一株纤维素降解菌的筛选与鉴定

doi:10.11924/j.issn.1000-6850.casb2021-0131

中国农学通报 ›› 2021, Vol. 37 ›› Issue (17): 26-31.doi: 10.11924/j.issn.1000-6850.casb2021-0131

所属专题：生物技术

一株纤维素降解菌的筛选与鉴定

杨娜¹^,²(), 何鑫³, 杜春梅¹^,²()

¹黑龙江大学农业微生物技术教育部工程研究中心,哈尔滨 150500
²黑龙江大学生命科学学院/黑龙江省普通高校微生物重点实验室,哈尔滨 150080
³河北环境工程学院/河北省农业生态安全重点实验室,河北秦皇岛 066102

收稿日期:2021-02-05 修回日期:2021-05-05 出版日期:2021-06-15 发布日期:2021-06-29
通讯作者: 杜春梅
作者简介:杨娜,女,1996年出生,黑龙江哈尔滨人,研究生,研究方向：微生物资源挖掘与利用。通信地址：150080 黑龙江省哈尔滨市南岗区学府路74号黑龙江大学生命科学学院501室,Tel：0451-86609134,E-mail：840328012@qq.com。
基金资助:
河北省重点研发计划项目“多种新型生物肥料产品生产菌种及技术研究”(18222906D);国家自然科学基金“侧孢短芽孢杆菌与枯草芽孢杆菌共培养抗菌活性增强的生态学机理”(31370511);国家重点研发计划项目子课题“枯草芽孢杆菌脂肽高产菌株的选育及发酵条件优化”(2017YFD0201101)

Screening and Identification of a Cellulose Degrading Strain

Yang Na¹^,²(), He Xin³, Du Chunmei¹^,²()

¹Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150500
²Key Laboratory of Microbiology, College of Heilongjiang Province, School of Life Sciences, Heilongjiang University, Harbin 150080
³Hebei Key Laboratory of Agroecological Safety, Hebei University of Environmental Engineering, Qinhuangdao Hebei 066102

Received:2021-02-05 Revised:2021-05-05 Online:2021-06-15 Published:2021-06-29
Contact: Du Chunmei

摘要/Abstract

摘要：

为了解决玉米秸秆降解率低下问题,本研究从以氨基酸尾液为氮源的自然发酵堆肥中筛选稳定的纤维素降解菌,为制备高效的秸秆腐熟剂奠定基础。采用刚果红染色法进行初筛、采用纤维素酶活测定及玉米秸秆降解率测定来进行复筛,并通过形态学及分子生物学方法对高效降解菌株进行鉴定。结果表明筛选得到一株高效降解纤维素的菌株SC2,其滤纸酶活、内切葡聚糖酶活、外切葡聚糖酶活和β-葡聚糖苷酶活分别为17.70、58.97、16.85和79.26 U/mL,对玉米秸秆的降解率达到33.07%,根据其菌落特征、产孢结构、孢子形态及ITS序列鉴定SC2为黑曲霉(Aspergillus niger)。菌株SC2具有较好的纤维素降解能力,能够有效的促进秸秆的降解,可以用于制备玉米秸秆腐熟剂。

关键词: 玉米秸秆, 玉米秸秆纤维素, 纤维素降解菌, 降解, 酶活, 筛选, 鉴定

Abstract:

To solve the problem of low degradation rate of corn straw, stable cellulose degrading bacteria were screened from natural fermentation compost with amino acid tail liquor as the nitrogen source, aiming to lay a foundation for the preparation of efficient straw decomposition agent. Congo red staining method was adopted for preliminary screening, the determination of cellulase activity and the loss rate of corn straw weight were used for re-screening, and morphological and molecular biological methods were used to identify the efficient degrading strain. The results showed that an efficient cellulose degrading strain, SC2, was obtained. The filter paper enzyme activity, endoglucanase activity, exoglucanase activity and β-glucosidase activity of SC2 was 17.70 U/mL, 58.97 U/mL, 16.85 U/mL and 79.26 U/mL, respectively. The degradation rate of corn straw reached 33.07%. According to the colony characteristics, sporulation structure, spore morphology and ITS sequence, SC2 was identified as Aspergillus niger. Strain SC2 has good cellulose degradation ability, can effectively promote the degradation of straw and be used to prepare corn straw decomposing agent.

