高效利用玉米秸秆的产氢菌种及其产氢性能研究
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摘要
随着能源危机、粮食短缺、环境污染等问题越来越严重,如何使废弃物无害化、资源化已成为世界性亟待解决的关键问题。秸秆是一种丰富的农业废弃物、因其具有充足的供应和低廉的价格,若利用微生物技术将其转化为氢气,既能降低产氢成本,又能使废弃物得到资源化,从清洁能源开发和废物利用的角度都具有极大的社会效益和经济效益。秸秆主要由纤维素、半纤维素和木质素构成,其中纤维素和半纤维素是可发酵性碳水化合物,可在微生物或酶的作用下将其转化为糖后再供微生物利用生成燃料氢气。但目前以秸秆为原料制备生物燃料研究中多数只强调对纤维素组分的转化利用,而半纤维素组分则作为废弃物处理,既造成环境污染,又造成资源浪费。同时,在纤维素物质转化过程中,纤维素酶解技术存在的主要障碍是纤维素酶活力较低、生产成本较高。针对目前制约秸秆木质纤维原料生物转化产氢研究中的主要技术瓶颈,本研究进行了戊糖发酵产氢菌和纤维素降解产氢菌的分离筛选,并分别考察了其利用半纤维素水解液和纤维素物质发酵产氢性能,同时结合预处理技术建立了玉米秸秆高效利用组合体系,论文取得主要研究成果如下:
分离获得一株高效戊糖发酵产氢菌Thermoanaerobacterium thermosaccharolyticum W16,该菌在碳源浓度10g/L,温度60oC,初始pH 6.5条件下,利用葡萄糖和木糖的最大比产氢率和产氢速率分别为2.42mol H2/mol glucose、12.9mmol H2/L·h和2.19mol H2/mol xylose、10.7 mmol H2/L·h。同时探讨了该菌同步发酵葡萄糖木糖混合糖产氢代谢特性,研究表明菌株W16能同时利用葡萄糖和木糖发酵产氢,但葡萄糖的代谢速率快于木糖的代谢速率。预处理水解液中常见抑制物影响试验表明菌株W16具有较强的乙酸钠和酚类化合物香兰素的耐受能力,而对呋喃衍生物糠醛、羧甲基糠醛和酚类化合物紫丁香醛较敏感。
利用响应曲面法优化玉米秸秆半纤维素稀酸水解条件,在酸浓度1.69%、处理时间117min最优水解条件下,玉米秸秆中半纤维素的脱除程度达80.5%,水解液中木糖、阿拉伯糖和葡萄糖含量分别为9.11g/L、1.85g/L和0.88g/L。半纤维素水解液产氢性能表明菌株W16能有效利用半纤维素水解液进行发酵产氢,水解液含有的乙酸糠醛等副产物没有对其产生抑制作用。
建立了嗜热厌氧纤维素降解产氢菌的筛选方法,并获得了一株具有良好纤维素降解产氢能力的菌株Thermoanaerobacterium thermosaccharolyticum M18。该菌能有效利用微晶纤维素、木聚糖、纤维素滤纸发酵产氢,也能利用未经处理的天然木质纤维原料玉米秸秆、稻草和玉米芯产氢。在碳源浓度为5g/L的微晶纤维素粉和玉米秸秆粉培养基中其产氢能力及对底物的降解率分别为44.5mmol/L(243.7ml/g-微晶纤维素)、8.45mmol/L(80.2ml/g-玉米秸秆)和81.9%、47.2%。纤维素酶学特性分析表明菌株M18所产纤维素酶为诱导酶,只有在纤维素类物质的诱导下才能产生,不同细胞组分酶活性检测表明其所产纤维素酶主要分布在细胞外。
借助SEM、FTIR、XRD、13C-NMR、GC-MS分析手段及纤维素、半纤维素分解酶活测定全面、系统的解析了菌株M18对玉米秸秆的降解特性,得出了菌株M18诱导产酶的过程也是秸秆降解的过程,明确了其在秸秆降解过程中对纤维素和半纤维素的降解优势,并且半纤维素的降解优先于纤维素的降解,在降解过程中,纤维素分子内氢键发生断裂,纤维素结晶区和非结晶区都遭到破坏,纤维素和半纤维素在纤维素酶和半纤维素酶的作用下被降解成小分子物质后而被菌株M18直接利用并进一步发酵产生氢气,同时也表明木质素的存在是制约秸秆生物转化的主要障碍。
针对秸秆中木质素的存在影响了菌株M18对纤维素和半纤维素的生物转化效率针,通过比较不同预处理方法的特点及作用机理,采用氢氧化钠预处理技术对玉米秸秆进行脱木素,预处理后得到纤维素固体残渣和碱溶半纤维素糖类水溶液两部分。利用戊糖发酵菌W16和纤维素降解菌M18各自优势将碱溶半纤维素糖类的回收利用与固体残渣中纤维素和半纤维素的氢气转化进行有机整合,从而实现了玉米秸秆的高效转化。与未经处理的玉米秸秆相比,其产氢量提高了36.8%。
With the problem of energy crisis, food shortages, enviroment pollution become more and more serious, how to make the waste harmless and resources has become the urgent issue to solve worldwide. Straw is an aboudant agricultural waste, because of its sufficient supply and low price, if convert it to hydrogen by microorganisms, it can not only reduce the cost of hydrogen production, but can also reclaim waste materials, which has remarkable social and economic benefits on the aspects of energy development and waste ultization. Straw was mainly composed of cellulose, hemicellulose and lignin, cellulose and hemicellulose are fermentable carbohydrates, which can be converted to sugars by microorganisms or enzymes before their use for biohydrogen generation. But take straw as raw materials, biofuels researches in most cases are emphasis on the conversion of cellulose, hemicellulose is disposed as waste, which not only cause environmental pollution but also wastes of resources. Meanwhile, in cellulose conversion process, the major obstacles of cellulase technology are lower cellulase activity and higher production costs. Aiming at current main technical bottleneck of conversion straw to biohydrogen, the pentose fermentation bacteria and cellulose degrading bacteria for hydrogen production were isolated and the performance of hydrogen production from hemicellulose hydrolysate and cellulosic materials were investigated respectively. Simultaneously, associated with pretreatment technology, a combined system for efficient utilization of corn stover for hydrogen production was established. The main achievements obtained by this research were as follows:
