一株降解纤维素的低温放线菌Streptomyces azureus及产酶条件优化

doi:10.11924/j.issn.1000-6850.casb2021-0107

中国农学通报 ›› 2021, Vol. 37 ›› Issue (32): 25-33.doi: 10.11924/j.issn.1000-6850.casb2021-0107

所属专题：生物技术

一株降解纤维素的低温放线菌Streptomyces azureus及产酶条件优化

李婷¹^,²(), 王玥¹^,², 刘中珊³, 刘奇³, 徐赫男³, 李冲伟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 150080
²黑龙江大学生命科学学院黑龙江省寒地生态修复与资源利用重点实验室,哈尔滨 150500
³哈尔滨华美亿丰复合材料有限公司,哈尔滨 150500

收稿日期:2021-02-01 修回日期:2021-04-13 出版日期:2021-11-15 发布日期:2022-01-07
通讯作者: 李冲伟
作者简介:李婷,女,1996年出生,黑龙江哈尔滨人,在读硕士,研究方向：生态修复。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,Tel：0451-86609453,E-mail： 761983600@qq.com。
基金资助:
黑龙江省自然科学基金团队项目“寒区农业土壤面源污染微生物调控技术的研究”(TD2019C002);校企合作项目“利用卧旋式好氧连续发酵设备对有机废弃物进行好氧发酵的机理及应用”(0926-09019应用)

A Novel Low Temperature Cellulose-degrading Strain Streptomyces azureus and Its Enzymatic Production Condition Optimization

Li Ting¹^,²(), Wang Yue¹^,², Liu Zhongshan³, Liu Qi³, Xu Henan³, Li Chongwei¹^,²()

¹Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150080
²Heilongjiang Key Laboratory of Ecological Restoration and Resource Utilization for Cold Region, School of Life Sciences, Heilongjiang University, Harbin 150500
³Harbin Huamei Yifeng Composite Materials Co., Ltd., Harbin 150500

Received:2021-02-01 Revised:2021-04-13 Online:2021-11-15 Published:2022-01-07
Contact: Li Chongwei

摘要/Abstract

摘要：

为了筛选一株可在低温堆肥中高效降解纤维素的放线菌,提高低温条件下纤维素的降解率,本实验采用刚果红染色法（水解圈法）和3,5-二硝基水杨酸(DNS)法从红松混交林凋落物层筛选一株低温纤维素降解菌,并对该微生物进行生理生化和分子生物学鉴定。结果表明,筛选到一株低温降解纤维素效果较好的菌种,命名为T23-B,经鉴定该菌株为远青链霉菌(Streptomyces azureus),可以在15℃生长。响应面法优化了该菌株纤维素酶最佳生产条件为甘露糖15.6 g/L,酵母浸粉7.8 g/L,初始pH 5.2,培养时间96 h,摇床转速160 r/min,培养温度35℃,最适产酶条件下纤维素酶活高达123.43 U/mL,较优化前提高了4.4倍。因此,本研究为T23-B应用于北方冬季堆肥发酵提供了理论基础和实践依据,对提高农业废弃物资源利用和保护寒地生态环境具有重要意义。

关键词: 纤维素降解菌, 低温菌, 堆肥, 纤维素酶, 条件优化

Abstract:

To screen an actinomycete which can efficiently degrade cellulose in low temperature composting, and improve the degradation rate of cellulose under low temperature, a strain from litter layer of mixed forest of Korean pine was screened with Congo red staining (hydrolysis circle method) and 3,5-Dinitrosalicylic acid (DNS) method. The physiological, biochemical and molecular identification of the strain was carried out. The results showed that a strain which could degrade cellulose effectively at low temperature were named T23-B, identified as Streptomyces Azureus, and could grow at 15℃. Response surface method was performed to optimize production condition of cellulose as mannose of 15.6 g/L, yeast extract of 7.8 g/L, initial pH of 5.2, culture time of 96 h, shaking speed of 160 r/min, and culture temperature of 35℃. The cellulase activity could reach 123.43 U/mL which was 4.4 times higher than that before the optimization. Therefore, this study provides a theoretical and practical basis for applying T23-B to winter compost fermentation. It is of great significance for improve the utilization of agricultural waste resources and protect the ecological environment in cold region.

Key words: cellulose degrading bacteria, low temperature bacteria, composting, cellulase, condition optimization

中图分类号:

李婷, 王玥, 刘中珊, 刘奇, 徐赫男, 李冲伟. 一株降解纤维素的低温放线菌Streptomyces azureus及产酶条件优化[J]. 中国农学通报, 2021, 37(32): 25-33.

