响应面法优化赤红球菌HDRR2Y发酵培养参数  

Optimization of Ferment Cultivation Parameters of Rhodococcus ruber HDRR2Y by Response Surface Method

作　　者：张淞 胡晓娟[2] 徐煜[2,3] 徐创文 杨铿[2] 苏浩昌[2] 徐武杰[2,3] 文国樑[1,2] 张建设 曹煜成 ZHANG Song;HU Xiao-juan;XU Yu;XU Chuang-wen;YANG Keng;SU Hao-chang;XU Wu-jie;WEN Guo-liang;ZHANG Jian-she;CAO Yu-cheng(Nat′l Engin.Res.Ctr.for Marine Aquacul.,ZhejiangOcean Uni.,Zhoushan 316022;S.China Sea Fishery Res.Inst.,Chinese Acad.of Fish.Sci./Key Lab.of S.China Sea Fishery Res.Exploi.&Util.,Minist.of Agric.&Rural Affairs/Key Lab.of Fishery Eco.&Environm′t,Guangzhou 510300;Maoming Branch ofGuangdong Lab.of Lingnan Modern Agric.Sci.&Technol.,Maoming 525000)

机构地区：[1]浙江海洋大学国家海洋设施养殖工程技术研究中心,浙江舟山316022 [2]中国水产科学研究院南海水产研究所/农业农村部南海渔业资源开发利用重点实验室/广东省渔业生态环境重点实验室,广东广州510300 [3]岭南现代农业科学与技术广东省实验室茂名分中心,广东茂名525000

出　　处：《微生物学杂志》2022年第5期50-57,共8页Journal of Microbiology

基　　金：国家重点研发计划项目(2020YFD0900401);财政部和农业农村部国家现代农业产业技术体系项目(CARS-48);中国水产科学研究院基本科研业务费专项(2020TD54);中国水产科学研究院南海水产研究所中央级公益性科研院所基本科研业务费专项(2021SD08);广东省现代农业产业技术体系创新团队建设专项(2019KJ149)。

摘　　要：通过响应面法对硝化菌——赤红球菌(Rhodococcus ruber)HDRR2Y的发酵培养参数进行优化,以提高其活菌数。首先通过单因素实验筛选出赤红球菌HDRR2Y的最优碳、氮源,并采用Plackett-Burman实验得到影响活菌数的显著因素,然后进行响应面实验,经最陡爬坡及回归分析得出最佳培养参数,最后以摇瓶实验检验其合理性。结果显示,赤红球菌HDRR2Y的最优碳、氮源分别为乙酸钠和酵母膏+蛋白胨+氯化铵(1:1:1,质量比),显著影响活菌数的因素有碳、氮源及温度,经回归分析得到的最优培养参数为乙酸钠5.48 g/L、酵母膏+蛋白胨+氯化铵4.96 g/L、温度29.24℃、pH 7.0、转速200 r/min、MgSO_(4)0.2 g/L、KH_(2)PO_(4)0.5 g/L、NaCl 9 g/L、CaCl_(2)0.5 g/L、MnSO_(4)0.025 g/L、FeSO_(4)0.05 g/L、C_(5)H_(9)NO_(4)0.002 g/L、接种活菌数1×10^(4)cfu/mL、装液量40%(体积分数)、培养时间36 h。优化后的实际活菌数为1.54×10^(9)cfu/mL,远高于优化前的活菌数(1.8×10^(8)cfu/mL)(P<0.01)。因此,采用响应面法优化赤红球菌HDRR2Y的发酵培养参数能大幅提高其发酵活菌数,为硝化菌剂的工业化生产提供数据参考。Fermentation parameters of nitrifying bacterium(Rhodococcus ruber HDRR2Y) were optimized by response surface method to improve bacterial quantity. First, the carbon sources and nitrogen sources that were most conducive to the growth of R.ruber HDRR2Y were selected by single factor experiment;second, Plackett-Burman experiment was used to screen out the factors that significantly affected the bacterial quantity;then, steepest climbing experiment and Box-Behnken experiment were used to determine the optimal level of each significant factor;finally, the rationality of the parameters was tested by shaking flask culture experiment. The results showed that the optimal carbon source and nitrogen source were sodium acetate, yeast extract+peptone+ammonium chloride(1:1:1), respectively. Sodium acetate, yeast extract+peptone+ammonium chloride and temperature were set as factor variables. The optimal culture parameters were sodium acetate 5.48 g/L, yeast extract+peptone+ammonium chloride 4.96 g/L, MgSO_(4)0.2 g/L, KH_(2)PO_(4)0.5 g/L, NaCl 9 g/L, CaCl_(2)0.5 g/L, MnSO_(4)0.025 g/L, FeSO_(4)0.05 g/L, C_(5)H_(9)NO_(4)0.002 g/L, pH 7.0, temperature 29.24 ℃, speed 200 r/min, the amount of inoculation at 1%, the filling volume of liquid was 40%(V/V), and culture for 36 h. After optimization, the actual bacterial quantity(1.54×10^(9) cfu/mL) was far higher than the bacterial quantity before optimization by response surface method(1.8×10^(8) cfu/mL)(P<0.01). Therefore, the fermentation culture parameters optimized by the response surface can be optimized to increase the bacterial quantity of R. ruber HDRR2Y, and provide data support for industrial production of nitrobacterial preparation.

关 键 词：赤红球菌 硝化菌 培养参数 BOX-BEHNKEN设计 响应面分析 

分 类 号：Q939.97[生物学—微生物学]