A moderately thermophilic bacterium, Thermoanaerobacterium thermosaccharolyticum W16, was isolated for efficiently producing hydrogen from pentose. Under the conditions of carbon source 10g/L, temperature 60°C, and initial pH 6.5, the maximum cumulative H2 yield and H2 production rate in glucose and xylose are 2.42mol H2/mol glucose, 12.9mmol H2/L·h and 2.19mol H2/mol xylose, 10.7mmol H2/L·h, respectively. The metabolic characteristics under mixed glucose and xylose are also discussed. It discovered that strain W16 could simultaneously uptake glucose and xylose, although, the consumption rate of glucose was faster than the consumption rate of xylose. The effect of common inhibitors derived from hydrolysate showed that strain W16 had high tolerance to acetate sodium and vanillin, however, it behaved more sensitive to furfural, hydroxymethylfurfural (HMF) and syringaldehyde.
By using response surface methodology, the hydrolysis conditions were optimized to produce hemicellulosic hydrolysates. Under the optimal hydrolysis conditions of acid concentration 1.69%, processing time 117min, the removal efficiency of hemicellulose was up to 80.%,and the content of xylose, arobinose and glucose were 9.11g/L、1.85g/L和0.88g/L, respectively. The performance of hydrogen production from such hydrolysates by the strain W16 was also investigated. The results indicated that the strain W16 could effectively utilize hemicellulosic hydrolysates for fermentative hydrogen production; the inhibitors derived from hydrolysate such as acetate sodium, furfural didn’t have inhibitory effect on the strain W16.
The screening method was constructed to isolate thermophilic anaerobic cellulolytic hydrogen-producing bacteria, and a good cellulytic hydrogen-producing bacteria Thermoanaerobacterium thermosaccharolyticum M18 was obtained. This strain could efficiently uptake microcrystalline cellulose, xylan, filter paper, and sodium carboxymethylcellulose for hydrogen production, as well as unpretreated corn stover , rice straw, and corn cob. While inoculated to the medium containing 5g/L microcrystalline cellulose or corn stover, the hydrogen production yield and the substrate degradation efficiency were 44.55mmol/L (243.7ml/g-microcrystalline cellulose), 8.45mmol/L (80.2ml/g-corn stover) and 81.9% , 47.2%, respectively. The cellulases characteristics analysis indicated that the cellulases produced by the strain M18 were inducible enzymes, which only be induced in the existence of cellulosic materials. The activity assays on different enzymes of respective component show that the distribution of produced cellulases is mainly outside the cell.
By virtue of SEM, FTIR, XRD, 13C-NMR, GC-MS, and the detection of cellulase and hemicelllase activities, the cellulolytic characteristics of the strain M18 were systematically analyzed. It can be concluded that the process of induced enzyme production coupled with corn stover degradation. The strain M18 exhibited degradation superiority on cellulose and hemicellulose and the degradation of hemicellulose was priority than the degradation of cellulose. In the degradation process, the intramolecular hydrogen bonds of the cellulose molecule were ruptured and the areas of crystal and amorphous were both destroyed. Cellulose and hemicellulose were decomposed into small molecule substances under the effect of cellulases and hemicellulases, and then fermented into hydrogen. It also suggested that lignin is the main barrier to restrict bioconversion of corn stover.
