微生物降解水稻秸秆及沼气发酵的研究
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摘要
我国的纤维素资源非常丰富,仅农作物秸秆,每年产量就达7亿多吨。我国三大秸秆中玉米秸占(35%)、小麦秸占(21%)、稻草占(19%)。除少量用于造纸、建筑、纺织等行业,或用作粗饲料、薪柴外,大部分秸秆未被有效利用而白白烂掉或烧掉,甚至还造成环境污染。充分、合理、有效地利用农作物秸秆资源,有益于生态环境的改善和循环经济的发展。
在可再生物质能中,CO_2和H_2O通过光合作用固定在农作物秸秆中的碳氢化合物—纤维素被认为是发展生物质能最廉价的原料。但由于秸杆纤维素中碳氢比过低导致秸秆中能源密度较低,不能像化石燃料一样直接用于体现现代文明的运输工具和经济活动。通过生物和化学的方法将秸秆中纤维素降解并产生五碳糖、六碳糖、甲醇、己醇、CO_2、甲烷、氢气等低分子化合物,就能够提高碳氢比并增加能源密度。
在玉米秸、小麦秸和稻草三大秸秆中,稻草中富含蜡质且纤维素聚合度更高,比玉米秸秆和小麦秸秆更难被降解。本文就降解水稻秸秆的相关微生物菌株的筛选和育种、甲烷菌的筛选及沼气发酵等方面进行相关研究,主要包括3个方面的内容:
(1)降解水稻秸秆的好氧性丝状真菌菌株的筛选、育种和初步鉴定。从湖南农业大学实验田和牛奶场腐烂稻草秸秆中分离筛选到7株可降解玉米和稻草秸秆的微生物菌株,它们的编号分别为:3号、5号、20号、22号、23号、48号、59号,其中以22号、59号降解秸秆纤维素能力较强;23号、48号降解秸秆半纤维素能力较强。
通过对所选项菌株的菌落形态特征、个体形态特征的观察并结合《真菌鉴定手册》进行鉴定,所选优势菌株鉴定结果如下:22号菌株属半知菌类丛梗孢目丛梗孢科单胞亚科卵形孢族卵形孢属梨卵形孢组;59号菌株属菌株属半知菌类丛梗孢目丛梗孢科单胞亚科卵形孢族卵形孢属梨卵形孢组;23号菌株属藻菌纲毛霉目毛霉科毛霉属总状枝毛霉组;48号菌株属半知菌类丛梗孢目丛梗孢科曲霉族曲霉属烟色曲霉组。
(2)初筛好氧性丝状真菌菌株的细胞融合育种及融合子菌株和发酵条件优化。将初筛菌株22号和48号经过紫外线诱变和原生质体融合,得到遗传稳定性较强的5株融合子菌株,分别编号为:22481～22485号。其中22483号菌株产葡萄糖有明显提高,其发酵醪液中葡萄糖含量达到0.7712mg/mL,而其产木糖的能力基本与48号菌株接近,其发酵醪液中木糖含量达到0.0902mg/mL。经发酵条件优化后,在30℃、108h发酵时间、加入0.5%硫酸铵、0.3%氯化钴,其发酵醪液中葡萄糖含量达到2.2712mg/mL。
(3)降解水稻秸秆的厌氧性菌株和甲烷菌的筛选、鉴定及其沼气发酵的研究。从沼气渣滓和下水道污泥中分离筛选到13株可降解稻草秸秆产生甲酸、乙酸、丁酸、乙醇、CO2及甲烷的厌氧性细菌菌株,将它们的分别编号为:1～13号。
通过对所选厌氧性细菌菌株的菌落形态特征、个体形态特征的观察及生化试验结果并结合《伯杰氏鉴定手册》进行鉴定,所选厌氧性细菌菌株鉴定结果如下:1号菌株属芽孢杆菌梭菌属中生孢子明胶不水解群未知种;3号菌株属芽孢杆菌梭菌属端生孢子明胶水解群未知种;12号菌株属芽孢杆菌梭菌属端生孢子明胶水解群生孢梭菌;8号菌株属芽孢杆菌梭菌属次端生孢子有特殊生长要求群热纤梭菌种;6号芽孢杆菌梭菌属端生孢子有特殊生长要求群克氏菌种;7号芽孢杆菌梭菌属端生孢子明胶水解群腐化梭菌。4号菌株属甲烷菌科甲烷杆菌属甲酸甲烷杆菌;5号菌株属甲烷菌科甲烷杆菌属索氏甲烷杆菌;8号菌株属甲烷菌科甲烷杆菌属反刍甲烷杆菌;2号菌株属甲烷菌科甲烷杆菌属热自养甲烷杆菌;10号菌株属甲烷菌科甲烷球菌属马氏甲烷球菌。
本试验将所选降解水稻秸秆的厌氧性细菌菌株和甲烷菌菌株制备成发酵剂,并将这些发酵剂按适当比例配合进行了发酵秸秆产沼气试验。此混合发酵剂在气温低于20℃时可正常发酵产沼气,并在发酵第3天即开始产沼气并能燃烧,第5天所产沼气燃烧时间最长,第15天后所产沼气较小无法正常燃烧。
China's rice straws and husks (which is rich in cellulose) production is more than 700 million ton per annum, in which the occupancy rate of maize straws is about 35 percent, wheat straws 21 percent and rice straws take up 19 percent—the 3 main native straw resources. Most of them were rotten spontaneously or burned up instead of using efficiently, thus, polluting the environment. People compelled to take steps to look for new reproducible substitute energy owing to the rising international crude oil price which approached to $150 per barrel several times especially in the recent years.
