Isolation,Identification of a Cellulose-degrading Bacterium with IAA-producing Ability and Optimization of Its Culture Conditions

doi:10.13560/j.cnki.biotech.bull.1985.2020-0454

Abstract

Abstract:

This study aims to screen strains with the ability of both degrading straw and promoting crop growth from Shajiang black soil for solving problems such as difficult survival status,weak rot-promoting effect and poor crop growth caused by exogenous rot straw. Screening with sodium carboxymethyl cellulose plate and combing with CMC enzyme（endo-1,4-β-D-glucanase）- and IAA（Indole-3-acetic acid）-producing capacity,the potential straw rot- and plant growth-promoting were evaluated. Species identification of strains were conducted via morphological,physiological and biochemical and 16S rDNA sequence analysis,then straw degradation shake flask and corn pot test were used to determine the actual rot- and growth-promoting effect of the strain,and a single factor test was set up to explore the optimal condition for growth and enzyme production of the strain. The screened strain n3 demonstrated the high activity of CMC enzyme（20.60 U/mL）and IAA synthesis（20.15 mg/L）,and was identified as Azospirillum zeae. The results showed that inoculation with n3 increased the rate of straw rot-promoting under liquid fermentation by 54.71%（P <0.01）compared with the control treatment;the root length,average diameter and SPAD value（Soil and plant analyzer development）of maize under soil culture conditions increased by 18.3%,22.0% and 5.24%,respectively（P<0.05）.The optimal nitrogen source and liquid volume of n3 growth,yielding CMC enzyme and IAA were the same as that of yeast powder and 25 mL/250mL,but the optimal pH was 5.0 for CMC enzyme production,and 6.0 for growth and IAA production. Conclusively,the screened A. zeae may be used to produce multi-functional straw-decomposing agent,accelerating the rot-decomposing of straw and promoting the growth of crops in the field,which has broad agricultural application prospect.

Key words: Shajiang black soil, Azospirillum zeae, straw rot-promoting, corn growth-promoting, optimization of conditions

WU Jing, NIE Cai-e, ZHU Yuan-yuan, HUANG Wei, MA Chao, JIANG Ying, ZHU Lin, GAO Hong-jian. Isolation,Identification of a Cellulose-degrading Bacterium with IAA-producing Ability and Optimization of Its Culture Conditions[J]. Biotechnology Bulletin, 2020, 36(12): 54-63.

