基于响应面法优化莱氏绿僵菌SZCY200812的液相发酵条件

doi:10.16409/j.cnki.2095-039x.2022.03.033

摘要/Abstract

摘要： 为提高莱氏绿僵菌菌株SZCY200812液相发酵时孢子萌发率和生物量，为固相发酵产孢提供高质发酵液，本研究采用单因素及Box-Behnken试验设计结合的方法对该菌液相发酵过程中培养液种类、装液量、pH、摇床转速及培养温度等条件进行优化，确定最佳的液相发酵培养条件。结果表明，通过单因素试验初步确定利用SDY培养液、装液量100/250mL、pH7、摇床转速180r/min及温度25℃，第120h每10mL液态发酵生物产量为0.8433g。通过响应面法建立了莱氏绿僵菌SZCY200812液体发酵培养菌丝生物量与自变量回归模型，根据模型所得结果得出装液量为100.82/250mL，摇床转速178.85r/min，温度24.80℃，pH6.73条件下为理论最佳液态发酵培养条件。为便于生产，得出该菌株SZCY200812在SDY培养液中最佳的培养条件为：装液量101/250mL、pH6.7、摇床转速179r/min以及温度25℃，第120h每10mL液相发酵生物产量达0.8637g。综合以上结果，影响莱氏绿僵菌SZCY200812液相发酵的因素由强至弱依次为pH（D）、温度（C）、摇床转速（B）和装液量（A）。

关键词: 莱氏绿僵菌, 液相发酵, 培养条件优化, 响应面法

Abstract: It is important to increase the liquid fermentation germination rate and biomass of Metarhizium rileyi strain SZCY200812 and provide high quality fermentation broth for solid phase fermentation. The best liquid fermentation conditions were determined in this study using the single factor and Box-Behnken experimental design to optimize the culture medium, liquid volume, pH, rotating speed, and culture temperature in the liquid fermentation process. The results showed a biomass yield of 0.8433 g per 10 mL at 120 h of fermentation employing conditions of SDY culture medium, liquid volume of 100/250 mL, pH of 7, rotating speed of 180 r/min and temperature of 25 ℃. The regression models of mycelial biomass in liquid fermentation culture of M. rileyi SZCY200812 with independent variables were established by response surface methodology. According to the results of the model, the optimal liquid fermentation conditions were liquid volume of 100.82/250 mL, rotation speed of 178.85 r/min, temperature of 24.80 ℃, and pH 6.73, which produced a biomass of liquid phase fermentation of 0.8637 g per 10 mL at 120 h. The RSM analysis also indicated that the influence on biomass from high to low were pH, temperature, rotating speed, liquid volume.

Key words: Metarhizium rileyi, liquid-state fermentation, optimization of culture conditions, response surface methodology

中图分类号:

S476.1

薛锐, 付玉飞, 邓细周, 沈绍运, 杜广祖, 陈斌. 基于响应面法优化莱氏绿僵菌SZCY200812的液相发酵条件[J]. 中国生物防治学报, 2022, 38(6): 1484-1494.

XUE Rui, FU Yufei, DENG Xizhou, SHEN Shaoyun, DU Guangzu, CHEN Bin. Optimization of Liquid-state Fermentation Conditions of Metarhizium rileyi SZCY200812 by Response Surface Methodology[J]. Chinese Journal of Biological Control, 2022, 38(6): 1484-1494.