Rice straw is an annually renewable, abundant, and cheap source for naturalcellulose fibers. Cellulose is an organic compound, a polysaccharide consisting of a linear chain of several hundred to over ten thousandβ(1→4) linked D-glucose units, is also considered as the cheapest raw material for developing biological energy among the reproducible energy. However, due to the very low carbon-hydrogen ratio of straw cellulose it results in lower energy density in straws, which cannot be used directly like fossil fuel in transportation and economic activity. The ratio can be improved to increase the energy density from biological and chemical method of decomposing the cellulose in straws into low-molecule compounds such as pentoses, hexoses, methanol, hexanol, CO_2, methane and hydrogen. As such, this study was conducted to determine the following: (1) identification of aerobic filamentous fungi strains degrading rice straw through sifting and breeding technique; (2) selection of aerobic filamentous fungi strains by cell fusion and optimization of fermentation conditions; and (3) selection and identification of the anaerobic strains degrading rice straws and the methanobacteria.
During the rice straw degradation process, 13 microbial strains were isolated from experimental plot (applied with strains 3, 5, 20, 22, 23, 48 and 59) of the Hunan Agriculture University and rotten rice straws from dairy. Strains 22 and 59 showed that degradation of cellulose was better, while strains 23 and 48 are better at degrading hemicellulose of straws. Colonies and individual morphological characteristics of those strains were observed and identified using the Manual of Determinative Fungi. Results showed that 22nd strain belongs to fungi imperfecti, moniliales, monilliella, pseudomonas subfamily, ovoidites, and pear ovoidites; 59th strain belongs to fungi imperfecti, moniliales, monilliella, pseudomonas subfamily, ovoidites, and pear ovoidites; 23rd strain belongs to phycomyceteae, mucorales, mucoraceae, mucor, and racemousus; and 48th strain belongs to fungi imperfecti, moniliales, monilliella, aspergillus, and aspergillus furnigatus.
Moreover, application of ultraviolet radiation mutagenesis and protoplast fusion method for the preliminarily selection of strains 22nd and 48th, 5 fusant strains of high genetic stability numbered 22481-22485 were obtained. The ability of producing glucose of 22483 strains has an obvious improvement, wherein glucose content of fermented liquor was 0.7712mg/ml. And the ability of producing xylose is basically close to the 48th parent strain. The xylose content of fermented liquor obtained was 0.0902mg/ml. After the optimization of fermenting conditions, at 30℃, fermenting time of 108 hours, addition of 0.5% ammonium sulfate and 0.3% cobalt chloride, the content of glucose of fermented liquor had increased to 2.2712mg/ml.
