生物能源面临的重要问题,选择能源作物木薯（Manihot esculenta Crantz）和废弃物厨余垃圾为底物,进行暗-光耦联两步法生物制氢的可行性研究。通过暗发酵产H₂,木薯、厨余垃圾及对照蔗糖三种底物产H₂量分别为212,226和223 mL/g-底物。再通过类球红细菌（Rhodobacter sphaeroides）ZX-5利用三组暗发酵液厌氧光照产H₂,产H₂量分别达到611±11 mL/g、451±20 mL/g和565±13 mL/g。通过暗-光耦联两步法产H₂,产H₂量比仅暗发酵高出2-3倍,并且发酵液COD去除率达80%以上。结果表明,木薯粉和厨余垃圾是暗-光耦联两步法生物制氢理想的生物质资源,所建立的暗光耦联两步法产H₂体系,不仅可以把包括废弃物在内的生物质等更加彻底地转化成清洁能源氢气,同时也避免了暗发酵产生的有机酸所带来的环境污染。2.解析10L连续搅拌式罐式反应器（CSTR）暗发酵产H₂过程中优势微生物的演替规律。通过变性梯度凝胶电泳（DGGE）分析16S-rRNA组成的多态并测序分析相关DGGE条带,以及采用实时定量PCR（qrt-PCR）和基于铁氢酶hydA的DNA及cDNA克隆建库等技术解析系统菌群结构与产H₂效率之间的关系。分析结果表明:从接种到产H₂结束的不同时期,系统中优势菌群存在明显的演替,产H₂前期为好氧的芽胞杆菌（Bacillus）,产H₂初期开始出现拜氏梭菌（Clostridium beijerinckii）,而产H₂旺期以拜氏梭菌和产气荚膜梭菌（Clostridium perfringens）为优势菌,产H₂末期又逐渐出现了另一些能够利用蛋白的梭菌,而且反应器中氧化还原电位（ORP）的变化与优势菌群的演替及产H₂速率一致。基于hydA基因构建的DNA和cDNA文库,得到一个反映不同时期产H₂菌种类和丰度的动态变化图。通过qrt-PCR研究不同时段hydA基因的表达,发现系统中总hydA的表达水平在稳定期达到峰值,而单位产H₂菌的相对hydAmRNA表达水平却在指数期达到峰值。上述结果表明采用分子生态学技术能有效地监测产H₂系统中混合菌群的优势种群及其动态演替规律。3.从暗发酵产H₂系统中分离产H₂功能菌。在解析暗发酵产H₂系统中微生物演替规律的基础上,针对不同产H₂阶段的优势菌设计分离方案。从运行8h的10LCSTR所采集的样品中分离纯化出08-2优势菌株,经16S rRNA基因序列鉴定,为蜡质芽孢杆菌（Bacillus cereus）。从运行16 h的10 L CSTR所采集的样品中分离纯化出14株产H₂菌菌株。并通过基于ERIC（肠杆菌基因间共有多重序列）-PCR技术将14株菌快速鉴别为3种不同的菌株。其中08-1菌株通过形态特征和分子生物学鉴定,初步鉴定为Clostridiumbeijerinckii。对该菌株的产H₂性能进行研究,发现同葡萄糖相比,该菌株更适宜利用蔗糖、生物质木薯粉以及成分更为复杂的厨余垃圾生长及产H₂。在间歇发酵中,实现最大产H₂速率为245 mL H₂/（L·h）,产H₂量达到3.09 mol-H₂/（mol-蔗糖）。而08-11菌株是能够产H₂的兼性厌氧菌,与亲缘关系最近的地衣芽孢杆菌菌株（Bacillus lichenformisAnBa7）相似度为95%,因此,该菌株可初步判断为杆菌中的一个新种。对10L CSTR运行24 h阶段采集的样品用选择性培养基分离出能够快速生长并产H₂的产气荚膜梭菌（Clostridium perfringens）08-31。另外,从其他产H₂系统中分离获得产H₂菌株128株,通过ERIC-PCR及16S rRNA基因序列分析,获得丁酸梭菌5株,肠细菌属（Enterobacter）3株,产气荚膜梭菌,拜氏梭菌,地衣芽孢杆菌和凝结芽孢杆菌各1株。结果表明,从10 L CSTR反应器中成功分离出多株菌株,进一步证实了基于分子标记的监测系统预测菌群结构的有效性;另外,首次将ERIC-PCR技术成功地用于暗发酵产H₂菌株的快速鉴别。4.分离与筛选光合产H₂菌株。从不同废水中分离纯化出13株光合细菌,以30mM苹果酸为底物进行产H₂能力研究,发现最大产H₂速率为47-103 mL/（L·h）,底物转化效率为27.98-72.00%。从中挑出4株产H₂能力较好的菌株,分别以乙酸和丁酸做碳源进行光发酵产H₂试验,其中HL-1菌株能利用这两种碳源生长并产H₂;SC-6和SC-7菌株不能利用这两种碳源产H₂;HL-5菌株在乙酸培养基中能生长但不产气,在丁酸培养基中能生长并产H₂。这些菌株的获得,对于研究光合细菌乙酸和丁酸代谢途径提供了极好的材料。另外,从37株光合细菌中筛选出一株能够利用甘油为碳源进行有效产H₂的菌株。根据菌株的形态、生理生化特征、16S rDNA序列和ERIC-PCR结果分析,初步鉴定DB803菌株为Rhodobacter sphaeroides,同时也研究了DB803菌株在30℃,4000 lux光照厌氧条件下利用不同浓度的生物柴油生产废水产H₂能力,当培养基中起始COD值为11.5g/L时,其在对数生长期平均产H₂速度为38 mL/（L·h）,同时,废水COD去除率达91.2%,结果表明该菌株在生物柴油生产废水的处理及利用其进行生物产H₂中具有潜在的应用价值。此外,不同的类球红细菌菌株利用甘油产H₂能力的差异显著,筛选出的DB803菌株为进一步研究光合细菌利用甘油产H₂的代谢途径提供了材料。5.优化光发酵产H₂条件及初步研究自然光光发酵产H₂。在光合产H₂实验中,通过设置两种条件下制成的接种物和三种装液量,发现光照厌氧种子产H₂性能优于黑暗好氧种子;反应器顶部空间小的其底物转化效率高;顶部空间填充氮气可能促进光合细菌菌体的代谢活性。另外,还研究了太阳光对6 L光发酵制氢系统的影响,在白天利用户外太阳辐射能晚上进行人工补光的产H₂实验中,光合细菌的产H₂速度与阳光光强成正比,白天和晚上的平均产H₂速率分别为12.4 mL/（L·h）和9.0 mL/（L·h）,整个试验的底物转化效率达72.4%,与之对应的一次实验,即白天利用阳光晚上不补光的试验组,底物转化效率为45.02%。光反应器体积放大后,产H₂时间的延长是径高比值大的柱状反应器其比表面积小所致。更多还原

【Abstract】 Biological hydrogen production,which is carried out at normal temperature,normal pressure and low energy-consuming as well as environmental-friendly,has been attracting the common attentions.Among the several main biological hydrogen-production pathways （photolysis of water,photo-fermentation of small molecular organic compounds,and light-independent anaerobic fermentation from relatively large molecular organic compounds）,light-independent anaerobic（dark-） fermentation and photo-fermentation are currently the closest to application,owing to thier higher hydrogen production rate.Many recent investigations demonstrated that photo-fermetnation further converted the metabolites from dark-fermentation to hydrogen and the two-step process of sequential dark- and photo-fermentation could not only improve the hydrogen yield but also remove the most COD in the dark-fermentational effluent.It provides a new promising approach to produce biohydrogen from waste biomass.To further improve the hydrogen producing efficiency of the two-step process of sequential dark- and photo-fermentation,the succession of microbial composition during the hydrogen producing process of darkfermentation was investigated,the bacterial strains with high hydrogen producing efficiency were screened and the conditions for photo-fermentation