Key Gene Experssion And Bioconversion Process From Lignin To PHA

Lignin is a kind of renewable aromatic polymers,which can be biotransformd for producing high-value chemicals and fuels.In this study,Pseudomonas putida KT2440 was employed to convert lignin to polyhydroxyalkanoate(PHA).The structure of acid-alkali treated lignin,alkali treated lignin,and lignosulfonates were characterized using FITR,GPC,and 2D-HSQC NMR.Microbial growth,PHA yield,and the expression of key genes in PHA synthesis were investigated to reveal the lignin bioconversion process.The main results are as follows:1.Lignin was extracted by acid-alkali treatment and alkali treatment,respectively.The acid-alkali treated lignin,alkali treated lignin and ligninsulfonate were analyzed.The S/G value of acid-alkali treated lignin was 3.17,the relative content of pCA was 96.38%,and the relative content ofβ-O-4 was 31.11%,the weight-average molecular weight(M_w)is 3410g/mol and the polydispersity index is 2.53.The S/G of alkali treated lignin was 3.58,the relative content of pCA was 73.60%,andβ-O-4 was 33.81%,the M_wis 2186 g/mol and the polydispersity index is 1.95.Lignosulfonate was introduced sulfonic groups in pulping process.Therefore,it showed better water solubility than the other two lignins.Its weight-average molecular weight was 628 g/mol,and its polydispersity index was 2.31.There was no pCA-related signal was detected in HSQC characterization.2.The expression levels of phaC1 and phaC2 genes encoding PHA polymerase and phaJ4 gene encoding enoyl-CoA hydratase in P.putida KT2440 were significantly different in fermentation utilizing different lignins.Gene expression in acid-alkali treated lignin for at fermentation for12 h was set as control group(the expression level was set as 1).After fermentation for 24 h,the acid-alkali treated lignin was 1.21,which was1.32 times of alkali treated lignin and 1.25 times of that treated by lignosulfonate,respectively.The expression of phaC2 in acid-alkali treated lignin was 5.18 and 3.36 times higher than that of alkali treated lignin and lignosulfonate,respectively.The expression of phaJ4 gene was at a low level in the whole fermentation processes.The expression of phaJ4 gene in acid-alkali treated lignin was the highest at 12 h,which was 1.56 and 2.70times higher than that of alkali treated lignin and lignosulfonate,respectively.The gene expression study showed that the acid-alkali treatment was more conducive to the producion of PHA from lignin.3.The P.putida KT2440 cell number reached 2.57×10~8CFU/m L in the acid-alkali treated lignin medium at 12 h,which was 1.36 times and2.14 times of alkali treated lignin and lignosulfonate,respectively.The PHA accumulation of acid-alkali treated lignin reached the maximum value of 0.34 g/L at 24 h,which was 1.39 times and 1.85 times higher than that of alkali treated lignin and lignosulfonate,respectively.Biomass and PHA accumulation indicated that the conversion efficiency of acid-alkali treated lignin was higher than that of the other two lignins.Through structural characterization,expression levels of related genes,and PHA production,it can be concluded that acid-alkali treatment facilitates biological lignin valorization to PHA.