Key words: corn straw, corn straw cellulose, cellulose degradation, degradation, enzyme activity, screening, identification

中图分类号:

S182

杨娜, 何鑫, 杜春梅. 一株纤维素降解菌的筛选与鉴定[J]. 中国农学通报, 2021, 37(17): 26-31.

Yang Na, He Xin, Du Chunmei. Screening and Identification of a Cellulose Degrading Strain[J]. Chinese Agricultural Science Bulletin, 2021, 37(17): 26-31.

图/表 5

菌株编号	透明圈直径(D)/mm	菌落直径(d)/mm	透明圈直径/菌落直径(D/d)
ND9	5.32±0.12	2.81±0.10	1.89 b
ND19	10.26±0.30	3.62±0.20	2.83 a
NC2	8.02±0.37	6.79±0.22	1.18 e
NC4	10.79±0.38	7.68±0.19	1.4 d
SC2	31.80±0.36	21.77±0.27	1.46 c
SC4	55.71±0.51	47.01±0.26	1.19 e

表1. 刚果红染色试验结果

图1. 不同菌株4种纤维素酶的活力

图2. 不同菌株对玉米秸秆的降解率

图3. 菌株SC2显微形态及菌落形态 A-菌落正面;B-菌落反面;C-孢子梗及孢子(×400)