were optimized in this study.The dissertation includes the following five parts:1.Hydrogen production from different substrates by the sequential dark- and photo-fermnetation.One of the main problems on developing bioenergy is to seek suitable feedstock.A Feasible study of sequential dark- and photo-fermentation hydrogen production from cassava and food waste was testified.In dark-fermentation,the average yield of hydrogen was approximately 212 mL H₂/g-cassava and 226 mL H₂/g-food waste. In subsequent photo-fermentation,the average yield of hydrogen from the effluent of dark-fermentation was approximately 611±11 mL H₂/g-cassava and 451±20 mL H₂/g-food waste.The process of sequential dark-fermentation and photo-fermentation could reach two-four fold hydrogen yields that dark-fermentation did.And meanwhile,the COD decreased greatly with a removal efficiency of 84.3%in cassava batch and 80.2%in food waste batch.These results demonstrate that cassava and food waste could be ideal substrates for biohydrogen production.And a two-step process of sequential dark-fermentation and photo-fermentation was highly efficient on both preventing the environmental issue and improving biohydrogen production capacity.2.The succession of dominant bacteria during the hydrogen producing process by dark-fermentation in a 10 L continuous stirred tank reactor was investigated.To elucidate the bacterial community structure-function relationships of 10 L continuous stirred tank reactor（CSTR） system possessing relatively high hydrogen producing potential,molecular marker of 16S-rRNA and hydA gene,denaturing gradient gel electrophoresis（DGGE）, qrt-PCR and hydA gene-based DNA and cDNA clone library were applied.The results of DGGE described a general profile of bacterial population dynamics:Bacillus spp.grew in the lag phase;Clostridium perfringens and Clostridium beijerinckii became dominant when hydrogen production was initiated;other Clostridium spp.began to appear when hydrogen production stopped and hydrogen uptake was observed.Deatailed dynamic profiles about categories and abundances of hydrogen producers in different stage were acquired with respect to hydA gene-based DNA and cDNA clone library.Expression level of hydA mRNA in different stages were studied by qrt-PCR,the results were that total hydA mRNA expression level in the culture reached the peak at stationary phase and is positively related to the instant hydrogen production rate,but relative hydA mRNA expression level of one hydrogen-producer reached the peak during exponential phase.The above results demonstrated that predominant bacterial communities and specific profile of succession can be monitored by molecular ecology technologies adopted.3.Isolation of the dominant hydrogen producers in the dark-fermentation system.The strain 08-2 was isolated from the 8h-sample of 10L CSTR,and identified as Bacillus cereus according to16S rRNA gene sequences.14 strains of hydrogen producers were isolated from 16h-sample of 10L CSTR,and then were merged as 3 different strains quickly by ERIC-PCR.Among them,the strain 08-1 was identified as Clostridium beijerinckii based on its phenotype and 16S rRNA gene.Sucrose,biomass and wastes which components complicated were favorite carbon sources for growth and hydrogen production by the strain 08-1.The optimum temperature was 40℃and pH was 5.5 for hydrogen production of strain 08-1.Under batch fermentative hydrogen production conditions:concentration of sucrose 20 g/L,temperature 40℃,pH 5.5 controlled and stirred rate 100 rpm,the maximal hydrogen yield and volumetric hydrogen production rate for strain 08-1 were 3.09 mol H₂/（mol-sucrose） and 245 mL/L.h,respectively.The results demonstrate that the strain possesses potential application in biohydrogen production from wastes.The strain 08-11 was a facultative anaerobic hydrogen producer and was judged as a new species based on its 16S rRNA gene.Clostridium perfringens were counted and