Preliminary Study On The Effect Of Phosphate On Biosurfactant Fermentation And Product Extraction

Biosurfactant has many advantages such as low toxicity and biodegradability,but its actual production is limited by low yield and high production cost.At present,researches mainly through optimizing the upstream fermentation process and downstream purification process to increase yield and reduce costs.In this study,Ochrobactrum sp.was used as the experimental strain,the effects of phosphate types and increase the concentration of carbon source,nitrogen source and phosphorus source on the fermentation of biosurfactants were preliminarily explored.Besides the extraction methods of biosurfactants were compared.Under the same total phosphorus concentration of 0.035 mol/L,KH₂PO₄+Na₂HPO₄,KH₂PO₄,and Na₂HPO₄ was respectively used as the phosphorus source of biosurfactant fermentation at 30℃and 160 rpm.The results showed that the bacteria grew slowly and the maximum emulsifying activity of fermentation broth reached 52.95%with KH₂PO₄+Na₂HPO₄as phosphorus source,and large bacterial growth and 35.9%maximum emulsifying activity with KH₂PO₄,small bacterial growth and 24.54%maximum emulsifying activity with Na₂HPO₄.Using KH₂PO₄+Na₂HPO₄ as the phosphorus source,the biosurfactants were produced by fermentation in low-carbon low-nitrogen low-phosphorus（sucrose 10 g/L,Na NO₃ 4 g/L,KH₂PO₄ 3.4 g/L+Na₂HPO₄ 1.5 g/L）,high-carbon high-nitrogen low-phosphorus（sucrose 35g/L,Na NO₃ 14 g/L,KH₂PO₄ 3.4 g/L+Na₂HPO₄ 1.5 g/L）,high-carbon high-nitrogen high-phosphorus（sucrose 35 g/L,Na NO₃ 14 g/L,KH₂PO₄ 11.5 g/L+Na₂HPO₄ 5.25 g/L）culture medium respectively.The results showed that the high-carbon high-nitrogen low-phosphorus group was more conducive to the accumulation of biosurfactants,while the high-carbon high-nitrogen high-phosphorus group was more conducive to the growth of bacteria.The maximum emulsifying activity of the three:high-carbon high-nitrogen low-phosphorus group（59.2%,on the 5th day of fermentation）>high-carbon high-nitrogen high-phosphorus group（55.3%,on the 6th day of fermentation）>low-carbon low-nitrogen low-phosphorus group（51.9%,on the 5th day of fermentation）;Cell growth:high-carbon high-nitrogen high-phosphorus group>high-carbon high-nitrogen low-phosphorus group>low-carbon low-nitrogen low-phosphorus group.Increasing the concentration of carbon and nitrogen sources had no effect on the appearance time of maximum emulsifying activity,while increasing the concentration of phosphorus source would delay the appearance time of maximum emulsifying activity.Increasing the concentration of phosphorus source under high carbon and nitrogen conditions might inhibit the synthesis of biosurfactants or reduce the activity of biosurfactants,but promote the growth of bacteria.The effects of pH adjustment method,solvent extraction method and solvent precipitation method were compared,and then the pH adjustment method and ethanol precipitation method were combined to extract biosurfactants.The results showed that the biosurfactant couldn’t be extracted by the pH adjustment method and the solvent extraction method,and the ethanol precipitation method could obtain a product with high emulsification activity.In the extraction process of using ethanol precipitation method,low temperature could significantly increase the yield,the stirring speed and stirring time had no influence on the extraction,and the pH of the fermentation broth had a great influence on the extraction.The pH of the fermentation broth was adjusted to 13 to remove the precipitate,and then three times the volume of cold ethanol was added for extraction,the yield（6.69 g/L）was 1.45 times that of adding 3 volumes of ethanol to the original fermentation broth for extraction.Extraction by this method could obtain a large molecular weight biosurfactant containing a small amount of sugar,protein,lipid and more phosphate.Using KH₂PO₄+Na₂HPO₄ as the phosphorus source,the biosurfactants were produced by fermentation in high-carbon high-nitrogen low-phosphorus medium for 5 days,the fermentation broth could be stored at-20℃for at least 7 weeks,and the emulsion could be stable for at least 8 months.The pH of the fermentation broth was adjusted to 13 to remove the precipitate,and then three times the volume of cold ethanol was added for extraction,the crude product was easily soluble in water,5%hydrochloric acid and 5%sodium bicarbonate,the activity was stable in the pH range of 6-14,and the critical micelle concentration was 4 mg/mL.