参考文献

[1] 杜广祖, 郑亚强, 陈斌, 等. 几种植物油对莱氏野村菌分生孢子萌发的影响[J]. 微生物学通报, 2015, 42(4): 690-698.
[2] Kish L P, Samson R A, Allen G E. The genus Nomuraea maublance[J]. Journal of Invertebrate Pathology, 1974, 24(2): 154-158.
[3] Kish L P, Allen G E. The biology and ecology of Nomuraea rileyi and a program for predicting its incidence on Anticarsia gemmatalis in soybean[J]. Florida. Agricultural Experiment Station. Bulletin (USA), 1978, 795.
[4] Kepler R M, Humber R A, Bischoff J F, et al. Clarification of generic and species boundaries for Metarhizium and related fungi through multigene phylogenetics[J]. Mycologia, 2014, 106(4): 811-829.
[5] Visalakshi M, Varma P, Kishore S V, et al. Studies on mycosis of Metarhizium (Nomuraea) rileyi on Spodoptera frugiperda infesting maize in Andhra Pradesh, India[J]. Egyptian Journal of Biological Pest Control, 2020, 30(1): 135-145.
[6] 龙秀珍, 高旭渊, 曾宪儒, 等. 一株莱氏绿僵菌的筛选及其对草地贪夜蛾的毒力[J]. 中国生物防治学报, DOI: 10.16409/j.cnki.2095-039x.2021.05.015.
[7] Edegar F, Alexandre S, Horacio H, et al. Metarhizium (Nomuraea) rileyi as biological control agent[J]. Biocontrol Science and Technology, 2017, 27(11): 1-22
[8] 陶新娉, 王彬, 管清真, 等. 莱氏绿僵菌对斜纹夜蛾的致病力及生理效应[J]. 微生物学通报, 2017, 44(6): 1349-1357.
[9] Kim S, Kim J C, Lee S J, et al. Beauveria bassiana ERL836 and JEF-007 with similar virulence show different gene expression when interacting with cuticles of western flower thrips, Frankniella occidentalis[J]. BMC genomics, 2020, 21(1): 836-836.
[10] Rosana A R R, Pokorny S, Klutsch J G, et al. Selection of entomopathogenic fungus Beauveria bassiana (Deuteromycotina:Hyphomycetes) for the biocontrol of Dendroctonus ponderosae (Coleoptera:Curculionidae, Scolytinae) in Western Canada[J]. Applied Microbiology and Biotechnology, 2021, 105(6): 1-17.
[11] 许天委, 张世清, 黄俊生. 金龟子绿僵菌的液固双相发酵研究[J]. 河南农业科学, 2013, 42(2): 93-97.
[12] 蔡守平. 绿僵菌MaZPTR-01菌株固体发酵条件筛选研究[J]. 西南林业大学学报, 2016, 36(5): 100-105.
[13] 李姝江, 彭娇洋, 朱涵明月, 等. 莱氏野村菌(Nomuraea rileyi)固液双相发酵配方研究[J]. 四川林业科技, 2017, 38(5): 33-37.
[14] 张璐璐. 防治西花蓟马的球孢白僵菌液固双相发酵条件优化[D]. 北京: 中国农业科学院, 2016.
[15] Kang S W, Lee S H, Yoon C S, et al. Conidia production by Beauveria bassiana (for the biocontrol of a diamondback moth) during solid-state fermentation in a packed-bed bioreactor[J]. Biotechnology Letters, 2005, 27(2): 135-139.
[16] Devi P S V. Conidia production of the entomopathogenic fungus Nomuraea rileyi and its evaluation for control of Spodoptera litura (Fab) on Ricinus communis[J]. Academic Press, 1994, 63(2): 145-150.
[17] 林健文, 罗志兵, 骆星丹, 等. 莱氏野村菌固体发酵培养基筛选与发酵条件优化[J]. 植物保护学报, 2009, 36(5): 437-442.
[18] 唐维媛, 邢丛丛, 董永刚, 等. 对斜纹夜蛾高致病性莱氏野村菌Nr19固体培养条件的优化[J]. 中国生物防治学报, 2016, 32(2): 180-188.
[19] 张功营, 杨华, 董丽红, 等. 响应面法优化绿僵菌产绿僵菌素A的培养条件[J]. 华南农业大学学报, 2018, 39(1): 76-82.
[20] 杜广祖, 郑亚强, 陈斌, 等. 莱氏野村菌Nr120815菌株在不同稻谷基质上的产孢特性研究[J]. 云南农业大学学报(自然科学版), 2016, 31(5): 791-798.
[21] 唐维媛, 彭小东, 王欢, 等. 不同地域莱氏野村菌的培养性状及对斜纹夜蛾的毒力[J]. 湖南农业大学学报(自然科学版), 2013, 39(3): 279-281.
[22] 陈剑翔, 张庆庆, 金鑫强, 等. 液态发酵细脚拟青霉培养基及其发酵条件的优化[J]. 安徽工程大学学报, 2013, 28(2): 7-10.
[23] Hong T D, Gunn J, Ellis R H, et al. The effect of storage environment on the longevity of conidia of Beauveria bassiana[J]. Mycological Research, 2001, 105(5): 597-602.
[24] 李尊华, 林健文, 马金成, 等. 变温干燥固体发酵产物对球孢白僵菌分生孢子性能的影响[J]. 微生物学报, 2008(7): 887-892.
[25] 安卫娟, 钟敏, 张庆华, 等. 响应面分析法优化沙雷氏菌(Serratia sp.) AXJ-M对己烯雌酚的降解[J]. 微生物学通报, DOI: https://doi.org/10.13344/j.microbiol.china.210077.
[26] 郑亚强, 胡惠芬, 付玉飞, 等. 草地贪夜蛾莱氏绿僵菌的分离鉴定[J]. 植物保护, 2019, 45(5): 65-70.
[27] 雷妍圆, 王德森, 薛志洪, 等. 广州地区一株绿僵菌的鉴定及其对草地贪夜蛾的致病力测定[J]. 南方农业学报, 2020, 51(6): 1265-1273.
[28] 王育花, 赵森, 陈芬, 等. 利用实时荧光定量PCR法检测转基因水稻外源基因拷贝数的研究[J]. 生命科学研究, 2007(4): 301-305.
[29] James P D A, Edwards C. The effects of cultural conditions on growth and secondary metabolism in Streptomyces thermoviolaceus[J]. FEMS Microbiology Letters, 1988, 52(1-2): 1-5.
[30] 张誉荠, 许永华, 文湘穗, 等. 响应面法优化北五味子褐斑病内生生防真菌淡紫拟青霉WG9发酵工艺及发酵产物稳定性研究[J]. 菌物学报, 2021, 40(6): 1561-1574.
[31] 程红艳, 郭伟, 常明昌, 等. 蛹虫草菌丝体液体发酵培养条件的优化研究[J]. 山西农业大学学报(自然科学版), 2011, 31(1): 66-72.
[32] 李义华, 刁红亮, 周稳稳, 等. 碳源对玫烟色虫草菌丝生长、芽生孢子产生及其耐热性的影响[J]. 菌物学报, 2022, 41(1): 51-58.