大熊猫粪便微生物多样性分析及纤维素降解产氢菌的筛选鉴定
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摘要
利用宏基因组测序技术对大熊猫粪便微生物进行了多样性分析,发现大熊猫粪便样品中含有大量可以降解纤维素的厌氧微生物种类,如梭菌科、瘤胃菌科和毛螺菌科等,因此,将熊猫粪便样品作为降解纤维素微生物菌种分离的来源具有可行性。采用刚果红染色法从大熊猫粪便样品中筛选具有降解纤维素产氢能力的菌株,分离获得1株纤维素降解产氢菌株Cel10,根据菌株的形态和生理生化特征并结合分子生物学,鉴定菌株Cel10属于缓纤维梭菌(Clostridium lentocellum),其16S rDNA序列的同源性为98%。菌株Cel10在pH 4.0～8.0、温度25～50℃范围内可以利用纤维素进行生长,其最适pH 7.0,最适生长温度37℃。本文丰富了大熊猫肠道纤维素降解菌的种类,为木质纤维素类农产品加工废弃物的开发和综合利用提供了良好的菌种资源和科学依据。
Microbial diversity analysis of giant panda excrement was carried out using metagenomics sequencing technology. The result showed that a lot of anaerobic microbial species which could degrade and use cellulose were in giant panda excrements, such as Clostridiaceae, Ruminococcaceae and Lachnospiraceae. It was feasible to make giant panda excrement as a source to isolate the microorganism of cellulose degradation. Using CMC-Na as the only carbon source, the strain of cellulose degradation hydrogenogens named Cel10 was screened from giant panda excrement by using Congo red staining method. According to the morphological characteristics and physiological and biochemical characteristics of the strain, the new strain was identified as Clostridium lentocellum by molecular biology, while the homology of its 16 S rDNA sequence was 98%. The strain Cel10 could use cellulose to grow at pH 4.0 ～ 8.0 under 25 ～ 50 ℃. The optimal pH was 7.0 and the optimal growth temperature was 37 ℃. This study not only enriched the cellulose degradation bacteria species of panda guts but also provided good strain resources and scientific basis for development and comprehensive utilization of wastes during the processing of lignocellulosic agricultural products.
Microbial diversity analysis of giant panda excrement was carried out using metagenomics sequencing technology. The result showed that a lot of anaerobic microbial species which could degrade and use cellulose were in giant panda excrements, such as Clostridiaceae, Ruminococcaceae and Lachnospiraceae. It was feasible to make giant panda excrement as a source to isolate the microorganism of cellulose degradation. Using CMC-Na as the only carbon source, the strain of cellulose degradation hydrogenogens named Cel10 was screened from giant panda excrement by using Congo red staining method. According to the morphological characteristics and physiological and biochemical characteristics of the strain, the new strain was identified as Clostridium lentocellum by molecular biology, while the homology of its 16 S rDNA sequence was 98%. The strain Cel10 could use cellulose to grow at pH 4.0 ～ 8.0 under 25 ～ 50 ℃. The optimal pH was 7.0 and the optimal growth temperature was 37 ℃. This study not only enriched the cellulose degradation bacteria species of panda guts but also provided good strain resources and scientific basis for development and comprehensive utilization of wastes during the processing of lignocellulosic agricultural products.
引文
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[22] 杨林丽. 纤维素降解菌筛选及混合菌种纤维素降解能力测定 [D]. 杨凌: 西北农林科技大学, 2013.
[23] 李春燕, 于琦, 冯露, 等. 低温纤维素降解菌分离鉴定及产酶条件优化 [J]. 东北农业大学学报, 2015, 46(10): 74-81.
[24] 王燚, 何延美, 钟志军, 等. 不同季节亚成体大熊猫肠道菌群ERIC-PCR指纹图谱分析 [J]. 中国兽医科学, 2011, 41(8): 778-783.
[25] 何廷美, 崔婷婷, 钟志军, 等. 成年大熊猫肠道菌群多样性的16S rDNA-RFLP分析 [J]. 中国兽医科学, 2012, 42 (11): 1121-1127.
[26] 崔明全, 何廷美, 钟志军, 等. 大熊猫粪便菌群ERIC-PCR 指纹图谱的分析及优势菌群的鉴定 [J]. 畜牧与兽医, 2013, 45(9): 6-11.
[27] 彭广能, 熊焰. 大熊猫肺炎克雷伯氏菌的研究现状 [J]. 四川畜牧兽医, 2000, 27(增刊): 86-87.
[28] 荣华, 邱成书, 胡国全, 等. 一株大熊猫肠道厌氧纤维素菌的分离鉴定、系统发育分析及生物学特性的研究 [J]. 应用与环境生物学报, 2006, 12(2): 239-242.
[29] 樊程, 李双江, 李成磊. 大熊猫肠道纤维素分解菌的分离鉴定及产酶性质 [J]. 微生物学报, 2012, 52(9): 1113-1121.
[30] 蒋芳, 赵婷, 刘成君, 等. 兼性厌氧纤维素菌的分离与系统发育分析 [J]. 微生物学通报, 2006, 33(3): 109-113.
[2] KUMAR G, SIVAGURUNATHAN P, SEN B, et al. Research and development perspectives of lignocellulose-based biohydrogen production [J]. International Biodeterioration and Biodegradation, 2017, 119: 225-238.
[3] WU Y R, ZHANG M M, ZHONG M Q, et al. Synergistic enzymatic saccharification and fermentation of agar for biohydrogen production [J]. Bioresource Technology, 2017, 241: 369-373.
[4] LI D, YUAN Z H, SUN Y, et al. Hydrogen production characteristics of the organic fraction of municipal solid wastes by anaerobic mixed culture fermentation [J]. International Journal of Hydrogen Energy, 2009, 34(2): 812-820.