Li Ting, Wang Yue, Liu Zhongshan, Liu Qi, Xu Henan, Li Chongwei. A Novel Low Temperature Cellulose-degrading Strain Streptomyces azureus and Its Enzymatic Production Condition Optimization[J]. Chinese Agricultural Science Bulletin, 2021, 37(32): 25-33.

图/表 9

参考文献 32

[1]	Wen Y C, Kit W C, Cheng F L, et al. Sustainable utilization of biowaste compost for renewable energy and soil amendments[J]. Environmental Pollution, 2020, 267:115662. doi: 10.1016/j.envpol.2020.115662 URL
[2]	Zheng J X, Liu J B, Han S H, et al. N₂O emission factors of full-scale animal manure windrow composting in cold and warm seasons[J]. Bioresource technology, 2020, 316:123905. doi: 10.1016/j.biortech.2020.123905 URL
[3]	Chen X M, Cheng W T, Li S Z, et al. The “quality” and “quantity” of microbial species drive the degradation of cellulose during composting[J]. Bioresource Technology, 2021, 320(PB):124425. doi: 10.1016/j.biortech.2020.124425 URL
[4]	Zhao L, Gu W M, Shao L M, et al. Sludge Bio-drying Process at Low Ambient Temperature: Effect of Bulking Agent Particle Size and Controlled Temperature[J]. Drying Technology, 2012, 30(10):1037-1044. doi: 10.1080/07373937.2012.665113 URL
[5]	Strom P F. Effect of temperature on bacterial species diversity in thermophilic solid-waste composting[J]. Applied and environmental microbiology, 1985, 50(4):899-905. doi: 10.1128/aem.50.4.899-905.1985 pmid: 4083885
[6]	Yousif A Y, Li T Z, Xi C, et al. Role of psychrotrophic fungal strains in accelerating and enhancing the maturity of pig manure composting under low-temperature conditions[J]. Bioresource Technology, 2021, 320(PB):124402. doi: 10.1016/j.biortech.2020.124402 URL
[7]	Gerday C, Aittaleb M, Bentahir M, et al. Cold-adapted enzymes: from fundamentals to biotechnology[J]. Trends in Biotechnology, 2000, 18(3):103-107. pmid: 10675897
[8]	Xie X Y, Zhao Y, Sun Q H, et al. A novel method for contributing to composting start-up at low temperature by inoculatng cold-adapted microbial consortium[J]. Bioresource Technology, 2017, 238:39-47. doi: 10.1016/j.biortech.2017.04.036 URL
[9]	Li C N, Li H Y, Yao T, et al. Effects of microbial inoculation on enzyme activity, available nitrogen content, and bacterial succession during pig manure composting[J]. Bioresource Technology, 2020, 306:123167. doi: 10.1016/j.biortech.2020.123167 URL
[10]	Yao Y, Huang G, An C J, et al. Anaerobic digestion of livestock manure in cold regions: Technological advancements and global impacts[J]. Renewable and Sustainable Energy Reviews, 2020, 119:109494. doi: 10.1016/j.rser.2019.109494 URL
[11]	Zhao Y, Zhao Y, Zhang Z C, et al. Effect of thermo-tolerant actinomycetes inoculation on cellulose degradation and the formation of humic substances during composting[J]. Waste Management, 2017, 68:64-73. doi: S0956-053X(17)30463-4 pmid: 28647221
[12]	Wei Y Q, Wu D, Wei D, et al. Improved lignocellulose-degrading performance during straw composting from diverse sources with actinomycetes inoculation by regulating the key enzyme activities[J]. Bioresource Technology, 2019, 271:66-74. doi: 10.1016/j.biortech.2018.09.081 URL
[13]	Cuesta G, Rosana G, Abad M, et al. Isolation and identification of actinomycetes from a compost-amended soil with potential as biocontrol agents[J]. Journal of Environmental Management, 2012, 95:S280-S284. doi: 10.1016/j.jenvman.2010.11.023 URL
[14]	吴静. 高产纤维素酶霉菌的筛选及纤维素酶系的分离纯化[D]. 贵阳:贵州大学, 2020.
[15]	沈大春. 秸秆堆肥降解菌株分离及降解稻秆效果研究[D]. 南京:南京农业大学, 2016.
[16]	Li F, Xie Y J, Gao X, et al. Screening of cellulose degradation bacteria from Min pigs and optimization of its cellulase production[J]. Electronic Journal of Biotechnology, 2020, 48:29-35. doi: 10.1016/j.ejbt.2020.09.001 URL
[17]	Hussain A A, 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
[18]	Kelly J, Kutscher A H, Tuoti A F. Thiostrepton, a new antibiotic: tube dilution sensitivity studies[J]. Oral Surg. Oral Med. Oral Pathol, 1959, 12:1334. doi: 10.1016/0030-4220(59)90222-1 URL
[19]	Dutcher J D, Vandeputt J T. A new antibiotic. II. Isolation and chemical characterization[J]. Antibiotics Annual, 1955, 3:560-561.
[20]	Jambor W P, Steinberg B A, Suydam L O. Thiostrepton, a new antibiotic. III. In vivo studies[J]. Antibiotics Annual, 1955, 3:562-565.
[21]	Sakihara K, Maeda J, Tashiro K, et al. Draft genome sequence of thiostrepton-producing Streptomyces azureus ATCC 14921[J]. Genome Announc, 2015, 3(5):e01183-15.
[22]	汪健. 硫链丝菌素Thiostrepton的优化设计及其对口腔致病菌的抗菌活性检测[D]. 上海:上海交通大学, 2019.
[23]	陈单丹, 段盼盼, 刘文. 以活性结构单元的突变和修饰为思路的硫链丝菌素的生物合成途径改造[J]. 中国抗生素杂志, 2017, 42(03):189-197.
[24]	孙晓萌, 公维丽, 李欣, 等. 降解木质素放线菌的功能组学分析及工业应用前景[J]. 中国科学:生命科学, 2017(2):201-210.
[25]	李欣. 中药渣堆肥微生物群落演替机制研究[D]. 包头:内蒙古科技大学, 2019.
[26]	Cavicchioli R, Siddiqui K S, Andrews D, et al. Low-temperature extremophiles and their applications[J]. Current Opinion in Biotechnology, 2002, 13(3):253-261. pmid: 12180102
[27]	Imran M, Anwar Z, Irshad M, et al. Optimization of cellulase production from a novel strain of Aspergillus Tubingensis IMMIS2 through response surface methodology[J]. Biocatalysis and Agricultural Biotechnology, 2017, 12:191-198. doi: 10.1016/j.bcab.2017.10.005 URL
[28]	Li Q, Loman A A, Nicholas V C, et al. Leveraging pH profiles to direct enzyme production (cellulase, xylanase, polygalacturonase, pectinase, α-galactosidase, and invertase) by Aspergillus foetidus[J]. Biochemical Engineering Journal, 2018, 137:247-254. doi: 10.1016/j.bej.2018.06.008 URL
[29]	Sreena, Sebastian D. Augmented cellulase production by Bacillus subtilis strain MU S1 using different statistical experimental designs[J]. Journal of Genetic Engineering and Biotechnology, 2018, 16:9-16. doi: 10.1016/j.jgeb.2017.12.005 URL
[30]	Santosh K G, Kataki S, Chatterjee S, et al. Cold adaptation in bacteria with special focus on cellulase production and its potential application[J]. Journal of Cleaner Production, 2020, 258:120351. doi: 10.1016/j.jclepro.2020.120351 URL
[31]	Xu K W, Zou X T, Xue Y T, et al. The impact of seasonal variations about temperature and photoperiod on the treatment of municipal wastewater by algae-bacteria system in lab-scale[J]. Algal Research, 2021, 54:102175. doi: 10.1016/j.algal.2020.102175 URL
[32]	Chen L Y, Bai S Q, You M H, et al. Effect of a low temperature tolerant lactic acid bacteria inoculant on the fermentation quality and bacterial community of oat round bale silage[J]. Animal Feed Science and Technology, 2020, 269:114669. doi: 10.1016/j.anifeedsci.2020.114669 URL

