Tolerance Of Klebsiella Sp.2e To Different Impurities Of Crude Glycerol And Its Key Enzymes Of 1,3-PDO Conversion

In this study,a strain of crude glycerol-tolerant strain Klebsiella sp.2e was selected as the research object.The strain was tested by/the detection of various physiological and biochemical indicators of bacteria.The strain growth,glycerol consumption and yield of l,3-propanediol in impurity fermentation broth,which contained KCl,K2SO4,methanol and oleic acid,and in the fermentation liquid with crude glycerol and pure glycerol were also tested.Meanwhile,the effects of different impurities and their different concentration parameters on the growth and fermentation of 1,3-propanediol were determined,and its reaction process and trend was learnt./At the same time,combining the activity analysis of three key enzymes during glycerol conversion,which were respectively glycerol dehydrogenase(GDH),glycerol dehydratase(GDHt)and 1,3-propanediol oxidase(PDOR),the effect of different impurities on enzyme activity was explored and the further test for the tolerance of Klebsiella sp.2e to crude glycerol was done.The results showed that:1.Low concentration of KCl impurities had no effect on the growth and fermentation of the strain using different isolation screening media culture to determine the growth of Klebsiella sp.2e strain.When the ratio of liquid to liquid was higher than 5%,it would inhibit the growth of the strain.This demonstrates that the excess inorganic salt ions will affect the osmotic pressure outside the bacteria and affect its growth and reproduction.When methanol solution was lower than 10 g/L,it had no effect on the growth of the strain,which was similar with the growth in pure glycerol fermentation broth;enven low K2SO4 and oleic acid content inhibited the growth of the strain,which proved that-SO42-and organic acid had a great influence on the growth of Klebsiella sp.2e.The crude glycerol can be sampled before fermentation,and-SO42-and organic acid could be removed by pretreatment.2.During the fermentation of Klebsiella sp.2e with different separation and screening medium,the glycerol consumption in the fermentation broth was proportional to the amount of 1,3-propanediol.The glycerol conversion efficiency was related to the growth of the strain,and KCl impurities lower than 5%could promote the fermentation of strains to produce 1,3-propanediol.Above this value,inhibition began to occur;methanol impurities below 10 g/L had no effect on the fermentation of the strain,and the yield of 1,3-propanediol was close to the yield of pure glycerol fermentation;the conversion of glycerol to 1,3-PDO in pure glycerol medium was as high as 46.4%,which was higher than that of the general glycerol-transformed strain,while the conversion rate in crude glycerol medium was only 36.8%after 36 h.Some of the impurities in the crude glycerol could promot the growth of the strain,but inhibited the conversion of the glycerol reaction to 1,3-propanediol.3.In the enzyme activity analysis,glycerol dehydrogenase(GDH),glycerol dehydratase(GDHt)and 1,3-propanediol oxidoreductase(PDOR)were more active in the logarithmic phase,generally higher than the stationary phase and the recession phase.At this time,the strain had the highest activity,and the carbon source mainly flowed to the reduction pathway during the reaction,so that the high concentration of 3-hydroxypropionaldehyde could be maintained,and less reaction by-products such as acetic acid in the early stage of fermentation,which could promote the conversion of glycerol to 1,3-propanediol.However,in the later stage of fermentation,some metabolic enzymes of similar intermediates were formed,which led to an increase in the enzyme activity measurement,but in fact the enzyme activities of the three kinds of glycerol convertases were decreased.4.Klebsiella sp.2e was used to directly ferment with crude glycerin waste,which was a by-product produced by the production of biodiesel from Swida wilsoniana oil.After 36 h,the yield of 1,3-propanediol was close to that of fermentation liquid adding the same quality pure glycerin.The fermentation broth,strain Klebsiella sp.2e,was well tolerated by crude glycerol.