Utilization Of Lignocellulosic Biomass For Two-step Step Fermentation Of L-lactic Acid

In recent years, escalating global energy and environmental problems have stimulated researchers worldwide to develop methods for producing almost everything through green methods. Lactic acid producing from cheap lignocellulosic biomass is one of them and has attracted a great deal of attention. Biodegradable polylactic acid (PLA) which is processed from L-lactic acid is a green alternative to petroleum-derived plastics. However, high production costs have hindered the large-scale application of PLA because of the high price of lactic acid. Therefore, reduction of lactic acid production cost through utilization of inexpensive substrates and improvement of lactic acid production and productivity has become an important goal. At present, there are three major bottleneck problems that constraint high cost of L-lactic acid production: First, the high cost of raw materials. Secondly, low enzymatic hydrolysis yield in cellulase enzymatic hydrolysis stage, resulting in a low concentration of reducing sugar in the hydrolyzate. Thirdly, high cost of fermentative substrates of nutrients, expensive nitrogen sources and neutralizing agents.Agricultural wastes, sugarcane bagasse, were used for raw materials in this paper. The material was pretreated by physical chemical method and the applications of Compound Mutation breeding process was used to obtain high L-lactic acid production strains. Aspergillus niger was used for producing cellulose and xylanase in the first-step fermentation, then material was enzymes hydrolyzed to provide sugar for second-step fermentation, in which Lactobacillus casei was used for producing L-lactic acid. This study mainly includes the following aspects:(1) Fungus fermentation of lignocellulosic biomass. The optimal condition for the first-step fermentation was as follows: inoculum dosage of6%, solid-to-liquid ratio (w/v) of1:8, fermentation temperature of30℃, fermentation period of76hours.(2) The pretreatment process of lignocellulosic biomass. Dilute alkali rubbing pretreatment was studied to enhance hydrolysis. It was found that NaOH density of0.5%, dosage of2ml for every gram of sugarcane bagasse, with the temperature of45℃. Sugarcane bagasse was rubbed every4hours for12hours. Then it was neutralized with0.1mol/L HCl. Result showed that reducing sugar concentration in pretreated material hydrolysis was65.6%higher than that of unpretreated.(3) Enzymatic Hydrolysis of lignocellulosic biomass. Various factors were studied, including substrate concentration, pH, enzyme dosage, temperature, and period, in order to find a cost-effective and efficient enzymolysis process. Orthogonal test was carried out to study the three main factors, pH, enzyme dosage, temperature. Result showed maximum concentration of reducing sugar of54g/L was observed when substrate concentration was8%with the enzyme dosage6.9ml, pH4.3, temperature43.9℃and total period14h.(4) Compound Mutation and breeding of high L-lactic acid production strains. Diethyl sulfate(DES) treatment combined with UV mutagenesis technology was used and a mutant strain NE-19was achieved with lactic acid concentration of57.2g/L in the temperature of40℃after48hours fermentation, increased by34.6%than the original strain. under the optimal condition for UV and DES mutagenesis was as follows; mutation period of25seconds, DES concentration of1%, treatment period of20minutes.(5) Lactic acid fermentation process from sugarcane bagasse enzymatic hydrolyzate by mutant strain. Single factor of fermentation medium was optimized and the best fermentation medium was obtained: inoculum dosage of8%, reducing sugar density of110g/L, peptone15.9g/L, beef extract11.4g/L, yeast extract5.6g/L, anhydrous sodium acetate2g/L, MgSO47H2O0.2g/L, MnSO40.075g/L, CaCO345.5g/L, Twain-801ml and pH6.7. Under the above conditions, the lactic acid production reached to70.2g/L when120h under the temperature of41℃with the residual sugar13.6g/L only.