[5] REN N Q, GUO W Q, LIU B F, et al. Biological hydrogen production by dark fermentation: challenges and prospects towards scaled-up production [J]. Current Opinion in Biotechnology, 2011, 22(3): 365-370.
[6] LIN C Y, CHIANG C C, NGUYEN M L T, et al. Enhancement of fermentative biohydrogen production from textile desizing wastewater via coagulation-pretreatment [J]. International Journal of Hydrogen Energy, 2017, 42(17): 12153-12158.
[7] RAI P K, SINGH S P , ASTHANA P K, et al. Biohydrogen production from sugarcane bagasse by integrating dark- and photo-fermentation [J]. Bioresource Technology, 2014, 152(1): 140-146.
[8] REN N Q, WANG A J, CAO G L, et al. Bioconversion of lignocellulosic biomass to hydrogen: potential and challenges [J]. Biotechnology Advances, 2009, 27(6): 1051-1060.
[9] ZHANG L, CHUNG J S, REN N Q, et al. Effects of the ecological factors on hydrogen production and [Fe-Fe]-hydrogenase activity in Ethanoligenens harbinense YUAN-3 [J]. International Journal of Hydrogen Energy, 2015, 40(21): 6792-6797.
[10] ABBASI T, ABBASI S A. ‘Renewable’ hydrogen: prospects and challenges [J]. Renewable and Sustainable Energy Reviews, 2011, 15(6): 3034-3040.
[11] SHOW K Y, LEE D J, CHANG J S. Bioreactor and process design for biohydrogen production [J]. Bioresource Technology, 2011, 102(18): 8524-8533.
[12] HO K L, LEE D J. Harvesting biohydrogen from cellobiose from sulfide or nitrite-containing wastewaters using Clostridium sp. R1 [J]. Bioresource Technology, 2011, 102(18): 8547-8549.
[13] ZHANG Q H, TANG L, ZHANG J H, et al. Optimization of thermal-dilute sulfuric acid pretreatment for enhancement of methane production from cassava residues [J]. Bioresource Technology, 2011, 102(4): 3958-3965.
[14] WEI G, LU F, ZHOU Z H, et al. The microbial community in the feces of the giant panda (Ailuropoda melanoleuca) as determined by PCR-TGGE pro?ling and clone library analysis [J]. Microbial Ecology, 2007, 54(1): 194-202.
[15] ZHU L F, WU Q, DAI J Y, et al. Evidence of cellulose metabolism by the giant panda gut microbiome [J]. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(43): 17714-17719.
[16] HIRAYAMA K, KAWAMURA S, MITSUOKA T, et al. The fecal ?ora of the giant panda (Ailuropoda melanoleuca) [J]. Journal of Applied Microbiology, 1989, 67(4): 411-415.
[17] FANG W, FANG Z M, ZHOU P, et al. Evidence for lignin oxidation by the giant panda fecal microbiome [J]. Public Library of Science One, 2012, 7(11): e50312.
[18] 王旭, 颜其贵, 陈世界, 等. 大熊猫源停乳链球菌类马亚种的分离与鉴定 [J]. 中国兽医科学, 2011, 41(5): 464-469.
[19] 东秀珠, 蔡妙英. 常见细菌系统鉴定手册 [M] . 北京: 科学出版社, 2001: 349-418.
[20] BURRELL P C, O’SULLIVAN C, SONG H, et al. Identification, detection, and spatial resolution of Clostridium populations responsible for cellulose degradation in a methanogenic landfill leachate bioreactor [J]. Applied and Environmental Microbiology, 2004, 70(4): 2414-2419.
[21] VAN DYKE M I, MCCARTHY A J. Molecular biological detection and characterization of Clostridium in municipal landfill sites [J]. Applied and Environmental Microbiology, 2002, 68(4): 2049-2053.
[22] 杨林丽. 纤维素降解菌筛选及混合菌种纤维素降解能力测定 [D]. 杨凌: 西北农林科技大学, 2013.
[23] 李春燕, 于琦, 冯露, 等. 低温纤维素降解菌分离鉴定及产酶条件优化 [J]. 东北农业大学学报, 2015, 46(10): 74-81.
[24] 王燚, 何延美, 钟志军, 等. 不同季节亚成体大熊猫肠道菌群ERIC-PCR指纹图谱分析 [J]. 中国兽医科学, 2011, 41(8): 778-783.
[25] 何廷美, 崔婷婷, 钟志军, 等. 成年大熊猫肠道菌群多样性的16S rDNA-RFLP分析 [J]. 中国兽医科学, 2012, 42 (11): 1121-1127.
[26] 崔明全, 何廷美, 钟志军, 等. 大熊猫粪便菌群ERIC-PCR 指纹图谱的分析及优势菌群的鉴定 [J]. 畜牧与兽医, 2013, 45(9): 6-11.
[27] 彭广能, 熊焰. 大熊猫肺炎克雷伯氏菌的研究现状 [J]. 四川畜牧兽医, 2000, 27(增刊): 86-87.
[28] 荣华, 邱成书, 胡国全, 等. 一株大熊猫肠道厌氧纤维素菌的分离鉴定、系统发育分析及生物学特性的研究 [J]. 应用与环境生物学报, 2006, 12(2): 239-242.
[29] 樊程, 李双江, 李成磊. 大熊猫肠道纤维素分解菌的分离鉴定及产酶性质 [J]. 微生物学报, 2012, 52(9): 1113-1121.
[30] 蒋芳, 赵婷, 刘成君, 等. 兼性厌氧纤维素菌的分离与系统发育分析 [J]. 微生物学通报, 2006, 33(3): 109-113.