Figures/Tables 6

References 45

[1]	Johnson SE, Angeles OR, Brar DS, et al. Faster anaerobic decomposition of a brittle straw rice mutant:Implications for residue management[J]. Soil Biology and Biochemistry, 2006,38(7):1880-1892.
[2]	宋大利, 侯胜鹏, 王秀斌, 等. 中国秸秆养分资源数量及替代化肥潜力[J]. 植物营养与肥料学报, 2018,24(1):1-21.
	Song DL, Hou SP, Wang XB, et al. Nutrient resource quantity of crop straw and its potential of substituting[J]. Journal of Plant Nutrition and Fertilizers, 2018,24(1):1-21.
[3]	叶新新, 王冰清, 刘少君, 等. 耕作方式和秸秆还田对砂姜黑土碳库及玉米小麦产量的影响[J]. 农业工程学报, 2019,35(14):112-118.
	Ye XX, Wang BQ, Liu SJ, et al. Influence of tillage and straw retention on soil carbon pool and maize-wheat yield in Shajiang black soil[J]. Transactions of the Chinese Society of Agricultural Engineering, 2019,35(14):112-118.
[4]	Qin S, Jiao K, Lyu D, et al. Effects of maize residue and cellulose-decomposing bacteria inocula on soil microbial community, functional diversity, organic fractions, and growth of Malus hupehensis Rehd[J]. Archives of Agronomy and Soil Science, 2015,61(2):173-184. doi: 10.1080/03650340.2014.928927 URL
[5]	陈欢, 曹承富, 孔令聪, 等. 长期施肥下淮北砂姜黑土区小麦产量稳定性研究[J]. 中国农业科学, 2014,47(13):2580-2590. doi: 10.3864/j.issn.0578-1752.2014.13.010 URL
	Chen H, Cao CF, Kong LC, et al. Study on wheat yield stability in Huaibei lime concretion black soil area based on long-term fertilization experiment[J]. Scientia Agricultura Sinica, 2014,47(13):2580-2590.
[6]	马超, 周静, 郑学博, 等. 秸秆促腐还田对土壤养分和小麦产量的影响[J]. 土壤, 2012,44(1):30-35.
	Ma C, Zhou J, Zheng XB, et al. Effects of returning rice straw into field on soil nutrients and wheat yields under promoting decay condition[J]. Soils, 2012,44(1):30-35.
[7]	Sun S, Zhang Y, Liu K, et al. Insight into biodegradation of cellulose by psychrotrophic bacterium Pseudomonas sp. LKR-1 from the cold region of China:optimization of cold-active cellulase production and the associated degradation pathways[J]. Cellulose, 2020,27(1):315-333. doi: 10.1007/s10570-019-02798-y URL
[8]	Jha B, Gontia I, Hartmann A. The roots of the halophyte Salicornia brachiata are a source of new halotolerant diazotrophic bacteria with plant growth-promoting potential[J]. Plant Soil, 2012,356(1-2):265-277. doi: 10.1007/s11104-011-0877-9 URL
[9]	贺军军, 罗萍, 李勤奋, 等. 甘蔗渣淀粉功能降解菌筛选及s2g5-1和s3g4-8的鉴定[J]. 安徽农业科学, 2011,39(13):7711-7714.
	He JJ, Luo P, Li QF, et al. Screening of starch-degrading strains in bagasse and identification of strains s2g5-1 and s3g4-8[J]. Journal of Anhui Agricultura Science, 2011,39(13):7711-7714.
[10]	王伏伟, 王晓波, 李金才, 等. 施肥及秸秆还田对砂姜黑土细菌群落的影响[J]. 中国生态农业学报, 2015,23(10):1302-1311.
	Wang FW, Wang XB, Li JC, et al. Effects of fertilization and straw incorporation on bacterial communities in lime concretion black soil[J]. Chinese Journal of Eco-Agriculture, 2015,23(10):1302-1311.
[11]	Li H, Dai M, Dai S, et al. Current status and environment impact of direct straw return in China’s cropland—A review[J]. Ecotoxicol Environ Saf, 2018,159(9):293-300.
[12]	张典利, 孟臻, 亓文哲, 等. 植物根际促生菌的研究与应用现状[J]. 世界农药, 2018,40(6):37-43, 50.
	Zhang DL, Meng Z, Qi WZ, et al. The research and application status of plant growth promoting rhizobacteria[J]. Word Pesticide, 2018,40(6):37-43, 50.
[13]	Spaepen S, Vanderleyden J, Remans R. Indole-3-acetic acid in microbial and microorganism-plant signaling[J]. FEMS Microbiology Reviews, 2007,31(4):425-448. URL pmid: 17509086
[14]	万兵兵, 刘晔, 吴越, 等. 烟草根际解磷解钾菌的筛选鉴定及应用效果研究[J]. 河南农业科学, 2016,45(9):46-51.
	Wang BB, Liu Y, Wu Y, et al. Screening, identification of phosphate-and potassium-solubilizing PGPR and its promoting effect on tobacco[J]. Journal of Henan Agricultural Sciences, 2016,45(9):46-51.
[15]	骆婷, 夏虹, 冯定胜, 等. 森林土壤中多功能降解菌的分离筛选及鉴定[J]. 西南农业学报, 2018,31(5):1032-1040.
	Luo T, Xia H, Feng DS, et al. Isolation and identification of multi-function degradation bacteria from forest soil[J]. Southwest China Journal of Agricultural Sciences, 2018,31(5):1032-1040.