Thirteen (13) strains of anaerobic bacteria which can degrade rice straws into methanoic acid, ethanoic acid, butyric acid, ethanol, CO_2 and methane were isolated from marsh gas off scouring and sewage sludge (with strain numbers 1 to 13). Colonies and individual morphological character of those anaerobic bacteria strains were observed and identified using the Bergeys Manual of Determinative Bacteriology. The following results showed that the 1st strain belongs to F. bacillus, endospore, and gelatin-unhydrolyzed unknown strain. The 3rd strain belongs to F. bacillus, terminal spore gelatin-hydrolyzed unknown strain. The 12th strain belongs to F. bacillus, terminal spore gelatin-hydrolyzed and ctostridium. The 8th strain belongs to F. bacillus, hypo-terminal spore, and special growing demand thermococcales. The 6th strain belongs to F. bacillus, terminal spore, and special growing demand Kluyveri species. The 7th strain belongs to F. bacillus, terminal spore, gelatin-hydrolyzed F. rotten. The 4th strain belongs to methanobacteriaceae, methanobacterium, and methanobacterium formicicum. The 5th strain belongs to methanobacteriaceae, methanobacterium, and methanobacterium sohngenii. The 9th strain belongs to methanobacteriaceae, methanobacterium, and methanobacterium ruminantium. The 2nd strain belongs to methanobacteriaceae, methanobacterium, and methanotherm obacterther mautotrophicus. The 10th strain belongs to methanobacteriaceae, methanococcus, and methanococcus Mazei.
In this experiment, starter prepared by anaerobic bacterial strain and methanebacteria strain, which have been selected before for degrading rice stalk was made and mixed in an appropriate proportion in order to produce biogas by straw fermentation. Consequently, the straw could ferment normally at temperature lower than 20℃to produce biogas using mixed starters. Biogas begun to produce and was burned on the third day of the fermentation. On the other hand, the burning time of the biogas produced in the fifth day lasts longer.
在可再生物质能中,CO_2和H_2O通过光合作用固定在农作物秸秆中的碳氢化合物—纤维素被认为是发展生物质能最廉价的原料。但由于秸杆纤维素中碳氢比过低导致秸秆中能源密度较低,不能像化石燃料一样直接用于体现现代文明的运输工具和经济活动。通过生物和化学的方法将秸秆中纤维素降解并产生五碳糖、六碳糖、甲醇、己醇、CO_2、甲烷、氢气等低分子化合物,就能够提高碳氢比并增加能源密度。
在玉米秸、小麦秸和稻草三大秸秆中,稻草中富含蜡质且纤维素聚合度更高,比玉米秸秆和小麦秸秆更难被降解。本文就降解水稻秸秆的相关微生物菌株的筛选和育种、甲烷菌的筛选及沼气发酵等方面进行相关研究,主要包括3个方面的内容:
(1)降解水稻秸秆的好氧性丝状真菌菌株的筛选、育种和初步鉴定。从湖南农业大学实验田和牛奶场腐烂稻草秸秆中分离筛选到7株可降解玉米和稻草秸秆的微生物菌株,它们的编号分别为:3号、5号、20号、22号、23号、48号、59号,其中以22号、59号降解秸秆纤维素能力较强;23号、48号降解秸秆半纤维素能力较强。
通过对所选项菌株的菌落形态特征、个体形态特征的观察并结合《真菌鉴定手册》进行鉴定,所选优势菌株鉴定结果如下:22号菌株属半知菌类丛梗孢目丛梗孢科单胞亚科卵形孢族卵形孢属梨卵形孢组;59号菌株属菌株属半知菌类丛梗孢目丛梗孢科单胞亚科卵形孢族卵形孢属梨卵形孢组;23号菌株属藻菌纲毛霉目毛霉科毛霉属总状枝毛霉组;48号菌株属半知菌类丛梗孢目丛梗孢科曲霉族曲霉属烟色曲霉组。