图4. 菌株SC2的系统发育树

参考文献 33

[1]	Li F H, Ding Z T, Ke W C, et al. Ferulic acid esterase-producing lactic acid bacteria and cellulase pretreatments of corn stalk silage at two different temperatures: Ensiling characteristics, carbohydrates composition and enzymatic saccharification[J]. Bioresource Technology, 2019,282:211-221. doi: 10.1016/j.biortech.2019.03.022 URL
[2]	Singh J, Suhag M, Dhaka A. Augmented digestion of lignocellulose by steam explosion, acid and alkaline pretreatment methods: A review[J]. Carbohydr Polym, 2015,117:624-631. doi: 10.1016/j.carbpol.2014.10.012 URL
[3]	Ma L L, Lu Y Y, Yan H, et al. Screening of cellulolytic bacteria from rotten wood of Qinling (China) for biomass degradation and cloning of cellulases from Bacillus methylotrophicus[J]. BMC Biotechnology, 2020,20(6):1-13. doi: 10.1186/s12896-019-0592-9 URL
[4]	Shakoor S, Aftab S, Rehman A. Characterization of cellulose degrading bacterium, Bacillus megaterium S3, isolated from indigenous environment[J]. Pakistan Journal of Zoology, 2013,45(6):1655-1662.
[5]	Krishna M, Guder D G. Isolation and characterization of potential cellulose degrading bacteria from sheep rumen[J]. Journal of Pure and Applied Microbiology, 2019,13(3):1831-1839. doi: 10.22207/JPAM URL
[6]	Sun S N, Sun S L, Cao X F, et al. The role of pretreatment in improving the enzymatic hydrolysis of lignocellulosic materials[J]. Bioresource Technology, 2016,199:49-58. doi: 10.1016/j.biortech.2015.08.061 URL
[7]	Carlos M, Bruna S, Betsy M, et al. A metagenomic advance for the cloning and characterization of a cellulase from red rice crop residues[J]. Molecules, 2016,21(7):1-12. doi: 10.3390/molecules21010001 URL
[8]	宋文芳, 胡国全, 徐彦胜. 一株低温厌氧纤维素降解细菌的分离、鉴定及其酶学性质的研究[J]. 西南农业学报, 2011,24(4):1317-1322.
[9]	Bansal N, Tewarir R, Soni R, et al. Production of cellulases from Aspergillus niger NS-2 in solid state fermentation on agricultural and kitchen waste residues[J]. Waste Management, 2012,32(7):1341-1346. doi: 10.1016/j.wasman.2012.03.006 URL
[10]	Egwuatu T F, Appeh O G. Isolation and characterization of filter paper degrading bacteria from the guts of coptotermes formosanus[J]. Journal of Bioremediation & Biodegradation, 2018,9(3):1-5.
[11]	Milala M A, Shugaba A, Gidado A, et al. Studies on the use of agricultural wastes for cellulase enzyme production by Aspegillus niger[J]. Research Journal of Agriculture and Biological Sciences, 2005,1(4):325-328.
[12]	Schwarz W H. The cellulosome and cellulose degradation by anaerobic bacteria[J]. Applied Microbiology & Biotechnology, 2001,56(5-6):634-649.
[13]	Rastogi G, Muooidi G L, Gurram R N, et al. Isolation and characterization of cellulose-degrading bacteria from the deep subsurface of the Homestake gold mine, Lead, South Dakota, USA[J]. Journal of Industrial Microbiology & Biotechnology, 2009,36(4):585-598.
[14]	Karthika A, Seenivasagan R, Kasimani R, et al. Cellulolytic bacteria isolation, screening and optimization of enzyme production from vermicompost of paper cup waste[J]. Waste Management, 2020,116:58-65. doi: 10.1016/j.wasman.2020.06.036 URL
[15]	Farjana I, Naranay R. Screening, purification and characterization of cellulase from cellulase producing bacteria in molasses[J]. BMC Research Notes, 2018,11(1):445-451. doi: 10.1186/s13104-018-3558-4 pmid: 29973263
[16]	张爱梅, 殷一然, 齐汝楠. 产纤维素酶沙棘根瘤内生放线菌的筛选,鉴定及其酶活性测定[J]. 西北师范大学学报:自然科学版, 2019,208(5):75-80,96.
[17]	刘晓飞, 宋洁, 马京求, 等. 产纤维素酶放线菌的筛选鉴定及其对玉米秸秆的降解[J]. 精细化工, 2020,37(8):1657-1664,1671.
[18]	张健, 李燕婷, 袁亮, 等. 氨基酸发酵尾液可促进樱桃番茄对水溶肥料氮素的吸收利用[J]. 植物营养与肥料学报, 2018,24(1):114-121.
[19]	陈莉. 绿色木霉固态发酵产纤维素酶条件研究[J]. 安徽农业科学, 2013,41(7):2823-2825.
[20]	李晓秀, 张盼, 刘琬瑜, 等. 玉米秸秆降解真菌的筛选及鉴定[J]. 饲料工业, 2017,38(3):37-42.
[21]	陈建爱, 贾梦莹, 陈为京. PDA中水含量对固体培养黄绿木霉T1010分生孢子生长发育的影响[J]. 中国农学通报, 2020,558(15):112-120.
[22]	李正风, 李岩, 朱杰, 等. 烟草秸秆中产纤维素酶细菌筛选,鉴定及酶活测定[J]. 西南农业学报, 2020,33(3):645-650.
[23]	许玉林, 郑月霞, 叶冰莹, 等. 一株纤维素降解真菌的筛选及鉴定[J]. 微生物学通报, 2013,40(2):220-227.
[24]	Azhar A H, Mohamed S A, Hoda H A, et al. Optimization and molecular identification of novel cellulose degrading bacteria isolated from Egyptian environment[J]. Journal of Genetic Engineering and Biotechnology, 2017,15:77-85. doi: 10.1016/j.jgeb.2017.02.007 URL
[25]	Gupta P, Samant K, Sahu A. Isolation of cellulose-degrading bacteria and determination of their cellulolytic potential[J]. International Journal of Microbiology, 2012,2012(6):1-5.
[26]	黄亚丽, 黄媛媛, 马慧媛, 等. 低温秸秆降解真菌的筛选及在秸秆还田中的应用[J]. 中国农学通报, 2020,36(21):53-60.
[27]	Sun Y, Qu J B, Li R L, et al. Optimization of the enzyme production conditions of Bacillus licheniformis and its effect on the degradation of corn straw[J]. Journal of Biobased Materials and Bioenergy, 2018,12(5):432-440. doi: 10.1166/jbmb.2018.1795 URL
[28]	张冬雪, 文亚雄, 罗志威, 等. 纤维素降解菌的分离筛选及其对水稻秸秆的降解效果分析[J]. 江西农业学报, 2020,32(1):76-80.
[29]	王勇, 张育铭, 朱洪磊, 等. 高效纤维素降解菌的筛选及产酶活力测定[J]. 江苏农业科学, 2020,48(23):255-260.
[30]	孙美娜, 张凡凡, 王永强, 等. 棉花秸秆纤维素降解菌的筛选鉴定与降解棉秆效果研究[J]. 新疆农业科学, 2018,55(1):16-23.
[31]	李思杨. 不同氮源和菌剂处理对水稻秸秆腐解规律及土壤养分的影响[D]. 沈阳:沈阳农业大学, 2019.
[32]	Gong X, Zou H, Qian C, et al. Construction of in situ degradation bacteria of corn straw and analysis of its degradation efficiency[J]. Annals of Microbiology, 2020,70(1):1-15. doi: 10.1186/s13213-020-01552-1 URL
[33]	李静, 张瀚能, 赵翀, 等. 高效纤维素降解菌分离筛选、复合菌系构建及秸秆降解效果分析[J]. 应用与环境生物学报, 2016,22(4):689-696.