ID	D/d	纤维素酶活力/(U/mL)
T13-3T	4.64±0.22b	21.12
N1-6L	3.00±0.16e	11.47
T23-B	5.40±0.37a	28.34
T13-5	4.20±0.24c	18.45
T23-3	3.73±0.19d	21.67
N4-3L	3.20±0.26e	14.32
T23-3L	3.00±0.21e	15.67

ID	D/d	纤维素酶活力/(U/mL)
T13-3T	4.64±0.22b	21.12
N1-6L	3.00±0.16e	11.47
T23-B	5.40±0.37a	28.34
T13-5	4.20±0.24c	18.45
T23-3	3.73±0.19d	21.67
N4-3L	3.20±0.26e	14.32
T23-3L	3.00±0.21e	15.67

生理生化特性	结果	生理生化特性	结果
革兰氏染色	+	硫化氢	-
糖酵解	-	甲基红	-
三糖解	+	靛基质	+
尿素酶	+	淀粉水解	+
明胶液化	+	Vp实验	+
触酶	-

生理生化特性	结果	生理生化特性	结果
革兰氏染色	+	硫化氢	-
糖酵解	-	甲基红	-
三糖解	+	靛基质	+
尿素酶	+	淀粉水解	+
明胶液化	+	Vp实验	+
触酶	-

