Optimization Of The L-lactic Acid Fermentation Conditions Of An Alkaliphile And Construction Of Its Genetic Transformation System

Lactic acid is an important platform chemical. Especially with rapid development of poly(lactic acid) industry, the demand for L-lactic acid is continuously increasing. This thesis focused on production of L-lactate by an alkaliphile Exiguobacterium sp.8-11-1, and try to build its genetic manipulation system. The results were as follows:1. When the additional NaCl concentrations varied from0to10%(w/v) in the L-lactic acid fermentation medium, the glucose consumption and lactic acid production rates by Exiguobacterium sp.8-11-1were almost consistent. So we choose sodium hydroxide as neutralizing agent.2. Fermentation conditions of strain8-11-1was optimized. Optimal temperature and pH were37℃and8.5, respectively. Optimized initial glucose concentration and yeast extract concentration for use in subsequent fermentations were80g/L and10g/L, respectively.3. In single-pulse fed-batch fermentation under sterile conditions,124.33g/L L-lactic acid was produced with a yield of98.01%and an average productivity of3.77g/L/h. No D-isomer was detected.4. Under nonsterile condition,125g/L L-lactic acid was produced with a slightly higher yield and average productivity of98.33%and3.79g/L/h, respectively, as compared to the data obtained under the sterile condition.5. Whole-genome sequencing of8-11-1was completed using Solexa paired-end sequencing technology. There were5,212,033filtered paired-end reads were obtained, with an average of61-fold coverage of the genome. After all reads were assembled, the genome sequence shows that it has a GC content of54%.2926coding sequences were annotated by COG, KEGG and nr databases. Exiguobacterium sp.8-11-1has the genes encoding xylose isomerase, L-lactate dehydrogenase, and permease, which indicates that strain8-11-1is a potential producer of L-lactic acid from cellulosic substrate. There are134genes for inorganic ion transport and metabolism, including1MnhB-type Na+/H+antiporter and5NhaC-type Na+/H+antiporters, which may contribute to pH homeostasis and the capacity of the cells to lower the cytoplasmic Na+concentration optimally at the alkaline pH.6. We tried to develop the genetic manipulation system of Exiguobacterium sp. strain8-11-1, by using electroporation transformation and protoplast transformation procedures.