| Potassium is one of the three essential nutrients which has an important impact on the plant growth,development of plants and sustainable development of agricultural production.Potassium feldspar is a typical representative of China's potassium resources which contains potassium quite rich.The insoluble potassium can be transformed into soluble potassium for absorption directly and utilization of plants to alleviate the shortage of soluble potassium resources in China.In recent years,China has used low-cost,low-power,green and environmentally friendly microbial solution in potassium production of microbial potassium in a large scale,but the dissolved potassium resources was dificient and thus limited the industrialization of microbial potash fertilizer in China.This study used genome shuffling techniques,through built the method about mutagenesis technology by physics and chemical,and gain the dissolved potassium,ultimate is to obtain efficient potassium feldspar decomposition strains by ‘recursive fusion',which helped insoluble potassium converted to soluble potassium in high efficiency,so that China's rich potassium feldspar resources are highly efficient used,providing the technical guidance and theoretical support to promote the rapid development of China's microbial industry.The starting strain was Bacillus cereus L18 KF494191 preserved in our laboratory with the construction of forward mutant library by traditional NTG and UV mutagenesis.The research shows that: that 0.6 g/L NTG reacts for 20 min with UV irradiation of bacteria on the 0.1 ml(107-108 cfu/mL)coated plate for 20 s was the best mutation conditions.The results showed that the two isolates were selected to improve the ability of the two strains,which were named L18-NTG-151 and L18-UV-68.The starting strain was L18-NTG-151 and L18-UV-68 to protoplast fusion,the optimum conditions for protoplast preparation and regeneration have been obtained: lysozyme concentration was 1.5 g/L,temperature was 37 oC,rotating speed was 85 r/min,enzymolysis lasted for 1 h.Its formation rate and regeneration rate were 97.56% and 36.46%.The protoplast inactivation conditions were screened by UV and heating respectively.The best conditions of UV inactivation are with 106 bacteria,irradiation of 5 min.Among them,the temperature remains 70 oC and the water bath lasts for 60 min was the best conditions of heating inactivation.With inactivating protoplasts as carriers,35% of PEG6000,temperature remained 20 oC,water bath lasted for 15 min and the fusion rate was 4.64% were the optimum conditions.After three rounds ‘recursive fusion' and continuous cultivation of 5 generations,the results showed that the fermentation medium was the most potent solution of L18-F3-46,and the highest potassium content was the highest in the fermentation broth,Which was 277.93 mg/L,2.58 times higher than that of the original wild strain L18 fermentation,1.23 times higher than that of the fermentation broth Of the passage of stability.we improve the culture conditions by univariate analysis and orthogonal test,the research shows that: the only carbon source was 9 g/L sucrose,pH was 7,potassium feldspar content was 3 g/L,temperature was 30 oC,rotating speed was 200 r/min,and the content of potassium in L18-F3-46 fermentation broth was 290.57 mg/L after 96 h incubation.In summary,we can substantial increase the dissolved potassium capacity of Bacillus cereus L18 through the use of genome shuffling techniques,and get the dissolved potassium strains with high efficiency.In addition,we improve the culture conditions of this strain by study on characteristics of dissolved potassium,give full play to its ability,it has broad industrial application prospect. |
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