利用秸秆水解液的微生物暗发酵产氢特性及机制研究
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摘要
秸秆类木质纤维素以其数量大、价格低廉、可再生、获取容易等诸多优点成为暗发酵生物制氢潜在的适宜底物。利用秸秆类物质进行生物制氢可以大大降低氢气的生产成本,使生物制氢在经济上更具有可行性,同时避免了秸秆类物质通常的焚烧处理导致的严重环境污染。然而,秸秆类物质具有高结晶度、高异质性的复杂结构,致使微生物对其的直接降解效率较低。因此,利用富含戊糖、己糖的秸秆水解液进行微生物发酵产氢成为生物制氢产业化应用的重要发展方向。目前,在混合微生物进行秸秆水解液发酵产氢过程中,存在着底物降解不充分,产氢效率较低等瓶颈问题,严重阻碍了秸秆水解液产氢的生产规模应用。另一方面,对秸秆水解液利用过程中的微生物产氢分子机制亦不清楚,难以从理论层面指导实际产氢工艺的运行过程。
基于此,本研究考察了种泥预处理和发酵温度对不同类型混合微生物利用秸秆水解液产氢过程的影响。为提高系统的底物降解率与产氢能力,采用对秸秆水解液具有高效降解、产氢能力的菌株Thermoanaerobacterium thermosaccharolyticum W16对混合微生物利用秸秆水解液产氢过程进行了生物强化。通过扩增片段长度多态性(AFLP)和木质纤维素降解基因分析阐述了T.thermosaccharolyticum类型微生物在秸秆类物质利用过程中的分子机制。对菌株W16的[FeFe]-氢化酶基因簇进行了基因克隆、特征分析与转录表达分析,解析了T. thermosaccharolyticum类型微生物的产氢机制。
结果表明,热、酸、碱、超声和紫外五种预处理方法中,热预处理对活性污泥、河流底泥和颗粒污泥中混合微生物的产氢过程具有较好提升效果,热预处理下的活性污泥接种达到了最大的氢气产量5.03mmol/g-sugar utilizedo在不同发酵温度(30℃、37℃、55℃、70℃)中,55℃时三种类型种泥均达到最高氢气产量,颗粒污泥接种时数值最大(7.74mmol/g-sugar utilized),而70℃时混合微生物对秸秆水解液的产氢效能显著低于55℃。预处理过程和高温条件显著改变了微生物群落结构,使之由Enterobacter spp.、Escherichia spp.、 Klebsiella spp.等兼性微生物的普遍存在,转变为Clostridium spp.、 Bacillus spp. Thermoanaerobacterium spp等高效厌氧产氢微生物的大量富集。
菌株W16的生物强化作用可以显著提升腐烂秸秆和厌氧污泥接种时混合微生物利用秸秆水解液过程的氢气产量,同时一定程度提高了底物降解率,生物强化下的腐烂秸秆接种达到最高的氢气产量9.90mmol-H2/g-sugar utilized。AFLP分析显示T. thermosaccharolyticum类型微生物具有较高的基因组DNA保守性,差异条带中的2-polyprenylphenol6-hydroxylase基因可能为T.thermosaccharolyticum类型微生物较强木质纤维素降解能力的关键因素。此外,在T. thermosaccharolyticum中的木质纤维素降解基因中,Cellulose1,4-beta-cellobiosidase基因中-10区RNA聚合酶结合位点处的单碱基差异可能与T. thermosaccharolyticum类型微生物对纤维素的利用效果密切相关。
克隆得到了菌株W16[FeFe]-氢化酶hyd和hfs基因簇的完整DNA序列,分别包含hyaⅡ (1308bp)、 hydC (480bp)、 hydD (363bp)、 hydB (1788bp)和hydA (1752bp),以及hfsA (243bp)、hfsB (1716bp)和hfsC (1512bp),其中hyaⅡ、hydB、 hydA、hfsB和hfsC为较重要的氢化酶基因。分析了hyd和hfs基因簇中各基因的DNA序列特征、对应蛋白质的物理化学性质、二级和三级结构、结构功能区与保守区。考察不同气相条件(起始N2、H2或CO2灌注)对氢化酶转录表达的影响。对hyd基因簇,hydA的转录过程在高H2或高CO2分压下被严重抑制；hyaB只对高H2分压较为敏感；hyaⅡ在高N2、高H2或高CO2分压下都表达量较低。对hfs基因簇,hfsB在高CO2分压下表现出较高的转录活性,在高H2分压下转录表达较低；hfsC基因在高CO2分压下的转录水平最低。
Due to the advantages of large quantities, lower cost, renewable and easy to obtain, stover-like lignocellulosic materials are considered as potential suitable substrates for dark-fermentation hydrogen (H2) production. Biohydrogen production by stover-like materials can largely decrease H2production cost, making biohydrogen production more economically available. In addition, serious environmental pollution can be avoided by utilizing stover for H2production instead of commonly adopted combustion disposal. However, the higher crystallinity and heterogeneity of stover biomass lead to the lower utilization efficiency of microbes. Therefore, biohydrogen production from stover hydrolysate, which is produced by various pretreatment methods towards stover materials, becomes important route for practical applications. At present, bottleneck problems, such as lower substrate utilization efficiency and H2yield, exist in biohydrogen production from stover hydrolysate by using mixed cultures, preventing the development of practical applications. Furthermore, it is not clear for the molecular mechanisms of substrate degradation and H2production during the process.