(2)初筛好氧性丝状真菌菌株的细胞融合育种及融合子菌株和发酵条件优化。将初筛菌株22号和48号经过紫外线诱变和原生质体融合,得到遗传稳定性较强的5株融合子菌株,分别编号为:22481～22485号。其中22483号菌株产葡萄糖有明显提高,其发酵醪液中葡萄糖含量达到0.7712mg/mL,而其产木糖的能力基本与48号菌株接近,其发酵醪液中木糖含量达到0.0902mg/mL。经发酵条件优化后,在30℃、108h发酵时间、加入0.5%硫酸铵、0.3%氯化钴,其发酵醪液中葡萄糖含量达到2.2712mg/mL。
(3)降解水稻秸秆的厌氧性菌株和甲烷菌的筛选、鉴定及其沼气发酵的研究。从沼气渣滓和下水道污泥中分离筛选到13株可降解稻草秸秆产生甲酸、乙酸、丁酸、乙醇、CO2及甲烷的厌氧性细菌菌株,将它们的分别编号为:1～13号。
通过对所选厌氧性细菌菌株的菌落形态特征、个体形态特征的观察及生化试验结果并结合《伯杰氏鉴定手册》进行鉴定,所选厌氧性细菌菌株鉴定结果如下:1号菌株属芽孢杆菌梭菌属中生孢子明胶不水解群未知种;3号菌株属芽孢杆菌梭菌属端生孢子明胶水解群未知种;12号菌株属芽孢杆菌梭菌属端生孢子明胶水解群生孢梭菌;8号菌株属芽孢杆菌梭菌属次端生孢子有特殊生长要求群热纤梭菌种;6号芽孢杆菌梭菌属端生孢子有特殊生长要求群克氏菌种;7号芽孢杆菌梭菌属端生孢子明胶水解群腐化梭菌。4号菌株属甲烷菌科甲烷杆菌属甲酸甲烷杆菌;5号菌株属甲烷菌科甲烷杆菌属索氏甲烷杆菌;8号菌株属甲烷菌科甲烷杆菌属反刍甲烷杆菌;2号菌株属甲烷菌科甲烷杆菌属热自养甲烷杆菌;10号菌株属甲烷菌科甲烷球菌属马氏甲烷球菌。
本试验将所选降解水稻秸秆的厌氧性细菌菌株和甲烷菌菌株制备成发酵剂,并将这些发酵剂按适当比例配合进行了发酵秸秆产沼气试验。此混合发酵剂在气温低于20℃时可正常发酵产沼气,并在发酵第3天即开始产沼气并能燃烧,第5天所产沼气燃烧时间最长,第15天后所产沼气较小无法正常燃烧。
China's rice straws and husks (which is rich in cellulose) production is more than 700 million ton per annum, in which the occupancy rate of maize straws is about 35 percent, wheat straws 21 percent and rice straws take up 19 percent—the 3 main native straw resources. Most of them were rotten spontaneously or burned up instead of using efficiently, thus, polluting the environment. People compelled to take steps to look for new reproducible substitute energy owing to the rising international crude oil price which approached to $150 per barrel several times especially in the recent years.
Rice straw is an annually renewable, abundant, and cheap source for naturalcellulose fibers. Cellulose is an organic compound, a polysaccharide consisting of a linear chain of several hundred to over ten thousandβ(1→4) linked D-glucose units, is also considered as the cheapest raw material for developing biological energy among the reproducible energy. However, due to the very low carbon-hydrogen ratio of straw cellulose it results in lower energy density in straws, which cannot be used directly like fossil fuel in transportation and economic activity. The ratio can be improved to increase the energy density from biological and chemical method of decomposing the cellulose in straws into low-molecule compounds such as pentoses, hexoses, methanol, hexanol, CO_2, methane and hydrogen. As such, this study was conducted to determine the following: (1) identification of aerobic filamentous fungi strains degrading rice straw through sifting and breeding technique; (2) selection of aerobic filamentous fungi strains by cell fusion and optimization of fermentation conditions; and (3) selection and identification of the anaerobic strains degrading rice straws and the methanobacteria.
