刺糖菌素产生菌的菌种选育及发酵条件优化研究
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摘要
绿色广谱生物杀虫剂刺糖菌素是由Saccharopolyspora spinosa经有氧发酵后的次级代谢产物，具有杀虫谱广，易降解，低残留，无抗药性，对人畜无害，环境污染少等特点。刺糖菌素在农牧业上有着广阔的应用前景，有望成为我国生物农药产业一个新的经济增长点，该产品的开发具有很好的经济效益和社会效益。本文进行了刺糖菌素产生菌Saccharopolyspora spinosa的菌种选育以及发酵工艺条件的优化，最后进行了10L罐小试。
根据刺糖菌素的生物合成途径和代谢调控原理，运用紫外线、γ射线，硫酸二乙酯、氯化锂和微波等多种诱变因子进行菌种诱变，并结合耐前体突变株、耐自身自身终产物结构类似物突变株等理性化筛选，使Saccharopolyspora spinosa产刺糖菌素的能力不断提高，最后选育出了一株刺糖菌素优良菌株NS-1。菌株NS-1的发酵单位为97.83μg／ml，比出发菌株的发酵单位提高了254.71％。同时，本文还对菌株NS-1进行了遗传稳定性试验，结果表明该菌株遗传性能比较稳定。
本文对发酵培养基以及培养条件进行了优化。考察了碳源、氮源，前体物质、微量元素等，最后对以上单因素进行了正交试验优化。培养条件主要进行了接种量、装量、pH、温度的优化。优化后的发酵工艺条件如下：发酵培养基为葡萄糖6.0％、麦芽糖3.0％、棉籽粉3.0％、鱼粉0.5％、玉米浆1.5％、NH_4Cl 0.1％、CoCl_20.003％、油酸甲酯1.0％、CaCO_3 0.5％，pH为7.0。接种量为10％、250mL三角瓶装量为30ml、30℃发酵72h后补加0.3％正丙醇，96h后温度变为28℃发酵。采用以上优化后的工艺条件进行了摇瓶间歇发酵，最终的发酵单位达到128.21μg／mL，较原始发酵工艺条件的发酵单位提高了31.05％。
最后，本文采用选育出的刺糖菌素优良菌株NS-1和优化后的发酵条件进行了10L罐小试，得到发酵过程中菌体干重、还原糖、pH及spionsad等参数的变化曲线。在10L罐刺糖菌素的发酵单位达到66.21μg／mL，比培养的发酵单位低62μg／mL。
Spinosad, which was a novel microbial broad-spectrum insecticide, was secondary metabolites from the aerobic fermentation of a naturally occurring actinomycetes bacterium, Saccharopolyspora spinosad. Spinosad, which was composed of a mixture of two most active naturally occurring metabolites (spinosyns A and D), was effective on a wide variety of crops. Attributes of spinosad included: unique chemistry and mode of action, high levels of activity against economically important pests, a short half-life, degradation to natural building blocks, and large margins of safety for mammals, birds, fish and even most beneficial insects. In this dissertation, strain improvement, optimization of spinosad fermentation process and scale-up of fermentation processes in 10 L fermentor. were studied.
According to the biosynthesis pathway and the metabolic regulation of spinosad, stram improvement was performed by mutation and rational screening to improve the productivity. Through treament by rational sceening and mutagens such as UV, DES, LiCl, r -ray and microwave etc, spinosad over-producing strain NS-1 was obtained. Productivity of strain NS-1 reached 97.83ug/mL, which was 254.71% higher than that of the original strain CT-20-I. Subculture test indicated that the hereditary character of strain NS-1 was stable.
In this dissertation, the medium composition and the fermentation conditions for spinosad production were investigated. The optimal medium composition, which was obtained through single factor experiments and orthogonal design experiments, was as
fbllows(g/L): glucose 0.6g, maltose 0.3g, cottonseed flour 0.3g, fish meal 0.05g, corn steep liquor 0.15g, NH4Cl 0.0lg, CoCl20.0003g, methyl oleate 0.1g, calcium carbonate 0.5g, precursor 0.3g (added at 72h for batch fermentation). The optimal fermentation conditions were as follows: initial pH valuer,of medium 7.0, the inoculum amount 10%, loading amount 30ml in 250ml shaking flask, culture temperature 30@ and after 96h at 28@. The productivity of the optimal fermentation
condition reached 128.21ug/mL, which was increased by 31.05% compare to that of the original fermentation condition.
