| Rhamnolipid is a kind of biosurfactants with excellent performance, mainly produced by pseudomonas aeruginosa fermentation. To solve the problems of low yield and high production cost, this subject research carried out the experiments in the laboratory from two aspects, one was raising the production capacity of fermentation strains, the other was optimizing fermentation conditions.First of all, through the method of blue gel tablet in combination with surface tension determination, the best natural strain was selected from the nature, named Pseudomonas aeruginosa-Z. This strain was used for fermentation experiments, after study, the following conclusions were got:(1) Centrifuging the fermented liquid, supernatant diluted20times with distilled water, then measured the surface tension of35.1mN/m, compared with unvaccinated medium of same diluted multiples, surface tension was decreased by42.7%;(2) The main Logarithmic growth phase was in12-22h, and in plateau the bacteria number could reached109/mL;(3) Extraction with equal volume VChloroform:VMethanol=2:1organic mixed solvent, the output of rhamnolipid coarse extraction product was3.43g/L, the CMC value was as low as20mg/L.Then, to acquire mutation strains, using UV and products dealed with the Pseudomonas aeruginosa-Z. Once again, using the method of blue gel tablet, the best one was picked out from the forward mutation strains, named Pseudomonas aeruginosa-ZCY. After study the strain was found as follows:(1) Centrifuging the fermented liquid, supernatant diluted20times with distilled water, then measured the surface tension of32.1mN/m, compared with fermentation diluted liquid of Pseudomonas aeruginosa-Z, the surface tension continued to be reduced by8.5%;(2) Extraction with equal volume VChioroform:VMethanol=2:1organic mixed solvent, the production of rhamnolipid coarse extraction was5.52g/L, compared with the Pseudomonas aeruginosa-Z, the production increased by60.9%, and, the stability of strain had good performance in the process of the wedding;(3) Compared with the Pseudomonas aeruginosa-Z, its growth curve was basically unchanged. When it was initial strain that vaccinated to simulate industrial culture medium, through the optimizing experiment could determine:the best quantity was3%;(4) The CMC value of the coarse products basically remained unchanged. Combined with the results of mass spectrometry analysis and the Rf values of TLC, its products were mainly double squirrel glycolipids lee could be concluded. Finally, Pseudomonas aeruginosa-ZCY fermentation conditions were optimized. Using single factor experiment and orthogonal experiment got the optimum medium formulas (g/L) that was as follows:Rapeseed oil30.0; Dried corn steep liquor powder6.0; NaNO36.0; K2HPO40.9; NaH2PO40.6; MgSO40.3; CaCl20.05; NaCl1.5. The optimum pH value was between6.9-7.0, the optimum temperature was:the first20h kept36℃, then34℃continued to ferment40h. Finally confirmed, the highest average rhamnolipid production of Pseudomonas aeruginosa-ZCY was34.52g/L. |
