木质纤维素分解复合菌群NSC-7菌种组成及种间协作机理
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摘要
地球上每年产生的植物光合作用产物可达2000亿吨,木质纤维素是超过其一半的主要成分,在木材、秸秆、农林产品加工副产品、畜禽粪便中都含有大量木质纤维素。由于木质纤维素分解难,它的分解成为限制含木质纤维素生物质利用及环境治理上的瓶颈环节。如,富含木质纤维素的生物质由于不占粮食而且能量密度较大,就成为全世界都在关注的生物质能源开发和利用的对象。但由于木质纤维素的分解糖化很难限制了该资源的进一步开发。秸秆还田是增加土壤有机质恢复土壤地力的有效途径,但在我国特殊的农作制度下,秸秆中的木质纤维素分解慢,影响下茬作物的生长,从而限制着秸秆直接还田的推广,采用化学方法处理木质纤维素也存在再次污染环境的问题,而利用微生物分泌的纤维素复合酶加快分解木质纤维素无论对生物质资源的转化利用还是环境治理都具有重要意义。
本研究在尊重自然界群体效应的前提下,利用限制性培养法筛选到一组具有分解木质纤维素和农药(林丹)双重功能的复合菌系NSC-7,本文对NSC-7的菌种组成多样性和种间协作机理进行深入研究。
NSC-7在14 d的培养过程中,可降解稻秆总干重的73.6%,其中降解纤维素58.2%,半纤维素82.1%,木质素5.4%。用GC-MS检测发酵液产物,发现有乙醇、乙酸、丙酸、丁酸、戊酸、甘油、尿素、乙二酸乙二脂、2-甲基丙酸、乙二醇等10种化合物,其中含量较大的依次为乙酸、甘油、丁酸、丙酸。复合系可同时分泌纤维素酶和半纤维素酶,且各种纤维素酶活性都呈单峰曲线,其中纤维素内切酶(CMC)、总纤维素酶(FPU)、外切酶(AC)和β-糖苷酶活性(CB)都在第8 d达到最大值,分别为4.48 U/mL、15.83 U/mL、7.51 U/mL和25.78 U/mL。半纤维素酶活性(XY)在第5 d达到最高值为280.9 U/mL。
在有氧条件下,利用传统的平板画线法分离到11株单菌,将获得的11株单菌按照体积比1:1重新组合后不具备分解纤维素的能力。利用单层和双层滤纸平板法检测NSC-7复合菌群的分解能力,发现只有双层平板上的滤纸能够分解,说明NSC-7复合系内降解纤维素的关键菌属于厌氧或微好氧菌。为了获取复合菌群内全部菌株的组成信息,本研究利用现代分子生态学技术对NSC-7构建克隆文库,获得195个16S rDNA片断,经DGGE筛选获得25个代表性克隆,其序列比对结果显示有60%的近缘种为已知菌,分别归属于Clostridium、Petrobacter、Bacteria、Paenibacillus,proteobacterium 5个属,其余40%菌的近缘种属于难培养菌。
从已分离的11株单菌株组合后不具备分解纤维素能力的实验结果推断,分解纤维素的关键菌很可能在没有分离到的Clostridium属内。根据Clostridium在分解纤维素过程中具有吸附于纤维素粉的特性,利用冲洗和离心沉淀等方法获得一组具备分解纤维素能力但无法分开的“3株梭菌组合”,将该组合与3株不具分解纤维素能力的单菌株分别重新组合,发现有两株单菌株Pseudoxanthomonas taiwanensis和Petrobacter sp.都有促进其分解的能力。通过理化指标测定发现,这两株菌具备高效利用纤维二糖的性质,消除纤维素的中间产物(纤维二糖)对纤维素分解的阻遏或抑制作用,很可能是这两株菌促进纤维素分解的一个重要原因。
NSC-7复合系经90～110℃高温处理30 min后,仍具备分解纤维素的能力,只是随着处理温度的升高,剩余菌群内菌株的数量和种类,以及发酵液中挥发产物种类也随之减少,分解能力也逐渐减弱,但随着继代次数的不断增加分解能力又有所恢复,说明复合系自身具备一定的抗逆境能力。
Cellulose account for more than 50 percent of the primary product of photosynthesis in terrestrial environments,and the most abundant renewable bioresource produced in the biosphere(200 billion dry tons/year),lignocellulose in wood,straw,agricultural waste, livestock dejecta and so on.The cellulose molecule is very stable,which restrict its widely used to produce sustainable biobased products and bioenergy to replace depleting fossil fuels.
