特基拉芽胞杆菌B05高密度发酵培养基与发酵条件优化
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摘要
特基拉芽胞杆菌(Bacillus tequilensis)B05是1株能显著提高小麦、玉米耐盐、耐旱能力的根际优良菌株,为提高其扩繁效率及使用效果,降低使用成本,以发酵液活菌数为指标,采用单因子预筛选和正交试验方法对菌株发酵培养基及发酵条件进行优化,在10 L发酵罐水平上进行扩大培养。结果表明,菌株B05最适发酵培养基(质量与体积分数)为麦芽浸粉1%、酵母浸粉2%、胰蛋白胨2%、复合无机盐0.3%(质量比为KH_2PO_4:K_2HPO_4:MnSO_4·H_2O=1:1:1)。最佳发酵培养条件为培养温度30℃、初始pH 6.5、摇床转速200 r/min、接种量7%、装液量40%,培养18 h菌液活菌数可达9.3×10~(10) cfu/mL,较优化前活菌数量提高了两个数量级。在含盐0.4%的盐碱土壤上进行了小麦盆栽试验,较空白对照植株生物量提高45.8%。
Bacillus tequilensis(B05) isolated from wheat rhizosphere was a fine strain that can significantly improve wheat and corn on salt and drought tolerance. In order to increase the strain cell proliferation, improve utilization efficiency and reduce utilization cost, in this study the living cells′ number in fermentation medium was taken as index, and single factor tests and orthogonal design method were used to optimize the fermentation medium and conditions, and the cultivation volume was expanded to 10 L in fermentation tank. The results showed that the optimal fermentation medium was 1% malt extract, 2% yeast powder, 2% peptone, 0.3% compound inorganic salt(KH_2PO_4:K_2HPO_4:MnSO_4·H_2O= 1:1:1). The optimum fermentation conditions was incubation temperature 30 ℃, initial pH 6.5, shaker speed 200 r/min, flask filling volume 40%, inoculation volume 7%. After 18 h of incubation, the number of viable bacteria reached 9.29×10~(10) cfu/mL, which were two orders higher than before optimization. The fermentation product was applied in wheat pot experiment soil containing 0.4% salt, and the biomass of wheat plants increased by 45.8% as compared with control plants.
Bacillus tequilensis(B05) isolated from wheat rhizosphere was a fine strain that can significantly improve wheat and corn on salt and drought tolerance. In order to increase the strain cell proliferation, improve utilization efficiency and reduce utilization cost, in this study the living cells′ number in fermentation medium was taken as index, and single factor tests and orthogonal design method were used to optimize the fermentation medium and conditions, and the cultivation volume was expanded to 10 L in fermentation tank. The results showed that the optimal fermentation medium was 1% malt extract, 2% yeast powder, 2% peptone, 0.3% compound inorganic salt(KH_2PO_4:K_2HPO_4:MnSO_4·H_2O= 1:1:1). The optimum fermentation conditions was incubation temperature 30 ℃, initial pH 6.5, shaker speed 200 r/min, flask filling volume 40%, inoculation volume 7%. After 18 h of incubation, the number of viable bacteria reached 9.29×10~(10) cfu/mL, which were two orders higher than before optimization. The fermentation product was applied in wheat pot experiment soil containing 0.4% salt, and the biomass of wheat plants increased by 45.8% as compared with control plants.
引文
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[18] 张文芝, 王云鹏,刘红霞, 等. 蜡质芽胞杆菌AR156 发酵培养基及发酵条件的优化[J]. 微生物学通报, 2010, 37(6): 803-810.
[19] 宫宇飞, 乔红萍, 魏国荣, 等. 内生枯草芽胞杆菌E1R-J 发酵条件的优化[J]. 西北农业学报, 2008, 17(1): 61-64.
