New Method For The Production Of L-lactic Acid By Pretreatment Of Lignocellulose

In this study,corn stover,a common waste product from agriculture,is composed of about 40%cellulose,15%hemicellulose and 20%lignin and is a potential source for the production of second generation biofuels and high value bio-chemicals.In this study,the microstructure of lignocellulose was broken by aqueous pretreatment with the inorganic alkali CaO and aqueous Na OH at high temperature and inorganic acid-base pretreatment,produceing L-lactic acid（L-LA）based on a simultaneous saccharification and fermentation process.In this process,single-step alkali pretreatment and two-step acid-base pretreatment showed different effects in promoting enzymatic saccharification.The experimental results showed that the black liquor component after the single-step alkali pretreatment could partially replace the water from the culture environment for reuse in the simultaneous saccharification and fermentation process,reducing the discharge of process effluent.Under optimised conditions 0.1 mol/L 180 ^oC CaO pretreatment of the residue yielded 442 g total sugar per kg of straw,culminating in 72 g/L L-LA,90%sugar-acid conversion and a 2.25 g/L·h temporal conversion by fed-batch of the simultaneous saccharification fermentation.Secondly,in order to achieve the objective of multi-stage utilisation of L-LA from lignocellulosic biomass,ultimately recovering 90%of the lignin component in the lignocellulosic biomass by adjusting the p H of the neutralised black liquor to be reused in the simultaneous saccharification fermentation additionally adopted inorganic acid-base sequential pretreatment to neutralise the acid-base black liquor and precipitate the lignin component to characterise it.This method reduces the discharge of black liquor from the pretreatment process by37%-40%.In order to improve the yield and purity of the final fermented L-LA and reduce the difficulty of the downstream isolation and purification process,this study attempted to improve the efficient conversion of the substrate carbon metabolic flow to the target product by metabolic engineering means to knock out a series of byproduct metabolic pathways existing in the bacterium,such as acetic acid ethanol and other small molecule products with chemical properties close to lactic acid.Since there is a mismatch between the optimal p H for strain growth and the optimal p H for enzymatic digestion in the synchronous saccharification fermentation process,we tried to improve the growth and metabolic capacity of the bacterium in acidic environment through appropriate metabolic engineering modification combined with the addition of a certain amount of exogenous amino acids.This approach improved the final cell density OD₆₀₀ to 6.17 at p H=5 and maintained the cellular conversion rate of L-LA production at 90%,reducing the addition of 60.6 g of neutralizer.In order to improve the enzymatic efficiency of the simultaneous enzymatic clear liquor fermentation process,so that the cells can obtain sufficient substrate carbon source to maintain the normal growth of cells and improve the resistance of cells under the harsh environment.In this study,an attempt was made to enhance the enzymatic yield by adding three different types of cellulase active reagents.The final enzymatic sugar concentration was increased from 30 g/L to 47 g/L at Li Cl:Tween 80:BSA=2:2:0.And the Na OH pretreated residues was used to compare the enzymatic clear liquor fermentation and the simultaneous saccharification fermentation.The L-LA concentrations of 103 g/L and 105 g/L and 92%sugar-acid conversion were achieved in both ways,respectively,which verified that the organism could produce L-LA at a productivity of 0.97 g/L·h under batch replenishment simultaneous saccharification fermentation.