[16]	程鹏, 刘姗姗, 王玉, 等. 1株高产纤维素酶菌株的筛选鉴定及对稻秆降解的研究[J]. 华南农业大学学报, 2019,40(1):84-91.
	Chen P, Liu SS, Wang Y, et al. Screening and identification of a cellulase-producing strain and itsdegradation of rice straw[J]. Journal of South China Agricultural University, 2019,40(1):84-91.
[17]	陈俊梅, 李文鹏, 赵素雅, 等. 蠼螋肠道中碱性内切葡聚糖酶基因的克隆表达及功能分析[J]. 微生物学报, 2019,59(9):1798-1812.
	Chen JM, Li WP, Zhao SY, et al. Cloning, expression and function of an alkaline resistance endo-glucanase gene isolated from guts of earwig[J]. Acta Microbiologica Sinica, 2019,59(9):1798-1812.
[18]	陈晶晶, 王伏伟, 刘曼, 等. 土壤中纤维素降解真菌的筛选及其纤维素酶活性的研究[J]. 安徽农业大学学报, 2014,41(4):654-661.
	Chen JJ, Wang FW, Liu M, et al. Isolation of cellulose-decomposing fungi from the soil and analyzing its cellulase activity[J]. Journal of Anhui Agricultural University, 2014,41(4):654-661.
[19]	刘晔, 刘晓丹, 张林利, 等. 花生根际多功能高效促生菌的筛选鉴定及其效应研究[J]. 生物技术通报, 2017,33(10):125-134.
	Liu Y, Liu XD, Zhang LL, et al. Screening, identification of multifunctional peanut root-promoting rhizobacteria and its promoting effects on peanuts[J]. Biotechnology Bulletin, 2017,33(10):125-134.
[20]	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.
	Dong XZ, Cai MY. Common bacterial system identification manual[M]. Beijing: Science Press, 2001.
[21]	布坎南 RE, 吉本斯 NE. 伯杰细菌鉴定手册[M]. 第8 版. 北京: 科学出版社, 1984.
	Buchanan RE, Gibbons NE. Bergey's manual of systemaic bacteriology[M]. 8th Edition. Beijing: Science Press, 1984.
[22]	徐平, 李文均, 徐丽华, 等. 微波法快速提取放线菌基因组DNA[J]. 微生物学通报, 2003,30(4):82-84.
	Xu P, Li WJ, Xu LH, et al. A microwave-based method for genomic DNA extraction from actinomycetes[J]. Microbiology China, 2003,30(4):82-84.
[23]	卢云峰, 许航, 段华泰, 等. 叶面喷施含Bacillus amyloliquefa-ciens SQR9复合微生物液体肥料的肥效研究[J]. 土壤, 2019,51(5):903-909.
	Lu YF, Xu H, Duan HT, et al. Foliar spraying of liquid compound microbial fertilizer containing Bacillus amyloliquefaciens SQR9 promoted plant growth[J]. Soils, 2019,51(5):903-909.
[24]	韦中, 徐春淼, 郑海平, 等. “挂壁”法筛选常温稻秆腐解菌及其降解能力研究[J]. 农业环境科学学报, 2015,34(10):2027-2031.
	Wei Z, Xu CM, Zheng HP, et al. Degradation of rice straw by degrading strains isolated by a “hanging-enrichment” method[J]. Journal of Agro-Environment Science, 2015,34(10):2027-2031.
[25]	李林超, 张超, 董庆, 等. 堆肥过程中纤维素降解菌的分离与鉴定[J]. 生物技术通报, 2019,35(9):165-171.
	Li LC, Zhang C, Dong Q, et al. Isolation and identification of cellulose degrading microorganisms in composting process[J]. Biotechnology Bulletin, 2019,35(9):165-171.
[26]	Liang Y, Zhang Z, Wu M, et al. Isolation, screening, and identification of cellulolytic bacteria from natural reserves in the subtropical region of China and optimization of cellulase production by Paenibacillus terrae ME27-1[J]. Biomed Research International, 2014: 1-13.
[27]	王奇, 王林风, 闫德冉, 等. 玉米秸秆酶水解过程中的酶复配条件优化[J]. 生物技术通报, 2016,32(3):171-177.
	Wang Q, Wang LF, Yan DR, et al. The optimization of enzyme complex formulation for enzymatic hydrolysis of corn stalk[J]. Biotechnology Bulletin, 2016,32(3):171-177.
[28]	于慧娟, 郭夏丽. 秸秆降解菌的筛选及其纤维素降解性能的研究[J]. 生物技术通报, 2019,35(2):58-63.
	Yu HJ, Guo XL. Screening of straw-degrading bacteria and study on their cellulose-degrading performance[J]. Biotechnology Bulletin, 2019,35(2):58-63.
[29]	苑博华, 廖祥儒, 郑晓洁, 等. 吲哚乙酸在植物细胞中的代谢及其作用[J]. 生物学通报, 2005,40(4):21-23.
	Yue BH, Liao XR, Zheng XJ, et al. Metabolism and function of indole acetic acid in plant cells[J]. Biological Bulletin, 2005,40(4):21-23.
[30]	刘小龙, 芦云, 罗明, 等. 3种旱生禾草内生固氮菌的分离及促生性能测定[J]. 草地学报, 2012, 20(4):759-761,763, 765-767.
	Liu XL, Lu Y, Luo M, et al. Isolating endophytic diazotrophic bacteria from three xerophil gramineae grasses to determine their nitrogen fixation and plant growth-promoting[J]. Acta Agrestia Sinica, 2012, 20(4):759-761,763, 765-767..
[31]	孙雪, 董永华, 王娜, 等. 耐盐碱促生菌的筛选及性能[J]. 生物工程学报, 2020,36(7):1356-1364.
