Screening And Fermentation Of 1,3-propanediol From Glycerol By Mixed Cultures Of Klebsiella Pneumoniae

Microbial fermentation of 1,3-PDO has attracted much attention because of its green production. Up to date, most researches have focused on fermentation optimization of 1,3-PDO using pure culture. However, pure culture has the problems of poor substrate tolerance, low product conversion rate, multi-product, difficult product separation and susceptible to phage infection, which limit the large-scale industrial production of 1,3-PDO. While the mixed fermentation has the advantage of multiple bacterial symbiosis and complementary enzymes, but it is mostly limited to the traditional areas of food, waste water treatment and biogas production. The literature on mixed fermentation of 1,3-PDO is few. To solve these problems, this paper focused on production of 1,3-PDO from fermentation of glycerol with mixed cultures. Mixed culture was screened from Dalian sea samples, then purification and identification were carried out, and the fermentation characteristics of strains were explored.Firstly,40 samples of barnacles, seaweed, sponges and sea mud were collected from Dalian, and then 16 groups of strains were selected using glycerol as the sole carbon source. Among them the mixed culture NHN8 selected from sea mud had a relatively high yield of 1,3-propanediol and stable fermentation performance.Secondly, isolation, purification of identification of NHN8 were carried out. The results showed that mixed culture NHN8 was composed of three single strains:W3, Y1 and Y5, which were all K. pneumoniae. They were collected in China General Microbiological Culture Collection Center (CGMCC) and the accession numbers were CGMCC NO.10440, 10439 and 10438, respectively.Thirdly, the morphology and fermentation characteristics of three pure culture were observed and studied. K. pneumoniae W3 had smooth and milky white colonies, growed faster, and produced 1,3-PDO as main product and a small amount of 2,3-butanediol as byproduct. K. pneumoniae Yl’s colonies contained a single large bubble, its growth was relatively slow, and the main product was ethanol. K. pneumoniae Y5’s colonies contained small and dense bubbles, the growth rate was between W3 and Y1, the main products were 1,3-PDO and ethanol. Neither of Yl and Y5 produced 2,3-butanediol.Finally, the effects of glycerol concentration and inoculum ratio on the fermentation of mixed culture were investigated. The results showed that NHN8 had a good substrate tolerance. The initial glycerol concentration of mixed fermentation was 40～200 g/L, preferably 120 g/L. The inoculum ratio of K. pneumoniae W3, Y1 and Y5 was 150～250: 10～20:50～100, preferably 208:17:82.Experiments showed that mixed fermentation overcame the shortcomings of poor substrate tolerance for pure culture, improved production efficiency, and no 2,3-butanediol was detected in fermentation broths, which was benefitful for the next separation process. Mixed culture could tolerate 200 g/L glycerol concentration, and produce 81.40 g/L 1,3-PDO. This method was expected to provide simple and economical fermentation process for the industrialization of microbial fermentation of 1,3-PDO.