一株纤维素降解菌的筛选与鉴定

Screening and Identification of a Cellulose Degrading Strain

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 33

相关文章 15

编辑推荐

Metrics

本文评价

关于本刊

作者中心

审者中心

在线期刊

联系我们

[1]	巩永永, 端木慧子. 甜菜TIFY基因家族的全基因组鉴定与生物信息学分析[J]. 中国农学通报, 2022, 38(8): 17-24.
[2]	刘颖, 耿丹丹, 韩永胜, 魏敏, 刘柳. 环保型农林保水剂研制、性能与应用[J]. 中国农学通报, 2022, 38(7): 86-90.
[3]	吴佳佳, 朱佳红, 曹坳程, 颜冬冬, 王秋霞, 张春华, 李园. 云南省草果主要病原真菌鉴定及防治药剂筛选[J]. 中国农学通报, 2022, 38(6): 121-128.
[4]	陈瑞英, 赵培荣, 刘宏金, 张雷, 郭晓宇. 可降解地膜在马铃薯上的应用效果研究[J]. 中国农学通报, 2022, 38(6): 37-41.
[5]	商娜, 任爱芝, 刘冰, 赵培宝. 白三叶草一种叶斑病病原菌的分离鉴定[J]. 中国农学通报, 2022, 38(4): 81-85.
[6]	黄磊, 李艺萌, 钟帅, 金保锋, 高晋军, 王晓园, 关罗浩, 王初亮, 黄妧婷, 李敏. 低氧贮存片烟解封后理化指标和质量变化研究[J]. 中国农学通报, 2022, 38(4): 107-112.
[7]	肖阳, 李庆荣, 邢东旭, 杨琼. 高温胁迫对耐受性不同的家蚕品种幼虫抗氧化酶活与基因表达的影响[J]. 中国农学通报, 2022, 38(35): 111-118.
[8]	吴芳, 冯立国, 姜性坚, 石跃龙, 王春晖, 黄晓辉, 喻初权. 洞庭湖区3种野生食用真菌生境调查与鉴定[J]. 中国农学通报, 2022, 38(34): 27-32.
[9]	程璐, 文永莉, 程曼. UV-B辐射增强对陆地生态系统温室气体排放影响的研究进展[J]. 中国农学通报, 2022, 38(33): 80-88.
[10]	杨艺炜, 刘晨, 魏佩瑶, 李英梅. 陕西省甘薯病毒病害(SPVD)发生现状及种类鉴定[J]. 中国农学通报, 2022, 38(33): 109-115.
[11]	努尔夏提·努尔买买提, 李焕宇, 杨成德. 辣椒根腐病菌拮抗链霉菌的筛选及16S rRNA基因系统发育分析[J]. 中国农学通报, 2022, 38(33): 116-123.
[12]	番华彩, 曾莉, 李卫雁, 丁云秀, 郭志祥, 李舒, 徐胜涛, 郑泗军, 王永斌. 香蕉细菌性软腐病防控药剂筛选及田间应用效果评价[J]. 中国农学通报, 2022, 38(31): 113-118.
[13]	曹永清, 刘艳, 张丽慧, 晋婷婷, 任嘉红. 荧光假单胞CLW17菌株对草甘膦的降解及其机制初探[J]. 中国农学通报, 2022, 38(30): 108-117.
[14]	魏日凤, 彭成彬, 陈美霞, 张政雄, 阮俊峰, 刘伟. 柳叶蜡梅真菌病害分离鉴定及生物学特性[J]. 中国农学通报, 2022, 38(3): 127-133.
[15]	刘淑娟, 张翠萍, 李淑英, 杨小燕, 周元清, 李元. 草本植物根际微生物降解地表水环境邻苯二甲酸酯的研究[J]. 中国农学通报, 2022, 38(3): 44-51.