This study investigated the effects of inoculum pretreatment and fermentation temperature on H2-production performances from stover hydrolysate by different kinds of mixed cultures. Thermoanaerobacterium thermosaccharolyticum W16, which was previously isolated and presented better utilization and H2production from stover hydrolysate, was introduced into mixed cultures for bioaugmentation. Amplified fragment length polymorphism (AFLP) and analysis of lignocellulose degradation genes were carried out to illustrate the molecular mechanisms of T. thermosaccharofyticum-kind microbes. For efficient H2-producing strain W16,[FeFe]-hydrogenase gene complexes were cloned, characterized, and analyzed for the transcriptional level to address the molecular mechanisms of H2production.
Among five pretreatment methods of heat, acid, alkali, ultrasonic, and ultraviolet, heat pretreatment presented the better efforts on the inoculums of activated sludge, river sediments and granular sludge, and activated sludge pretreated by heat reached the optimum H2yield of5.03mmol-H2/g-sugar utilized. Among different fermentation temperatures (30℃,37℃,55℃and70℃), H2production reached the optimum at55℃for the three inoculums, in which granular sludge achieved the highest H2yield (7.74mmol-H2/g-sugar utilized). In addition, Fb production at70℃was significantly lower than that at55℃. Inoculum pretreatment and thermophilic condition significantly changed the microbial community structure, from the universal existances of facultive anaerobes such as Enterobacter spp., Escherichia spp. and Klebsiella spp. to the enrichments of some efficient H2-producing microbes such as Clostridium spp., Bacillus spp. and Thermoanaerobacterium spp.
The bioaugmentation by the strain W16can significantly increase the H2production from stover hydrolysate by using rotten corn stover and anaerobic sludge, and increase the utilization efficiency to some extent. Bioaugmented rotten corn stover reached the highest H2yield of9.90mmol-H2/g-sugar utilized. AFLP analysis showed that the genomic DNA of T. thermosaccharolyticum-kind microbes was highly reserved, and the2-polyprenylphenol6-hydroxylase gene detected by AFLP might act a key role in the degradation of lignocellulose materials. In addition, the single base located in the-10region (RNA polymerase binding site) of Cellulose1,4-beta-cellobiosidase gene might be relevant to the utilization of cellulose by T. thermosaccharolyticum-kind microbes.
For the strain W16, the complete DNA sequences of [FeFe]-hydrogenase hyd and hfs gene complexes were cloned and analyzed, which contained hydⅡ (1308bp), hydC (480bp), hydD (363bp), hydB (1788bp), hydA (1752bp) and hfsA (243bp), hfsB (1716bp), hfsC (1512bp), respectively, in which hydⅡ, hydB, hydA, hfsB, and hfsC were important [FeFe]-hydrogenase genes. The study also discussed the physical and chemical characteristics, secondary and the third level structures of proteins, structural functional zones, and conservative zones of [FeFe]-hydrogenase hyd and hfs gene complexes. While investigating the effects of different gas-phase conditions (initial N2, H2, or CO2pouring) on transcriptional expression of important [FeFe]-hydrogenase genes, the transcription of hydA was significantly inhibited under high H2or CO2partial pressure, hydB was only susceptible for high H2partial pressure, and hydll preformed lower transcriptional activities for all the situations. For hfs complex, hfsB showed higher transcriptional level under high CO2partial pressure and lower transcriptional level under high H2partial pressure, and hfsC displayed the lowest transcriptional level under high CO2partial pressure.