[20] PEI Honglei, GUO Xiaojing, YANG Wenhan, et al. Directed evolution of abeta-1,3-1,4-glucanase from Bacillus subtilis MA139 for improving thermal stability and other characteristics[J]. Journal of Basic Microbiology, 2015, 55(7):869-878.
[21] 周映华, 吴胜莲,贺月林, 等.饲用枯草芽胞杆菌发酵条件的优化[J].湖南农业科学, 2010, (11):21-23.
[2] Zafarulhye M, Farooq HM, Hussain M. Bacteria in combination with Fertilizerspromote root and shoot growth of maize in saline-sodas soil[J]. Brazilian Journal of Microbiology, 2015, 46(1): 97-102.
[3] 樊润威, 崔志祥.内蒙古河套灌区盐碱土覆膜对土壤生态环境及作物生长的影响[J].土壤肥料, 1996,(3):10-12.
[4] 张万钧, 龙怀玉, 杨永利, 等.天津滨海园林绿化中盐土治理的理论及工艺[J].北京林业大学学报, 2000,22(5):41-42.
[5] 王春娜, 宫伟光.盐碱地改良的研究进展[J].防护林科技, 2004,62(5):38-41.
[6] 毛建华, 刘太祥, 马履, 等.中新天津生态城资源节约、环境友好的改土绿化技术[J].天津农业科学, 2008,14(4):1-3.
[7] 牛东玲, 王启基.盐碱地治理研究进展[J].土壤通报, 2002,33(6):449-451.
[8] 刘彩霞, 黄为一. 耐盐碱细菌与有机物料对盐碱土团聚体形成的影响[J]. 土壤, 2010, 42(1):111-116.
[9] 孙蓟锋, 王旭.土壤调理剂的研究和应用进展[J].中国土壤与肥料,2013,(1):1-7.
[10] 曲发斌, 于明礼, 张柱岐, 等.盐生植物根际耐盐碱微生物的筛选及其降解特性[J].贵州农业科学, 2015,43(3):121-124.
[11] 张海英, 黄叶梅, 吴小莉, 等. 一株抗高粱蚜芽胞杆菌的分离与鉴定[J].宜宾学院学报, 2017,17(6):111-114.
[12] 王雪山, 王善林, 杜秉海, 等. 牡丹根腐病拮抗菌的筛选与鉴定[J].山东农业科学, 2012,(7):90-94.
[13] 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.
[14] 周德庆. 微生物教程[M].北京:高等教育出版社, 2002:79-81.
[15] 方传记, 陆兆新, 孙力军,等. 淀粉液化芽胞杆菌抗菌脂肽发酵培养基及发酵条件的优化[J]. 中国农业科学, 2008, 41(2): 533-539.
[16] 章四平, 刘圣明, 王建新, 等. 枯草芽胞杆菌生防菌株NJ-18 的发酵条件优化[J]. 南京农业大学学报, 2010, 33(2): 58-62.
[17] 纪明山, 王毅婧. 地衣芽胞杆菌生防菌株SDYT-79 发酵条件优化[J]. 沈阳农业大学学报,2011, 42(2): 164-169.
[18] 张文芝, 王云鹏,刘红霞, 等. 蜡质芽胞杆菌AR156 发酵培养基及发酵条件的优化[J]. 微生物学通报, 2010, 37(6): 803-810.
[19] 宫宇飞, 乔红萍, 魏国荣, 等. 内生枯草芽胞杆菌E1R-J 发酵条件的优化[J]. 西北农业学报, 2008, 17(1): 61-64.
[20] PEI Honglei, GUO Xiaojing, YANG Wenhan, et al. Directed evolution of abeta-1,3-1,4-glucanase from Bacillus subtilis MA139 for improving thermal stability and other characteristics[J]. Journal of Basic Microbiology, 2015, 55(7):869-878.
[21] 周映华, 吴胜莲,贺月林, 等.饲用枯草芽胞杆菌发酵条件的优化[J].湖南农业科学, 2010, (11):21-23.
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