During the rice straw degradation process, 13 microbial strains were isolated from experimental plot (applied with strains 3, 5, 20, 22, 23, 48 and 59) of the Hunan Agriculture University and rotten rice straws from dairy. Strains 22 and 59 showed that degradation of cellulose was better, while strains 23 and 48 are better at degrading hemicellulose of straws. Colonies and individual morphological characteristics of those strains were observed and identified using the Manual of Determinative Fungi. Results showed that 22nd strain belongs to fungi imperfecti, moniliales, monilliella, pseudomonas subfamily, ovoidites, and pear ovoidites; 59th strain belongs to fungi imperfecti, moniliales, monilliella, pseudomonas subfamily, ovoidites, and pear ovoidites; 23rd strain belongs to phycomyceteae, mucorales, mucoraceae, mucor, and racemousus; and 48th strain belongs to fungi imperfecti, moniliales, monilliella, aspergillus, and aspergillus furnigatus.
Moreover, application of ultraviolet radiation mutagenesis and protoplast fusion method for the preliminarily selection of strains 22nd and 48th, 5 fusant strains of high genetic stability numbered 22481-22485 were obtained. The ability of producing glucose of 22483 strains has an obvious improvement, wherein glucose content of fermented liquor was 0.7712mg/ml. And the ability of producing xylose is basically close to the 48th parent strain. The xylose content of fermented liquor obtained was 0.0902mg/ml. After the optimization of fermenting conditions, at 30℃, fermenting time of 108 hours, addition of 0.5% ammonium sulfate and 0.3% cobalt chloride, the content of glucose of fermented liquor had increased to 2.2712mg/ml.
Thirteen (13) strains of anaerobic bacteria which can degrade rice straws into methanoic acid, ethanoic acid, butyric acid, ethanol, CO_2 and methane were isolated from marsh gas off scouring and sewage sludge (with strain numbers 1 to 13). Colonies and individual morphological character of those anaerobic bacteria strains were observed and identified using the Bergeys Manual of Determinative Bacteriology. The following results showed that the 1st strain belongs to F. bacillus, endospore, and gelatin-unhydrolyzed unknown strain. The 3rd strain belongs to F. bacillus, terminal spore gelatin-hydrolyzed unknown strain. The 12th strain belongs to F. bacillus, terminal spore gelatin-hydrolyzed and ctostridium. The 8th strain belongs to F. bacillus, hypo-terminal spore, and special growing demand thermococcales. The 6th strain belongs to F. bacillus, terminal spore, and special growing demand Kluyveri species. The 7th strain belongs to F. bacillus, terminal spore, gelatin-hydrolyzed F. rotten. The 4th strain belongs to methanobacteriaceae, methanobacterium, and methanobacterium formicicum. The 5th strain belongs to methanobacteriaceae, methanobacterium, and methanobacterium sohngenii. The 9th strain belongs to methanobacteriaceae, methanobacterium, and methanobacterium ruminantium. The 2nd strain belongs to methanobacteriaceae, methanobacterium, and methanotherm obacterther mautotrophicus. The 10th strain belongs to methanobacteriaceae, methanococcus, and methanococcus Mazei.
In this experiment, starter prepared by anaerobic bacterial strain and methanebacteria strain, which have been selected before for degrading rice stalk was made and mixed in an appropriate proportion in order to produce biogas by straw fermentation. Consequently, the straw could ferment normally at temperature lower than 20℃to produce biogas using mixed starters. Biogas begun to produce and was burned on the third day of the fermentation. On the other hand, the burning time of the biogas produced in the fifth day lasts longer.
引文
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