试验序号	A.pH	B.氮源浓度/(g/mL)	C.碳源浓度/(g/mL)	纤维素酶活力/(U/mL)
1	0	0	0	124.12±1.12
2	0	1	1	103.71±0.89
3	1	0	1	112.43±0.76
4	-1	-1	0	97.85±0.98
5	0	0	0	120.19±0.83
6	0	-1	-1	105.17±0.40
7	-1	0	1	101.49±0.91
8	0	1	-1	80.08±0.62
9	1	1	0	93.27±0.76
10	1	-1	0	112.19±0.89
11	-1	0	-1	86.39±0.33
12	1	0	-1	95.19±1.09
13	0	0	0	119.14±0.82
14	0	0	0	118.33±1.46
15	-1	1	0	87.27±0.93
16	0	0	0	120.74±0.88
17	0	-1	1	108.98±1.07

一株降解纤维素的低温放线菌Streptomyces azureus及产酶条件优化

A Novel Low Temperature Cellulose-degrading Strain Streptomyces azureus and Its Enzymatic Production Condition Optimization

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 32

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

水平	因素
水平	pH	氮源浓度/(g/L)	碳源浓度/(g/L)
-1	4	6	13
0	5	7	15
1	6	8	17

[1]	李祥, 王永平, 王耀凤, 褚春年, 孙喜军, 柯希恒, 曾桥. 枝条有机肥最佳堆肥参数及施用效果研究[J]. 中国农学通报, 2022, 38(6): 63-68.
[2]	任绪瑞, 王一辉, 杨红宇, 袁亮, 赵越. 农业有机废弃物集中回收处理后资源化利用体系研究——以甘南县兴鲜村为例[J]. 中国农学通报, 2022, 38(33): 74-79.
[3]	王海候, 程月琴, 金梅娟, 刘泽凯, 施林林, 陆长婴. 稻壳生物质炭对羊粪堆肥中氮素转化及固定的影响[J]. 中国农学通报, 2022, 38(27): 51-59.
[4]	聂晓瑀, 于春静, 卢倩, 崔继哲. 微生物在农作物秸秆好氧堆肥过程中的研究进展[J]. 中国农学通报, 2022, 38(26): 76-81.
[5]	宋芸, 樊平, 王敏, 王强, 薛鹏飞. 几种废弃物基质有氧堆肥腐解因素比较分析[J]. 中国农学通报, 2022, 38(11): 53-57.
[6]	朱海云, 马瑜, 柯杨, 李勃. 抗猕猴桃细菌性溃疡病蜡样芽孢杆菌MA23培养基及发酵条件优化[J]. 中国农学通报, 2021, 37(7): 112-118.
[7]	赵龙妹, 陈林, 杜东晓, 董惠心, 李旺, 李元晓, 何万领, 曹平华. 产纤维素酶细菌的筛选鉴定与特性分析[J]. 中国农学通报, 2021, 37(30): 83-88.
[8]	刘淑娟, 刘虎俊, 刘光武, 李银科, 万翔, 张芝萍, 刘开琳, 李菁菁. 施用2种牛粪发酵肥对甜高粱生长量的影响[J]. 中国农学通报, 2021, 37(19): 22-26.
[9]	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.
[10]	卢佳伟, 王铭泽, 汪棋, 魏宗友, 张艳丽, 王锋. 辅料及微生物菌剂对羊粪好氧堆肥腐熟度的影响[J]. 中国农学通报, 2021, 37(15): 39-46.
[11]	梁艳琼, 李锐, 吴伟怀, 谭施北, 习金根, 郑金龙, 陆英, 贺春萍, 易克贤. 基于HS-SPME-GC-MS的Bacillus subtilis Czk1挥发性物质的萃取条件优化[J]. 中国农学通报, 2021, 37(11): 24-31.
[12]	王玥, 刘中珊, 刘奇, 徐赫男, 袁润泽, 李冲伟, 宋福强. 木耳菌糠和鸡粪混合堆肥改良盐碱土壤效果评价[J]. 中国农学通报, 2020, 36(26): 77-82.
[13]	刘维维, 金晓, 辛寒晓, 史庆华, 姚强, 刘丽英, 孙中涛. 强化型EM菌剂对金针菇菌糠堆肥的影响[J]. 中国农学通报, 2020, 36(22): 78-85.
[14]	黄艳艳, 杨旭, 杨红竹, 贝美容, 茶正早, 罗微, 林清火. 碳氮比对热带地区鸡粪和蔗渣堆肥腐熟进程的影响[J]. 中国农学通报, 2020, 36(21): 61-68.
[15]	谢夏, 杨建兰, 刘新育. 土壤中细菌菌落总数的测定条件优化[J]. 中国农学通报, 2020, 36(11): 92-95.