基于此,本研究考察了种泥预处理和发酵温度对不同类型混合微生物利用秸秆水解液产氢过程的影响。为提高系统的底物降解率与产氢能力,采用对秸秆水解液具有高效降解、产氢能力的菌株Thermoanaerobacterium thermosaccharolyticum W16对混合微生物利用秸秆水解液产氢过程进行了生物强化。通过扩增片段长度多态性(AFLP)和木质纤维素降解基因分析阐述了T.thermosaccharolyticum类型微生物在秸秆类物质利用过程中的分子机制。对菌株W16的[FeFe]-氢化酶基因簇进行了基因克隆、特征分析与转录表达分析,解析了T. thermosaccharolyticum类型微生物的产氢机制。
结果表明,热、酸、碱、超声和紫外五种预处理方法中,热预处理对活性污泥、河流底泥和颗粒污泥中混合微生物的产氢过程具有较好提升效果,热预处理下的活性污泥接种达到了最大的氢气产量5.03mmol/g-sugar utilizedo在不同发酵温度(30℃、37℃、55℃、70℃)中,55℃时三种类型种泥均达到最高氢气产量,颗粒污泥接种时数值最大(7.74mmol/g-sugar utilized),而70℃时混合微生物对秸秆水解液的产氢效能显著低于55℃。预处理过程和高温条件显著改变了微生物群落结构,使之由Enterobacter spp.、Escherichia spp.、 Klebsiella spp.等兼性微生物的普遍存在,转变为Clostridium spp.、 Bacillus spp. Thermoanaerobacterium spp等高效厌氧产氢微生物的大量富集。
菌株W16的生物强化作用可以显著提升腐烂秸秆和厌氧污泥接种时混合微生物利用秸秆水解液过程的氢气产量,同时一定程度提高了底物降解率,生物强化下的腐烂秸秆接种达到最高的氢气产量9.90mmol-H2/g-sugar utilized。AFLP分析显示T. thermosaccharolyticum类型微生物具有较高的基因组DNA保守性,差异条带中的2-polyprenylphenol6-hydroxylase基因可能为T.thermosaccharolyticum类型微生物较强木质纤维素降解能力的关键因素。此外,在T. thermosaccharolyticum中的木质纤维素降解基因中,Cellulose1,4-beta-cellobiosidase基因中-10区RNA聚合酶结合位点处的单碱基差异可能与T. thermosaccharolyticum类型微生物对纤维素的利用效果密切相关。
克隆得到了菌株W16[FeFe]-氢化酶hyd和hfs基因簇的完整DNA序列,分别包含hyaⅡ (1308bp)、 hydC (480bp)、 hydD (363bp)、 hydB (1788bp)和hydA (1752bp),以及hfsA (243bp)、hfsB (1716bp)和hfsC (1512bp),其中hyaⅡ、hydB、 hydA、hfsB和hfsC为较重要的氢化酶基因。分析了hyd和hfs基因簇中各基因的DNA序列特征、对应蛋白质的物理化学性质、二级和三级结构、结构功能区与保守区。考察不同气相条件(起始N2、H2或CO2灌注)对氢化酶转录表达的影响。对hyd基因簇,hydA的转录过程在高H2或高CO2分压下被严重抑制；hyaB只对高H2分压较为敏感；hyaⅡ在高N2、高H2或高CO2分压下都表达量较低。对hfs基因簇,hfsB在高CO2分压下表现出较高的转录活性,在高H2分压下转录表达较低；hfsC基因在高CO2分压下的转录水平最低。
Due to the advantages of large quantities, lower cost, renewable and easy to obtain, stover-like lignocellulosic materials are considered as potential suitable substrates for dark-fermentation hydrogen (H2) production. Biohydrogen production by stover-like materials can largely decrease H2production cost, making biohydrogen production more economically available. In addition, serious environmental pollution can be avoided by utilizing stover for H2production instead of commonly adopted combustion disposal. However, the higher crystallinity and heterogeneity of stover biomass lead to the lower utilization efficiency of microbes. Therefore, biohydrogen production from stover hydrolysate, which is produced by various pretreatment methods towards stover materials, becomes important route for practical applications. At present, bottleneck problems, such as lower substrate utilization efficiency and H2yield, exist in biohydrogen production from stover hydrolysate by using mixed cultures, preventing the development of practical applications. Furthermore, it is not clear for the molecular mechanisms of substrate degradation and H2production during the process.