Based on the above experimental data, tests in 10L fermentor were also carried out. The fermentation kinetics of spinosad over-producing strain NS-1 was studied in this dissertation. Compared with shake-flask fermentation in 10L fermentor, a mase of foam were produced, which were controlled by adding an antifoam agent. Finally, the scale-up of spinosad fermentation from shaking flask to 10L fermentor was realized successfully, and productivity of spinosad high-producing strain NS-1 in 10L fermentor reached 66.21 ug/mL which was decreased by 62ug/mL compare to that of the original fermentation condition .
根据刺糖菌素的生物合成途径和代谢调控原理，运用紫外线、γ射线，硫酸二乙酯、氯化锂和微波等多种诱变因子进行菌种诱变，并结合耐前体突变株、耐自身自身终产物结构类似物突变株等理性化筛选，使Saccharopolyspora spinosa产刺糖菌素的能力不断提高，最后选育出了一株刺糖菌素优良菌株NS-1。菌株NS-1的发酵单位为97.83μg／ml，比出发菌株的发酵单位提高了254.71％。同时，本文还对菌株NS-1进行了遗传稳定性试验，结果表明该菌株遗传性能比较稳定。
本文对发酵培养基以及培养条件进行了优化。考察了碳源、氮源，前体物质、微量元素等，最后对以上单因素进行了正交试验优化。培养条件主要进行了接种量、装量、pH、温度的优化。优化后的发酵工艺条件如下：发酵培养基为葡萄糖6.0％、麦芽糖3.0％、棉籽粉3.0％、鱼粉0.5％、玉米浆1.5％、NH_4Cl 0.1％、CoCl_20.003％、油酸甲酯1.0％、CaCO_3 0.5％，pH为7.0。接种量为10％、250mL三角瓶装量为30ml、30℃发酵72h后补加0.3％正丙醇，96h后温度变为28℃发酵。采用以上优化后的工艺条件进行了摇瓶间歇发酵，最终的发酵单位达到128.21μg／mL，较原始发酵工艺条件的发酵单位提高了31.05％。
最后，本文采用选育出的刺糖菌素优良菌株NS-1和优化后的发酵条件进行了10L罐小试，得到发酵过程中菌体干重、还原糖、pH及spionsad等参数的变化曲线。在10L罐刺糖菌素的发酵单位达到66.21μg／mL，比培养的发酵单位低62μg／mL。
Spinosad, which was a novel microbial broad-spectrum insecticide, was secondary metabolites from the aerobic fermentation of a naturally occurring actinomycetes bacterium, Saccharopolyspora spinosad. Spinosad, which was composed of a mixture of two most active naturally occurring metabolites (spinosyns A and D), was effective on a wide variety of crops. Attributes of spinosad included: unique chemistry and mode of action, high levels of activity against economically important pests, a short half-life, degradation to natural building blocks, and large margins of safety for mammals, birds, fish and even most beneficial insects. In this dissertation, strain improvement, optimization of spinosad fermentation process and scale-up of fermentation processes in 10 L fermentor. were studied.
According to the biosynthesis pathway and the metabolic regulation of spinosad, stram improvement was performed by mutation and rational screening to improve the productivity. Through treament by rational sceening and mutagens such as UV, DES, LiCl, r -ray and microwave etc, spinosad over-producing strain NS-1 was obtained. Productivity of strain NS-1 reached 97.83ug/mL, which was 254.71% higher than that of the original strain CT-20-I. Subculture test indicated that the hereditary character of strain NS-1 was stable.
In this dissertation, the medium composition and the fermentation conditions for spinosad production were investigated. The optimal medium composition, which was obtained through single factor experiments and orthogonal design experiments, was as
fbllows(g/L): glucose 0.6g, maltose 0.3g, cottonseed flour 0.3g, fish meal 0.05g, corn steep liquor 0.15g, NH4Cl 0.0lg, CoCl20.0003g, methyl oleate 0.1g, calcium carbonate 0.5g, precursor 0.3g (added at 72h for batch fermentation). The optimal fermentation conditions were as follows: initial pH valuer,of medium 7.0, the inoculum amount 10%, loading amount 30ml in 250ml shaking flask, culture temperature 30@ and after 96h at 28@. The productivity of the optimal fermentation
condition reached 128.21ug/mL, which was increased by 31.05% compare to that of the original fermentation condition.
Based on the above experimental data, tests in 10L fermentor were also carried out. The fermentation kinetics of spinosad over-producing strain NS-1 was studied in this dissertation. Compared with shake-flask fermentation in 10L fermentor, a mase of foam were produced, which were controlled by adding an antifoam agent. Finally, the scale-up of spinosad fermentation from shaking flask to 10L fermentor was realized successfully, and productivity of spinosad high-producing strain NS-1 in 10L fermentor reached 66.21 ug/mL which was decreased by 62ug/mL compare to that of the original fermentation condition .
引文
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