The synergic mechanism and microbial component diversity of the microbial community NSC-7 with efficient cellulose and lindan degradating ability was determined.The results showed that NSC-7 could degrade 73.6%of rice staw in weight,including 82.1%of cellulose, 58.2%of hemicellulase,and 5.4%of lignin within fourteen days.In addition,ethanol,propiolic acid,acetic acid,ethanedioic acid diethyl ester,propionic acid 2-methyl,urea,pentanol, butanoic acid,ethanol,2,2-oxybis and glycerin the ten kinds of material were detected in volatile products by gas chromatography mass spectrometry(GC-MS).NSC-7 could produce cellulase and hemicellulase simultaneity.And the maximum of endoglucanases,exoglucanases,β-glucosidases and the total cellulase activity were 4.48 U/mL,15.83 U/mL,25.78 U/mL and 7.5 U/mL,respectively,on the day 8,and the maximum of hemicellulase activity were 280.6 U/mL on the day 5.
For determine the bacterial compose of the community,11 isolate strains were detected by plate isolation under aerobic condition,while the community reset by the 11 isolate strains without capacity of degrading cellulose.The capacity of degrading of the filter paper in double deck plate and monolayer plate were determined,only the filter paper in double deck plate were degraded,that means the main or key microbe are anaerobic.The Denaturing Gradient Gel Electrophoresis(DGGE) and construction of 16S rDNA clone library were used to identify the composition diversity of NSC-7 community,with 25 strains detected,in that about 60% closest relative among them was know the detailed information and they were belonged to Clostridium,Petrobacter,Bacteria,Paenibacillus,proteobacterium respectively.Furthermore, there were 40%closest relative belonged to uncultured bacterium clone.
Due to the isolate strains without capacity of degrading cellulose,a conclusion could be made that the key microbe may be in the uncultured bacterium of Clostridium sp.According to the character of Clostridium sp.with the capability of adhibit in the cellulose power in degradation process,therefore,washing by the PBS buffer and centrifuging the deposition were used to isolated the Clostridium sp.However,we obtained the microbial community "key microbial community " with three strains(Uncultured bacterium clone、Clostridium sp. PML14 16S、Clostridium straminisolvens)which could not be isolate until now.The "key microbial community" was assembled with three isolates which without the capability of degradation cellulose(Pseudoxanthomonas taiwanensis,Bacillus sp.Petrobacter sp.).The results showed that the two isolates(A-Pseudoxanthomonas taiwanensis and 6-Petrobacter sp.) could promot the degradation capability of "key microbial".More research found that the two isolates with the capability of high degradated and utilized cellobiose,a conclusion was deduce that utilized cellobiose maight be the main reason for isolate improve the cellulose degradation.
The microbial community NSC-7 was treated under 90℃,100℃,105℃and 110℃temperature about 30 min.After treated,each of them still with the cellulose degradating ability,however,the kinds and quantity of microbial and the kinds of volatile products became less than before,and the ability of degration cellulose became less efficient than before.While with the subculture times increased,the capability of the degradation resume.
本研究在尊重自然界群体效应的前提下,利用限制性培养法筛选到一组具有分解木质纤维素和农药(林丹)双重功能的复合菌系NSC-7,本文对NSC-7的菌种组成多样性和种间协作机理进行深入研究。
NSC-7在14 d的培养过程中,可降解稻秆总干重的73.6%,其中降解纤维素58.2%,半纤维素82.1%,木质素5.4%。用GC-MS检测发酵液产物,发现有乙醇、乙酸、丙酸、丁酸、戊酸、甘油、尿素、乙二酸乙二脂、2-甲基丙酸、乙二醇等10种化合物,其中含量较大的依次为乙酸、甘油、丁酸、丙酸。复合系可同时分泌纤维素酶和半纤维素酶,且各种纤维素酶活性都呈单峰曲线,其中纤维素内切酶(CMC)、总纤维素酶(FPU)、外切酶(AC)和β-糖苷酶活性(CB)都在第8 d达到最大值,分别为4.48 U/mL、15.83 U/mL、7.51 U/mL和25.78 U/mL。半纤维素酶活性(XY)在第5 d达到最高值为280.9 U/mL。
在有氧条件下,利用传统的平板画线法分离到11株单菌,将获得的11株单菌按照体积比1:1重新组合后不具备分解纤维素的能力。利用单层和双层滤纸平板法检测NSC-7复合菌群的分解能力,发现只有双层平板上的滤纸能够分解,说明NSC-7复合系内降解纤维素的关键菌属于厌氧或微好氧菌。为了获取复合菌群内全部菌株的组成信息,本研究利用现代分子生态学技术对NSC-7构建克隆文库,获得195个16S rDNA片断,经DGGE筛选获得25个代表性克隆,其序列比对结果显示有60%的近缘种为已知菌,分别归属于Clostridium、Petrobacter、Bacteria、Paenibacillus,proteobacterium 5个属,其余40%菌的近缘种属于难培养菌。