isolated from 24 h-samples of 10L CSTR by selective medium.Moreover,128 strains of hydrogen producers isolated from other hydrogen production system,and among them,5 strains of Clostridium butyricum,3 strains of Enterobacterium sp.,1 strain of Clostridium perfringens,Clostridium beijerinckii,Bacillus licheniformis and condensate bacillus. These important functional strains acquired from 10L CSTR further demonstrate validity on forecasting hydrogen production community structure by molecular marker-based monitoring system.Furthermore,ERIC-PCR method was successfully applied to quick identifying and classifying dark-fermentation hydrogen production strains for the first time.4.Screening photosynthetic dacteriat strains to produce hydrogen from wastewater of biodiesel manufacture.13 strains of photosynthetic bacterial isolated from different water bodies.Their maximum hydrogen production rates and substrate conversion efficiency from 30mM malate were 47-103 mL/l·h and 27.98-72.00%,respectively.Photo-hydrogen production was carried out from acetate and butyrate by 4 selected photosynthetic bacterial with higher biohydrogen efficiency from malate.The strain HL-1 can utilize both acetate and butyrate for growth and hydrogen production.Strain SC-6 and SC-7 can grow but can’t produce hydrogen from acetate and butyrate.The strain HL-5 can can grow but can’t produce hydrogen from acetate,and utilize butyrate for growth and hydrogen production. These strains would be excellent material for investigating different acetate and butyrate metablic pathway.A strain（designated as DB803） producing hydrogen efficiently from glycerol as the carbon source was screened from 37 photosynthetic bacterial strains.This strain was identified as a new strain of Rhodobacter sphaeroides based on its phenotype, physiological and biochemical characteristics,the sequence of its 16S rRNA gene and fingerprinting of ERIC-PCR.Its hydrogen yields from biodiesel wastewater at different dilution times were investigated under the anaerobic condition of 30℃,4000lux of illumination.The average hydrogen-producing rate of 38 mL/L·h was observed at log phase when the initial COD value of wastewater was diluted to 11.5 g/L.Meanwhile,the COD decreased greatly with a removal efficiency of 91.2%at batch test.The results demonstrate that the strain possesses potential application in hydrogen production from wastewater of biodiesel manufacture.And moreover,DB803 would be material for further studing metabolic pathway of glycerol for hydrogen production by photosynthetic bacterial. 5.The optimal conditions for photo-fermentation and investigation on hydrogen production of photo-fermentation under sunlight.The effects of different inoculum,culture volume and gas types on photohydrogen production were investigated.Hydrogen production potential inoculated by inoculum under photo and anaerobic conditions was superior to the inoculum under dark and aerobic conditions.Substrate conversion efficiency was high with smaller headspacein bioreactor.Nitrogen gas might promote the metabolic activities of photosynthetic bacterium.Moreover,Production of hydrogen by photosynthetic bacteria under sunlight was studied.In the experiment on hydrogen production by natural sunlight during daytime and supplement with artificial light during night,the hydrogen production rate by the photosynthetic bacteria depended on the sunlight intensity;the average hydrogen production rate during daytime and night was 12.4 mL/L·h and 9.0 mL/L·h,respectively.And the substrate conversion efficiency was relatively high,72.4%.Another experiment on hydrogen production by natural sunlight during daytime and not supplement with artificial light during night was also conducted,its substrate conversion efficiency was 45.02%.The cause of time prolonged in amplified photobioreactor would be contributed to smaller specific surface area of cylindrical photobioreactor with bigger diameter height ratio.更多还原