Lactate-tolerant Adaption Of An Engineered Escherichia Coli HBUT-L And Its L-lactic Acid Fermentation

As a monomer of polylactic acid which is an important alternate for plastic,there is a great demand for L-lactic acid. Recently,engineered E.coli strains has played an important role in L-lactic acid production.Escherichia coli strain has many advantages,such as a clear genetic background,shorter fermentation period and simple nutritional requirements,what’s more,it is also very easy to be genetically engineered.Thus it has played an important role in L-lactic acid production in recent years. However,the production of L-lactic acid is relatively lower in the fermentation by Escherichia coli compared with other srains.This is due to the glucose repression and the inhibition of lactic acid,as a result that the production of L-lactic acid has been limited.Early in our lab, E.coli HBUT-L16 was engineered to produce L-lactic acid with high optical purity in mineral salt medium.We try to improve L-lactic acid production and conversion rate by using adaptive evolution and feed methodology.The results are as follows:(1) A previously engineered L-lactic acid producing E.coli strain, HBUT-L, was adaptively evolved for 28 generation in the presence of 2%~12% sodium lactate.The evolved strain has been named HBUT-L16. The results showed that HBUT-L16 produced 36.6% more L-lactic acid and 42.3% more biomass compared to that of the parent strain HBUT-L in batch fermentation using a 16% initial glucose.(2) The influence of different neutralizers on the fermentation by HBUT-L16 has been studied.In this study, a 10% initial glucose has been used.The yield of L-lactic acid and the productivity was respectively for 98.23 g/L and 3.51 g/(L·h) using Ca(OH)2 as the neutralizer.While the yield and conversion rate and the productivity was respectively for 95.19 g/L and 2.64 g/(L·h) using KOH as the neutralizer.The yield and productivity of L-lactic acid was the lowest when NH4 OH was used as the neutralizer,respectively for 81.27 g/L and 1.69 g/(L·h).(3) Feed methodology was adopted to minimize glucose catabolite repress.The results showed that in the fed-batch fermentation using an 8%+8%+4% glucose feeding pattern,HBUT-L16 produced 186.8 g/L of L-lactic acid in 42 h, with a conversion rate of 93.4%,a productivity of 4.45 g/(L·h), and an optical puring of 99.6%. Compared to that of batch fermentation using an initial 20% glucose, fed-batch fermentation achieved an increase of 24% and 23.6%, respectively for product titer and conversion rate, and a more then 50% decreased fermentation time.(4) We compared the immobilization effects of five kinds of embedding methods with sodium alginate as embedding material. Finally,sodium alginate embedding method was choosen as the best embedding method. Sodium alginate embedding method has been optimized through the orthogonal experiment to produce L-lactic acid by E.coli HBUT-L16.The optimization results were as follows: the concentration of sodium alginate was 2%, the concentration of CaCl2 was 3%,and the hardening time was 4 h. Under this condition,the cell of E.coli HBUT-L16 has been immbolized,and the yield of L-lactic acid is 39.56 g/L in the fermentation. Compared with single factor optimization results, the yield of L- lactic acid has increased by 7.7%.The production of L-lactic acid kept 38.27 g/L when the immbolized gel beads had been used for 9 times.