Fermentation Production Of L-lactic Acid From Wastepaper By Recombinant Escherichia Coli

As biodegradable material, polylactic acid(PLA) has huge potential to be widely used and the applicaition prospect in the market is enormous. At present, the production cost and high market price of the optical pure L-lactic acid is an important factor to the development of PLA, which limits the widely use of L-lactic in the field of other important industries at the same time. Microbial fermentation is an important way to product high optical purity L-lactic acid, but it is in the lack of microbial strains which can use inexpensive lignocellulose to product high optical purity L-lactic acid fermentation at current. In this paper, a recombinant E.coli engineering bacterium WL302 was build, which can use pentose to product high purity L-lactic acid. E.coli WL302 has laid a solid foundation for the use of inexpensive lignocellulose to product high optical purity L-lactic acid fermentation.Firstly, a recombinant Escherichia coli strain for the production of high-purity L-lactic acid was constructed, using a homoethanol fermenting mutant E. coli RM10(Δfrd BC Δldh A Δack A Δfoc A-pfl B Δpdh R::pfl Bp6-pfl Brbs-ace EF-lpd Δrng HSR2) as the starting strain. By using homologous recombination strategies, we deleted the adh E gene from RM10 to obtained a mutant E. coli RM11, which could not grow under anaerobic conditions. Then the L-lactate dehydrogenase gene(ldh L) of Pediococcus acidilactici was cloned and inserted it into the chromosome of RM11 via electroporation to obtain a recombinant strain E. coli WL301. E.coli WL301 was domesticated in anaerobic tube, and the resulted strain E. coli WL302 was obtained.Secondly, the pretreatment conditions of wastepaper were further optimized by response surface method based on the single factor experiment, and obtained optimum conditions with solid-liquid ratio 1: 8.7, the concentration of sulfuric acid 0.4 mol/L, nder the condition of 12℃ and pretreatment 1.99 h. On this conditions, acid hydrolysis was adjusted to p H 4.8±0.1with Na OH, 30 FPU per g of substrate cellulase was added, and then hydrolysis 60 h at the condition of 50℃, 150 rpm, the total reducing sugar concentration in hydrolyzate is 55.86±0.91 g/L. Compared with the best conditions of the single factor experiments, the reducing sugar concentration was increased by 11% after the response surface optimization.Finally, the L-lactic acid production with different carbon source sand waste hydrolyzate of the recombinant strain E.coli WL302 in a 7 L fermenter was evaluated. When E.coli WL302 using glucose(60 g/L), xylose(60 g/L), mixed sugars of xylose(30 g/L) and glucose(30 g/L) as the carbon source fermentation, lactic acid production was 50.68 g/L, 43.62 g, 46.36 g/L respectively, mass yield was 86.72%, 80.49%, 80.71% respectively, less byproduct, the optical purity is up to 98%. Fermentation 72 h of the detoxified hydrolyzate of wastepaper with E.coli WL302, lactic acid production was43.96 g/L, and the strength of lactic acid productiom was 0.61 g/(L·h). Under these conditions, E. coli WL302 produced 31 g L-lactic acid from 100 g dry wastepaper and the lactic acid yield coefficient was 80.59%, the optical purity is up to 98%. These results demonstrate that E.coli WL302 has the potential for homofermentative production of L-lactic acid by making use of substrates derived cellulosic biomass.