Non-Sterilized Fermentation for Ethanol or L-Lactic Acid from Waste Substrate By Metabolic Engineering of Thermoanaerobacterium Sp. strain

Thermoanaerobacterium aotearoense SCUT27 is one kind of thermophilic, obligate anaerobic bacterium, which could assimilate a variety of carbohydrates from lignocellulosic biomass, to produce primarily L-lactic acid, ethanol, acetic acid and hydrogen with little carbon catabolite repression. Since its unique culture condition, this strain can ferment mixed substrate with non-sterilized condition. In our previous study, we have redesigned and engineered the carbon flux of T. aotearoense SCUT27 to generate some mutated strains which can achieve higher yields of products. Herein, we will show the distinct improvements of ethanol and lactic acid production from lignocellulosic derived sugars by different mutants derived from T. aotearoense SCUT27.

In the first place, we blocked the lactate metabolic pathway (deleted the ldh gene) and redistributed total cellular NADH, to acquire an increase of 2.37 folds for ethanol yield compared to the wild strain, accompanied by 2 folds of hydrogen yield. By using 80 g/L glucose plus 40 g/L xylose as substrate in 5 L fermentor, 31.5 g/L of ethanol concentration was obtained with the yield of 1.5 mol/mol. Meanwhile, there was no lactic acid formation during all the fermentation which can dramatically reduce the cost of purification of the final product and facilitate fermentation to industrial production.

In the second place, the acetic acid synthetic pathway of T. aotearoense SCUT27 was deleted by genetic manipulation to enhance the optically pure L-lactic acid production, and the mutated strain of which is named as LA1002. With 50 g/L of substrate in bioreactor, the maximum L-lactic acid yield of LA1002 could reach 0.93 g/g glucose with an optical purity of 99.3%, which is very close to the theoretical value (1.0 g/g of glucose). Moreover, an advanced biorefinery technology was established to convert mixed bakery waste as a nutrient rich feedstock to optically pure L-lactic acid. After hydrolyzation by A. awamori and A. oryzae, a nutrient-rich hydrolysate was obtained from the mixed bakery waste collected from Starbucks, which contained 83.6 g/L glucose, 9.5 g/L fructose and 612 mg/L free amino nitrogen. By using an evolved T. aotearoense LA1002-G40, the overall lactic acid produced from mixed bakery waste was 0.18 g/g under non-sterile conditions, and the titer of which was 78.5 g/L. Here we proposal an innovative procedure involving a complete bioconversion process for L-lactic acid production from mixed bakery waste with non-steriliztion.