This study investigated the effects of inoculum pretreatment and fermentation temperature on H2-production performances from stover hydrolysate by different kinds of mixed cultures. Thermoanaerobacterium thermosaccharolyticum W16, which was previously isolated and presented better utilization and H2production from stover hydrolysate, was introduced into mixed cultures for bioaugmentation. Amplified fragment length polymorphism (AFLP) and analysis of lignocellulose degradation genes were carried out to illustrate the molecular mechanisms of T. thermosaccharofyticum-kind microbes. For efficient H2-producing strain W16,[FeFe]-hydrogenase gene complexes were cloned, characterized, and analyzed for the transcriptional level to address the molecular mechanisms of H2production.
Among five pretreatment methods of heat, acid, alkali, ultrasonic, and ultraviolet, heat pretreatment presented the better efforts on the inoculums of activated sludge, river sediments and granular sludge, and activated sludge pretreated by heat reached the optimum H2yield of5.03mmol-H2/g-sugar utilized. Among different fermentation temperatures (30℃,37℃,55℃and70℃), H2production reached the optimum at55℃for the three inoculums, in which granular sludge achieved the highest H2yield (7.74mmol-H2/g-sugar utilized). In addition, Fb production at70℃was significantly lower than that at55℃. Inoculum pretreatment and thermophilic condition significantly changed the microbial community structure, from the universal existances of facultive anaerobes such as Enterobacter spp., Escherichia spp. and Klebsiella spp. to the enrichments of some efficient H2-producing microbes such as Clostridium spp., Bacillus spp. and Thermoanaerobacterium spp.
The bioaugmentation by the strain W16can significantly increase the H2production from stover hydrolysate by using rotten corn stover and anaerobic sludge, and increase the utilization efficiency to some extent. Bioaugmented rotten corn stover reached the highest H2yield of9.90mmol-H2/g-sugar utilized. AFLP analysis showed that the genomic DNA of T. thermosaccharolyticum-kind microbes was highly reserved, and the2-polyprenylphenol6-hydroxylase gene detected by AFLP might act a key role in the degradation of lignocellulose materials. In addition, the single base located in the-10region (RNA polymerase binding site) of Cellulose1,4-beta-cellobiosidase gene might be relevant to the utilization of cellulose by T. thermosaccharolyticum-kind microbes.
For the strain W16, the complete DNA sequences of [FeFe]-hydrogenase hyd and hfs gene complexes were cloned and analyzed, which contained hydⅡ (1308bp), hydC (480bp), hydD (363bp), hydB (1788bp), hydA (1752bp) and hfsA (243bp), hfsB (1716bp), hfsC (1512bp), respectively, in which hydⅡ, hydB, hydA, hfsB, and hfsC were important [FeFe]-hydrogenase genes. The study also discussed the physical and chemical characteristics, secondary and the third level structures of proteins, structural functional zones, and conservative zones of [FeFe]-hydrogenase hyd and hfs gene complexes. While investigating the effects of different gas-phase conditions (initial N2, H2, or CO2pouring) on transcriptional expression of important [FeFe]-hydrogenase genes, the transcription of hydA was significantly inhibited under high H2or CO2partial pressure, hydB was only susceptible for high H2partial pressure, and hydll preformed lower transcriptional activities for all the situations. For hfs complex, hfsB showed higher transcriptional level under high CO2partial pressure and lower transcriptional level under high H2partial pressure, and hfsC displayed the lowest transcriptional level under high CO2partial pressure.
引文
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