	Sun X, Dong YH, Wang N, et al. Screening and evaluation of saline-alkali-tolerant and growth-promoting bacteria[J]. Chinese Journal of Biotechnology, 2020,36(7):1356-1364.
[32]	韩如月, 李睿瑞, 杨帆, 等. 一株水稻根内生拮抗细菌SM13的分离及鉴定[J]. 微生物学通报, 2019,46(5):1030-1040.
	Han RY, Li RR, Yang F, et al. Isolation and identification of an endophytic and antagonistic bacterium from rice roots[J]. Microbiology China, 2019,46(5):1030-1040.
[33]	蒋先军, 黄昭贤, 谢德体, 等. 硅酸盐细菌代谢产物对植物生长的促进作用[J]. 西南农业大学学报:自然科学版, 2000,22(2):116-119.
	Jiang XJ, Huang ZX, Xie DT, et al. Promoting effects of the metabolites of silicate for plant growth[J]. Journal of Southwest Agricultural University, 2000,22(2):116-119.
[34]	吴凯朝, 梁俊, 韦莉萍, 等. 内生固氮菌对甘蔗伸长期光合生理特性的影响[J]. 广西植物, 2011,31(5):668-673.
	Wu KC, Liang J, Wei LP, et al. Effects of nitrogen fixing bacteria on photosynjournal and chlorophyll fluorescence in sugarcane at e longating stage[J]. Guihaia, 2011,31(5):668-673.
[35]	席琳乔, 姚拓, 杨俊基, 等. 联合固氮菌株分泌能力及其对燕麦的促生效应测定[J]. 草原与草坪, 2005(4):25-29.
	Xi LQ, Yao T, Yang JJ, et al. Porperty of associative nitrogen-fixing bacteria producing IAA and its promoting growth of oat[J]. Grassland and Turf, 2005(4):25-29.
[36]	张辉, 王会方, 李春华, 等. 外源压力对甘薯生长和生理特征的影响[J]. 江苏师范大学学报:自然科学版, 2018,36(4):36-43.
	Zhang H, Wang HF, Li CH, et al. Effect of exogenous pressure on growth and physiological characteristics of sweetpotat[J]. Journal of Jiangsu Normal University:Natural Science Edition, 2018,36(4):36-43.
[37]	喻江, 于镇华, 刘晓冰, 等. 植物根组织内生细菌多样性及其促生作用[J]. 中国农学通报, 2015,31(13):169-175.
	Yu J, Yu ZH, Liu XB, et al. Diversity and growth promoting effects of endophytic bacteria in plant root[J]. Chinese Agricultural Science Bulletin, 2015,31(13):169-175.
[38]	王辰, 张谷月, 张园园, 等. 白刺链霉菌(Streptomyces albospinus)CT205菌株发酵条件优化及其次生代谢产物性质研究[J]. 南京农业大学学报, 2015,38(2):304-310.
	Wang C, Zhang GY, Zhang YY, et al. The optimization of fermentation conditions of Streptomyces albospinus CT205 and study of secondary metabolite[J]. Journal of Nanjing Agricultural University, 2015,38(2):304-310.
[39]	Anandham R, Heo J, Krishnamoorthy R, et al. Azospirillum ramasamyi sp. nov., a novel diazotrophic bacterium isolated from fermented bovine products[J]. International Journal of Systematic and Evolutionary Microbiology, 2019,69(5):1369. doi: 10.1099/ijsem.0.003320 URL pmid: 30810523
[40]	Han LC, Hu P, Yang GQ, et al. Azospirillum humicireducens sp. nov., a nitrogen-fixing bacterium isolated from a microbial fuel cell[J]. Int J Syst Evol Microbiol, 2013,63(7):2618-2624.
[41]	王聪, 凌娟, 张燕英, 等. 海洋固氮菌和解磷菌的分离鉴定及发酵条件优化[J]. 微生物学报, 2018,58(5):817-829.
	Wang C, Ling J, Zhang YY, et al. Isolation, characterization and culture optimization of nitrogen-fixing and phosphate-solubilizing bacteria from rhizosphere sediments of Halophila ovalis[J]. Acta Microbiologica Sinica, 2018,58(5):817-829.
[42]	王霞, 华琳, 张海龙, 等. 纤维素降解菌CMC-4的分离鉴定、诱变和酶学特性研究[J]. 土壤, 2017,49(5):919-925.
	Wang X, Hua L, Zhang HL, et al. Isolation, identification, mutagenesis of cellulose-degrading bacterium CMC-4 and its enzymatic properties[J]. Soils, 2017,49(5):919-925.
[43]	张东艳, 刘晔, 吴越, 等. 花生根际产IAA菌的筛选鉴定及其效应研究[J]. 中国油料作物学报, 2016,38(1):104-110.
	Zhang DY, Liu Y, Wu Y, et al. Isolation and identification of IAA-producing strains from peanut rhizosphere and its promoting effects on peanut growth[J]. Chinese Journal of Oil Crop Sciences, 2016,38(1):104-110.
[44]	Sheng P, Huang S, Wang Q, et al. Isolation, screening, and optimization of the fermentation conditions of highly cellulolytic bacteria from the hindgut of Holotrichia parallela larvae(Coleoptera:Scarabaeidae)[J]. Applied Biochemistry and Biotechnology, 2012,167(2):270-284. URL pmid: 22544686
[45]	Jiang Y, Li P, Wu Y, et al. A new isolated indole-3-acetic acid producing Rhizobacteria Bacillus muralis HS4 from sandy soil:characterization and growth enhancement on peanuts[J]. J Comput Theor Nanosci, 2016,13(8):5171-5177.