Due to the existence of lignin influenced the bioconversion of cellulose and hemicellulose, the sodium hydroxide pretreatment was used to delignify corn stover. After pretreatment, the cellulosic solid residues and alkali-soluable hemicellulose sugar were obtained. During hydrogen production from corn stover, the bioconversion of cellulose and hemicellulose in NaOH-pretreated corn stover to hydrogen by M18 and alkali-soluable hemicellulose sugar recovery by W16 could be integrated into a biorefinery scheme, thus the maximum bioconversion efficiency of corn stover was acchieved. Compared with unpretreated corn stover, the hydrogen yield increased 36.8%.
分离获得一株高效戊糖发酵产氢菌Thermoanaerobacterium thermosaccharolyticum W16,该菌在碳源浓度10g/L,温度60oC,初始pH 6.5条件下,利用葡萄糖和木糖的最大比产氢率和产氢速率分别为2.42mol H2/mol glucose、12.9mmol H2/L·h和2.19mol H2/mol xylose、10.7 mmol H2/L·h。同时探讨了该菌同步发酵葡萄糖木糖混合糖产氢代谢特性,研究表明菌株W16能同时利用葡萄糖和木糖发酵产氢,但葡萄糖的代谢速率快于木糖的代谢速率。预处理水解液中常见抑制物影响试验表明菌株W16具有较强的乙酸钠和酚类化合物香兰素的耐受能力,而对呋喃衍生物糠醛、羧甲基糠醛和酚类化合物紫丁香醛较敏感。
利用响应曲面法优化玉米秸秆半纤维素稀酸水解条件,在酸浓度1.69%、处理时间117min最优水解条件下,玉米秸秆中半纤维素的脱除程度达80.5%,水解液中木糖、阿拉伯糖和葡萄糖含量分别为9.11g/L、1.85g/L和0.88g/L。半纤维素水解液产氢性能表明菌株W16能有效利用半纤维素水解液进行发酵产氢,水解液含有的乙酸糠醛等副产物没有对其产生抑制作用。
建立了嗜热厌氧纤维素降解产氢菌的筛选方法,并获得了一株具有良好纤维素降解产氢能力的菌株Thermoanaerobacterium thermosaccharolyticum M18。该菌能有效利用微晶纤维素、木聚糖、纤维素滤纸发酵产氢,也能利用未经处理的天然木质纤维原料玉米秸秆、稻草和玉米芯产氢。在碳源浓度为5g/L的微晶纤维素粉和玉米秸秆粉培养基中其产氢能力及对底物的降解率分别为44.5mmol/L(243.7ml/g-微晶纤维素)、8.45mmol/L(80.2ml/g-玉米秸秆)和81.9%、47.2%。纤维素酶学特性分析表明菌株M18所产纤维素酶为诱导酶,只有在纤维素类物质的诱导下才能产生,不同细胞组分酶活性检测表明其所产纤维素酶主要分布在细胞外。
借助SEM、FTIR、XRD、13C-NMR、GC-MS分析手段及纤维素、半纤维素分解酶活测定全面、系统的解析了菌株M18对玉米秸秆的降解特性,得出了菌株M18诱导产酶的过程也是秸秆降解的过程,明确了其在秸秆降解过程中对纤维素和半纤维素的降解优势,并且半纤维素的降解优先于纤维素的降解,在降解过程中,纤维素分子内氢键发生断裂,纤维素结晶区和非结晶区都遭到破坏,纤维素和半纤维素在纤维素酶和半纤维素酶的作用下被降解成小分子物质后而被菌株M18直接利用并进一步发酵产生氢气,同时也表明木质素的存在是制约秸秆生物转化的主要障碍。
针对秸秆中木质素的存在影响了菌株M18对纤维素和半纤维素的生物转化效率针,通过比较不同预处理方法的特点及作用机理,采用氢氧化钠预处理技术对玉米秸秆进行脱木素,预处理后得到纤维素固体残渣和碱溶半纤维素糖类水溶液两部分。利用戊糖发酵菌W16和纤维素降解菌M18各自优势将碱溶半纤维素糖类的回收利用与固体残渣中纤维素和半纤维素的氢气转化进行有机整合,从而实现了玉米秸秆的高效转化。与未经处理的玉米秸秆相比,其产氢量提高了36.8%。
With the problem of energy crisis, food shortages, enviroment pollution become more and more serious, how to make the waste harmless and resources has become the urgent issue to solve worldwide. Straw is an aboudant agricultural waste, because of its sufficient supply and low price, if convert it to hydrogen by microorganisms, it can not only reduce the cost of hydrogen production, but can also reclaim waste materials, which has remarkable social and economic benefits on the aspects of energy development and waste ultization. Straw was mainly composed of cellulose, hemicellulose and lignin, cellulose and hemicellulose are fermentable carbohydrates, which can be converted to sugars by microorganisms or enzymes before their use for biohydrogen generation. But take straw as raw materials, biofuels researches in most cases are emphasis on the conversion of cellulose, hemicellulose is disposed as waste, which not only cause environmental pollution but also wastes of resources. Meanwhile, in cellulose conversion process, the major obstacles of cellulase technology are lower cellulase activity and higher production costs. Aiming at current main technical bottleneck of conversion straw to biohydrogen, the pentose fermentation bacteria and cellulose degrading bacteria for hydrogen production were isolated and the performance of hydrogen production from hemicellulose hydrolysate and cellulosic materials were investigated respectively. Simultaneously, associated with pretreatment technology, a combined system for efficient utilization of corn stover for hydrogen production was established. The main achievements obtained by this research were as follows:
