The Production Of 1,3-propanediol From Crude Glycerol By Microbial Consortia

1,3-Propanediol(1,3-PD)is a versatile bulk chemical with numerous possible applications,in particular as a monomer to produce new type of polysesters,such as polytrimethylene terephthalate(PTT)and polypropylene furandicarboxylate(PTF).So far,1,3-PD can be produced by either chemical synthesis or microbial fermentation.Considering the advantages of using renewable resources,mild conditions and environmental protection,biological method has attracted more and more attention.Microbial production of 1,3-PD is mainly based on microbial pure culture technology,which needs strictly aseptic condition,substrates with a certain purity,and genetates many by-products,such as acetic acid,lactic acid,ethanol,2,3-butanediol(BD)and so on,leading to much difficulty in the downstreaming separation and purification and much cost of 1,3-PD.In order to solve the disadvantage of pure culture,microbial consortia were selected and used to produce 1,3-PD from crude glycerol in this work.The interaction relationship of single strain in the microbial consortium,the stability of microbial consortium and the effect of community composition of microbial consortium on its function have been investigated.The main results are obtained as follows:(1)Two microbial consortia DL38 and CJD-3 were selected form Dalian seashore,and used to produce 1,3-PD from glycerol in batch fermentations.The experimental results showed that the concentration and yield of 1,3-PD produced by microbial consortium DL38 were higher and the concentration of by-products were lower than those by microbial consortium CJD-3.The by-products of both consortia did not contain BD,which was beneficial for the purification of 1,3-PD and reducing the production cost.In batch fermentation,microbial consortium DL38 could grow well at an initial glycerol concentration of 200 g/L to produce 81.40 g/L of 1,3-PD with a yield of 0.63 mol/mol.The bacterial community compositions of microbial consortia DL38 and CJD-3 were analyzed by microbial 16S rRNA gene amplicon high-throughput sequencing and showed that a good relationship between fermentation performance and the composition of microbial consortium.The proportion of Enterobacteriaceae that mainly produces 1,3-PD in microbial consortium DL38(95.57%)was significantly higher than that in CJD-3(85.99%).Three strains of Klebsiella pneumonia were isolated from microbial consortium DL38.The primary product of isolated strain W3 was 1,3-PD(17.84 g/L),however,strain Y5 produced 1,3-PD(9.63 g/L)and ethanol(7.60 g/L),strain Y1 produced ethanol(12.69 g/L)as the main product,respectively,when 40 g/L glycerol was used as carbon resource.The batch fermentation performance with the natural proportion(W3:Y5:Y1 = 208:82:17)was superior to that with other proportions and single strain W3,which demonstrated that microbial consortium under the natural state had the superiority and rationality and the synergy relationship between the strains improved the substrate tolerance and 1,3-PD production.(2)The key factors influencing on microbial consortium stability and fermentation performance were investigated by batch fermentations and community composition analysis.The experimental results showed that the fermentation performance of microbial consortium DL38 changed with the increasing number of subcultures(microbial consortium DL38 was renamed as DL38-BH),such as the longer fermentation time,the lower concentration and yield of 1,3-PD,and the higher concentration of lactic acid and ethanol.The community composition of microbial consortium DL38 also had chaged dramatically,such as the proportion of Enterobacteriaceae in microbial consortium DL38 significantly reduced from 95.57%to 71.46%,and that of Enterococcaceae and Moraxellaceae markedly increased from 2.10%and 1.21%to 15.31%and 12.97%,respectively.Comparision of fermentation performance showed that the glycerol consumption prolonged,the yield of 1,3-PD reduced,and the concentration of lactic acid and acetic acid increased in batch fermentations with strain W3-BH;the glycerol consumption promoted,the yield of 1,3-PD reduced,and the concentration of lactic acid and acetic acid increased in batch fermentations with strain Y5-BH;the glycerol consumption also promoted,the yield of 1,3-PD,lactic acid and acetic acid increased,but the yield of enthanol decreased in batch fermentations with strain Y5-BH.All of those mean that the changes of community composition of microbial consortium and the fermentation performance of single strains lead to the change of the fermentation performance by microbial consortium DL38-BH.(3)The effect of 1-ethyl-3-methylimidazolium trifluoromethanesulfonate([Emim][TfO])on the capacity of 1,3-PD production from crude glycerol was evaluated by microbial consortium DL38-BH.The experimental results demonstrated that the effect of ionic liquid on the fermentation performance of microbial consortium was not only related to the type of ionic liquid,but also to the concentration of ionic liquid and substrate.In the batch fermentation at 60 g/L crude glycerol and 10 g/L[Emim][TfO],the concentration and yield of 1,3-PD from glycerol increased from 23.14 g/L and 0.45 mol/mol to 31.17 g/L and 0.60 mol/mol,respectively.Compared to the control,the number and proportion of Klebsiella genus which could convert glycerol to 1,3-PD increased significantly from 51346 and 79.19%to 55514 and 89.49%,respectively,and the other genera that did not produce 1,3-PD were dramatically decreased(P<0.05)at the end of batch fermentation with[Emim][TfO]addition.The activities of three key enzymes(GDH,GDHt and PDOR)in glycerol metabolism were stimulated by the increase in content of Klebsiella in microbial consortium DL38-BH during the batch fermentation with[Emim][TfO]addition,leading to the improvement of concentration and yield of 1,3-PD.In the batch fermentation by single strain,the ionic liquid[Emim][TfO]influenced the kinds and yield of the by-products,but there was no significant effect on the concentration and yield of 1,3-PD.A conclusion was drawn that[Emim][TfO]influenced fermentation performance by adjusting community composition of microbial consortium.(4)The effect of community composition change on the fermentation performance of microbial consortium was investigated by antibiotics and dominant species addition.The experimental results showed that the proportion of Klebsiella in microbial consortium DL38-BH was improved from 85.86%to 94.98%during batch fermentation with ampicillin addition,the comsumption of glycerol was faster,but the yield of 1,3-PD did not increase.The reason may be that the proportion of strain Y5-BH and Y1-BH which mainly produced enthanol increased according to the increase of ethanol.By increasing the proportion of strain W3-BH and Y5-BH in microbial consortium DL38-BH to adjust community composition,the fermentation performance of microbial consortium DL38-BH was improved,such as the fermentation time was shorter(from 30 h to 26 h),and the concentration and yield of 1,3-PD increased from 21.31 g/L and 0.25 mol/mol to 37.16 g/L and 0.35 mol/mol,respectively.In conclusion,the fermentation technology by microbial consortium can substitute for pure culture technology to solve the industrialized problems,such as difficult utilization of cheap substrate,low conversion rate of substrate,many by-products,high cost of fermentation and separation and so on,which provides references for the production of other bio-based chemicals by microbial consortium.