| D-lactic acid is the monomer to synthesize poly(D-lactic acid)(PDLA).At present,Dlactic acid is mainly produced by microbial fermentation using starch crops.It is of great significance to produce D-lactic acid and synthesize cyclic D-lactide as the precursor of PDLA using lignocellulose with great abundance and wide sources as feedstock.However,the major obstacles for D-lactic acid production and D-lactide synthesis from lignocellulose are as follows:(1)A considerable number of inhibitors released from the pretreatment of lignocellulose will not only inhibit the growth of fermentation strains and the fermentation efficiency of D-lactic acid,but also adversely affect the recovery and purity of D-lactic acid;(2)Non-glucose sugars(xylose,galactose,mannose,arabinose)released after the pretreatment of lignocellulose accounted for 30-40%of the total monosaccharides.These sugars can not be completely consumed by the strain into a high concentration of D-lactic acid,and the residual sugars will affect the yield of D-lactic acid and the later separation and purification of D-lactic acid.The pH of fermentation broth decreased with the accumulation of lactic acid during the fermentation process of lactic acid,and results in the inhibition to the growth and metabolism of the strain.The addition of a large number of neutralization agents was required to keep a suitable pH environment for the cell growth,which results in a large number of high-salt wastes in the late separation and purification processes of lactic acid.The first part of this study is the production of D-lactic acid by lignocellulosic dry biological refining and its purification.A unique fungus Amorphotheca resinae ZN1 screened by our laboratory was applied for the biodetoxification of the dry acid pretreated lignocellulosic biomass.The furan and organic acid inhibitors were completely removed and a small amount of residual phenolic inhibitors are completely removed in the subsequent decolorization step.Simultaneous saccharification and co-fermentation of wheat straw was carried out at 30%(w/w)solids loading and high temperature(50℃)by the engineered D-lactic acid producing strain Pedioccocus Acidilactici ZY15.Finally,glucose and non-glucose monosaccharides(xylose,arabinose,mannose and galactose)were all converted into D-lactic acid with high concentration(128.1 g/L)and high chiral purity(99.07%),and the residual sugar content was less than 1.8g/L.Cellulosic D-lactic acid fermentation broth was conducted by the conventional separation and purification steps,including solid-liquid separation,secondary activated carbon decolorization,concentrated crystallization,acidification and adsorption of cation resin.The residual sugars,proteins,acetic acid and total phenols in the fermentation broth were completely removed.Mg2+was completely removed and the concetration of Ca2+,K+,Na+ and Cl-was reduced to 2.2-3.6 mg/g D-lactic acid.Chiral D-lactic acid with polymerization level was obtained.The second part of this study is the synthesis and characterization of D-lactide.The cellulosic D-lactic acid was a polymeric monomer,and D-lactide was successfully synthesized by the polycondensation and depolymerization of cellulosic D-lactic acid with the conventional stannous octoate catalyst.The structure and properties of D-lactide from two sources were characterized by elemental composition,melting point,molecular weight,H-NMR,IR and chiral purity with D-lactide synthesized from starch-derived D-lactic acid as control.The results showed that the characterization of D-lactide from the two sources were consistent and in agreement with that in relevant literature reports.This study verified the feasibility of producing high chiral purity of D-lactic acid and synthesizing D-lactide as PLDA precursor using lignocellulosic biomass,and provides a feasible method for the industrial application of PDLA production using lignocellulose in the future.In the third part of this study,adaptive evolution method was applied to improve the low pH tolerance and fermentation performance of P.acidilactici ZY271 under low pH condition.Lactic acid fermentation was carried out at pH 4.0 using synthetic medium and wheat straw feedstock by simultaneous saccharification and co-fermentation.The adaptively evolved strain produced 23.5 g/L and 31.4 g/L of L-lactic acid using the synthetic medium and wheat straw feedstock,respectively.The production of L-lactic acid increased by 34.7%and 88.5%compared with the starting strain.This study provides the reference value for improving the acid tolerance and fermentation performance of strains under low pH condition. |