A moderately thermophilic bacterium, Thermoanaerobacterium thermosaccharolyticum W16, was isolated for efficiently producing hydrogen from pentose. Under the conditions of carbon source 10g/L, temperature 60°C, and initial pH 6.5, the maximum cumulative H2 yield and H2 production rate in glucose and xylose are 2.42mol H2/mol glucose, 12.9mmol H2/L·h and 2.19mol H2/mol xylose, 10.7mmol H2/L·h, respectively. The metabolic characteristics under mixed glucose and xylose are also discussed. It discovered that strain W16 could simultaneously uptake glucose and xylose, although, the consumption rate of glucose was faster than the consumption rate of xylose. The effect of common inhibitors derived from hydrolysate showed that strain W16 had high tolerance to acetate sodium and vanillin, however, it behaved more sensitive to furfural, hydroxymethylfurfural (HMF) and syringaldehyde.
By using response surface methodology, the hydrolysis conditions were optimized to produce hemicellulosic hydrolysates. Under the optimal hydrolysis conditions of acid concentration 1.69%, processing time 117min, the removal efficiency of hemicellulose was up to 80.%,and the content of xylose, arobinose and glucose were 9.11g/L、1.85g/L和0.88g/L, respectively. The performance of hydrogen production from such hydrolysates by the strain W16 was also investigated. The results indicated that the strain W16 could effectively utilize hemicellulosic hydrolysates for fermentative hydrogen production; the inhibitors derived from hydrolysate such as acetate sodium, furfural didn’t have inhibitory effect on the strain W16.
The screening method was constructed to isolate thermophilic anaerobic cellulolytic hydrogen-producing bacteria, and a good cellulytic hydrogen-producing bacteria Thermoanaerobacterium thermosaccharolyticum M18 was obtained. This strain could efficiently uptake microcrystalline cellulose, xylan, filter paper, and sodium carboxymethylcellulose for hydrogen production, as well as unpretreated corn stover , rice straw, and corn cob. While inoculated to the medium containing 5g/L microcrystalline cellulose or corn stover, the hydrogen production yield and the substrate degradation efficiency were 44.55mmol/L (243.7ml/g-microcrystalline cellulose), 8.45mmol/L (80.2ml/g-corn stover) and 81.9% , 47.2%, respectively. The cellulases characteristics analysis indicated that the cellulases produced by the strain M18 were inducible enzymes, which only be induced in the existence of cellulosic materials. The activity assays on different enzymes of respective component show that the distribution of produced cellulases is mainly outside the cell.
By virtue of SEM, FTIR, XRD, 13C-NMR, GC-MS, and the detection of cellulase and hemicelllase activities, the cellulolytic characteristics of the strain M18 were systematically analyzed. It can be concluded that the process of induced enzyme production coupled with corn stover degradation. The strain M18 exhibited degradation superiority on cellulose and hemicellulose and the degradation of hemicellulose was priority than the degradation of cellulose. In the degradation process, the intramolecular hydrogen bonds of the cellulose molecule were ruptured and the areas of crystal and amorphous were both destroyed. Cellulose and hemicellulose were decomposed into small molecule substances under the effect of cellulases and hemicellulases, and then fermented into hydrogen. It also suggested that lignin is the main barrier to restrict bioconversion of corn stover.
Due to the existence of lignin influenced the bioconversion of cellulose and hemicellulose, the sodium hydroxide pretreatment was used to delignify corn stover. After pretreatment, the cellulosic solid residues and alkali-soluable hemicellulose sugar were obtained. During hydrogen production from corn stover, the bioconversion of cellulose and hemicellulose in NaOH-pretreated corn stover to hydrogen by M18 and alkali-soluable hemicellulose sugar recovery by W16 could be integrated into a biorefinery scheme, thus the maximum bioconversion efficiency of corn stover was acchieved. Compared with unpretreated corn stover, the hydrogen yield increased 36.8%.
引文
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