项目	结果	项目	结果
明胶液化	-	柠檬酸盐利用	-
V-P试验	-	淀粉水解	-
M.R.反应	-	好氧性试验	好氧
接触酶试验	+	革兰氏染色	-

项目	结果	项目	结果
明胶液化	-	柠檬酸盐利用	-
V-P试验	-	淀粉水解	-
M.R.反应	-	好氧性试验	好氧
接触酶试验	+	革兰氏染色	-

处理	秸秆降解率/%	玉米生长
处理	秸秆降解率/%	SPAD值	平均直径/mm	根长/cm	根表面积/cm²	株高/cm	地上部鲜重/g
CK	9.76±0.90	40.2±1.62	0.41±0.04	23.5±2.29	111.5±23.0	51.8±1.25	3.91±0.42
n3	15.1±0.12**	42.3±0.82*	0.50±0.05*	27.8±5.02*	124.3±43.9	54.4±1.71	4.12±0.25

处理	秸秆降解率/%	玉米生长
处理	秸秆降解率/%	SPAD值	平均直径/mm	根长/cm	根表面积/cm²	株高/cm	地上部鲜重/g
CK	9.76±0.90	40.2±1.62	0.41±0.04	23.5±2.29	111.5±23.0	51.8±1.25	3.91±0.42
n3	15.1±0.12**	42.3±0.82*	0.50±0.05*	27.8±5.02*	124.3±43.9	54.4±1.71	4.12±0.25