从已分离的11株单菌株组合后不具备分解纤维素能力的实验结果推断,分解纤维素的关键菌很可能在没有分离到的Clostridium属内。根据Clostridium在分解纤维素过程中具有吸附于纤维素粉的特性,利用冲洗和离心沉淀等方法获得一组具备分解纤维素能力但无法分开的“3株梭菌组合”,将该组合与3株不具分解纤维素能力的单菌株分别重新组合,发现有两株单菌株Pseudoxanthomonas taiwanensis和Petrobacter sp.都有促进其分解的能力。通过理化指标测定发现,这两株菌具备高效利用纤维二糖的性质,消除纤维素的中间产物(纤维二糖)对纤维素分解的阻遏或抑制作用,很可能是这两株菌促进纤维素分解的一个重要原因。
NSC-7复合系经90～110℃高温处理30 min后,仍具备分解纤维素的能力,只是随着处理温度的升高,剩余菌群内菌株的数量和种类,以及发酵液中挥发产物种类也随之减少,分解能力也逐渐减弱,但随着继代次数的不断增加分解能力又有所恢复,说明复合系自身具备一定的抗逆境能力。
Cellulose account for more than 50 percent of the primary product of photosynthesis in terrestrial environments,and the most abundant renewable bioresource produced in the biosphere(200 billion dry tons/year),lignocellulose in wood,straw,agricultural waste, livestock dejecta and so on.The cellulose molecule is very stable,which restrict its widely used to produce sustainable biobased products and bioenergy to replace depleting fossil fuels.
The synergic mechanism and microbial component diversity of the microbial community NSC-7 with efficient cellulose and lindan degradating ability was determined.The results showed that NSC-7 could degrade 73.6%of rice staw in weight,including 82.1%of cellulose, 58.2%of hemicellulase,and 5.4%of lignin within fourteen days.In addition,ethanol,propiolic acid,acetic acid,ethanedioic acid diethyl ester,propionic acid 2-methyl,urea,pentanol, butanoic acid,ethanol,2,2-oxybis and glycerin the ten kinds of material were detected in volatile products by gas chromatography mass spectrometry(GC-MS).NSC-7 could produce cellulase and hemicellulase simultaneity.And the maximum of endoglucanases,exoglucanases,β-glucosidases and the total cellulase activity were 4.48 U/mL,15.83 U/mL,25.78 U/mL and 7.5 U/mL,respectively,on the day 8,and the maximum of hemicellulase activity were 280.6 U/mL on the day 5.
For determine the bacterial compose of the community,11 isolate strains were detected by plate isolation under aerobic condition,while the community reset by the 11 isolate strains without capacity of degrading cellulose.The capacity of degrading of the filter paper in double deck plate and monolayer plate were determined,only the filter paper in double deck plate were degraded,that means the main or key microbe are anaerobic.The Denaturing Gradient Gel Electrophoresis(DGGE) and construction of 16S rDNA clone library were used to identify the composition diversity of NSC-7 community,with 25 strains detected,in that about 60% closest relative among them was know the detailed information and they were belonged to Clostridium,Petrobacter,Bacteria,Paenibacillus,proteobacterium respectively.Furthermore, there were 40%closest relative belonged to uncultured bacterium clone.
Due to the isolate strains without capacity of degrading cellulose,a conclusion could be made that the key microbe may be in the uncultured bacterium of Clostridium sp.According to the character of Clostridium sp.with the capability of adhibit in the cellulose power in degradation process,therefore,washing by the PBS buffer and centrifuging the deposition were used to isolated the Clostridium sp.However,we obtained the microbial community "key microbial community " with three strains(Uncultured bacterium clone、Clostridium sp. PML14 16S、Clostridium straminisolvens)which could not be isolate until now.The "key microbial community" was assembled with three isolates which without the capability of degradation cellulose(Pseudoxanthomonas taiwanensis,Bacillus sp.Petrobacter sp.).The results showed that the two isolates(A-Pseudoxanthomonas taiwanensis and 6-Petrobacter sp.) could promot the degradation capability of "key microbial".More research found that the two isolates with the capability of high degradated and utilized cellobiose,a conclusion was deduce that utilized cellobiose maight be the main reason for isolate improve the cellulose degradation.
The microbial community NSC-7 was treated under 90℃,100℃,105℃and 110℃temperature about 30 min.After treated,each of them still with the cellulose degradating ability,however,the kinds and quantity of microbial and the kinds of volatile products became less than before,and the ability of degration cellulose became less efficient than before.While with the subculture times increased,the capability of the degradation resume.
引文
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