Fermentation Conditions And Extraction Of Biosurfactant From Bacillus Licheniformis

Biosurfactant(BS)has high surface activity,foaming ability and biodegradability,and is widely applied.In our previous study,a bacterial strain producing biosurfactant,Bacillus licheniformis DGG-1-3-F was isolated.In this paper,the conditions of inocula cultivation and fermentation are optimized to produce biosurfactant,and the produced biosurfactant is extracted and qualitatively analyzed.By measuring the cell density in inocula culture broth,the cell density and surface tension of fermenting broth,the effects of temperature,ventilation and time of inocula cultivation and inoculation rate on cell growth and the surfactant production were studied.The results indicated that the inocula should be cultivated better at 50? and 180 r/min for 14 h in 100 mL/250 mL shaking flask sealed with 12-layer gauze.And when the fermentation was conducted for 24 h with 10%of inoculation rate,the surface tension of cell-free fermenting broth lowered to the minimum,22.6 mN/m,decreased by 64.1%.The time of inocula cultivation was shorten for 10 h than that before optimization.The surface tension of fermenting broth with the inocula could reached to the minimum 24 h earlier,the reduction was significantly increased by 7.1%(P<0.05).By using decane,tridecane,liquid paraffin,palm oil,soybean oil and rapeseed oil as carbon source,both cell growth and surface tension reduction of fermenting broth was small.Cell growth by using rapeseed oil and the reduction(26%)of fermenting broth surface tension by using palm oil were significantly the highest(P<0.05).Starch(1%)could promote the utilization of palm oil by the strain,but the surface tension and emulsifying activity of fermenting broth with starch and palm oil is less than that of sole starch(1%)significantly(P<0.05).Extracellular lipase related to substrate metabolism,but not directly to biosurfactant synthesis.By using 0.4%(mass fraction)sodium nitrate,potassium nitrate,ammonium chloride,ammonium nitrate and ammonium sulfate as nitrogen source respectively,it was found that the surface tension of fermenting broth using sodium nitrate decreased by 65.9%which is significantly higher than that by using other nitrogen source(P<0.05),and of which the emulsifying index(4%)wasn't significanty different from that by using potassium nitrate(P>0.05).By adjusting C/N ratio at 5:1,10:1,15:1,20:1,25:1 with starch as carbon source and 0.4%(mass fraction)sodium nitrate as nitrogen source,it was found that the surface tension of fermenting broth at 20:1 of C/N ratio reduced to the lowest 20.0 mN/m,significantly greater than that at other C/N ratio(P<0.05),the emulsifying index wasn't significantly different with that at 25:1 of C/N ratio(P>0.05).By fermenting biosurfactant with 2%,4%,6%wort and molasses,it was found that the surface tension of fermenting broth 2%wort or 2%molasses reduced to the lowest,20.3 mN/m and 20.4 mN/m respectively,decreased by 55.9%and 62.3%which was significantly higher than that at other concentration(P<0.05).There was no significant difference in the emulsifying index(about 4%)of fermenting broth of wort at various concentrations(P>0.05).The emulsifying index(42%)of fermenting broth with 2%molasses was significantly higher than that of molasses at other concentration(P<0.05).The waste residue and waste water produced in potato starch production was used as the culture medium,in which contained 2%,4%,6%,8%,10%waste residue in the waste water.The results showed that the surface tension of fermenting broth with 10%waste residue reduced to the lowest,21 mN/m,decreased by 59.1%which was obviously higher than that with waste residue at other concentration(P<0.05).There was no significant difference in the emulsifying index(about 4%)of fermenting broth with waste residue at various concentrations(P>0.05).By acidic precipitation at pH 2.0,extraction with chloroform:methanol(2:1,volume rate),crude biosurfactant could be obtained.The impurities of sugar and protein could be effectively removed by extraction with methanol:water(1:1,volume rate).The crude biosurfactant could be identified as glycolipid and lipopeptide by thin layer chromatography.The crude biosurfactant could form water-in-oil(W/O)emulsion layer with liquid paraffin,easily dissolve in chloroform,and dissolve in both water and methanol.