D-lactic acid production from starch by Lactobacillus plantarum D1X1 and purification

Abstract

Lactobacillus plantarum D1X1 is a genetically modified from an amylolytic lactic acid bacterium L. plantarum S21 by deletion of L-lactic acid producing genes. Investigation for growth and lactic acid production found that L. plantarum D1X1 has ability to produce optically pure D-lactic acid of 8.56 g/L which is calculated to be 0.85 gegs of lactic acid production efficiency and 0.97 of lactic acid yield, when cultivated at 37 *C for 48 hours in modified MRS broth containing 10 g/L of starch as carbon source. The maximum viable cell of 10' CFU/mL and maximum amylase activity of 8.4 U/mL were achieved at 12 hours and 48 hours of cultivation, respectively. The optimum incubation temperature and initial pH of medium for lactic acid production were 37*C and 6-7, respectively. The pH controlling value at 6-7 with NaOH during fermentation period showed the increasing of viable cell growth and amylase activity when compared with non-controlled condition. The appropriate low-cost carbon and nitrogen source to replace the high-cost ingredient composed in mMRS were cassava starch and com steep liquor powder (CSLP), respectively. The evaluation of the most significant medium compositions effecting on lactic acid production was conducted by Plackett-Burman design (PBD). It was found that only cassava starch and CSLP showed the significantly positive effect on lactic acid production. The low-cost production medium obtained from central composite design (CCD) composing of 140.5 g/L cassava starch and 120 g/L CSLP, respectively. The maximum lactic acid produced from low-cost production medium was 64.9 g/L with productivity at 24 and 48 hours of fermentation were 1.47 and 1.35 g/L-h, production efficiency 0.46 gp/gs, and 0.82 gp/gs of lactic acid yield. To improve the production efficiency, the effect of inoculum size and optimized medium for inoculum were investigated. The result found that 10% (v/v) of inoculum showed the highest amylase activity at 3.65 U/mL in low-cost production medium. The lactic acid production in modified MRS medium was used for comparison, the productivity at 24 and 48 hours of fermentation were 4.12 and 2.45 g/Lㆍh, lactic acid yield 0.88 gp/gs and generated more lactic acid at 118.88 g/L with production efficiency of 0.84 gpgs. This result was better than lactic acid production in low-cost production medium. Then, the low-cost inoculum medium was conducted with PBD revealed that cassava starch and CSLP were the two factors significantly influencing amylase activity and the low-cost inoculum medium composition obtained from CCD were 116 g/L cassava starch and 110 g/L CSLP. The reduction of 10 g/L CSLP in the improved medium made the decreasing of enzyme inhibitor in the medium and the amylase activity obtained from low-cost inoculum medium was raised to 7.97 U/mL which was higher than using low- cost production medium as inoculum around 2.5 times. The lactic acid fermentation in 1-L fermenter using the low-cost production medium with 10% (v/v) low-cost inoculum medium was carried out at 37 *C for 48 hours with pH controlled at 6.5. It was found that the maximum lactic acid was 110.12 g/L, 0.78 gp/gs production efficiency, and 0.82 gp/gs of lactic acid yield. The productivity at 24 and 48 hours of fermentation were 3.82 and 2.29 g/Lㆍh which closed to modified MRS medium at 4.12 and 2.45 g/L-h. Lactic acid fermentation in large scale was investigated in 10-L fermenter with the same medium in 1-L scale. The highest lactic acid was 109.67 g/L with productivity at 24 and 48 hours of fermentation 3.58 and 2.28 g/L h, production efficiency 0.78 gp/gs, and 0.83 ge/gs of lactic acid yield. Moreover, L. plantarum D1X1 showed the ability to produce lactic acid in repeated batch fermentation. The average lactic acid concentration was around 109.67 g/L with stable production efficiency and lactic acid yield around 0.78 giegs and 0.80 gpgs. The pure D-lactic acid was maintained at 99% along the 5 cycles of fermentation process. In the part of lactic acid purification, solvent extraction technique was used for preliminary extracting lactic acid from fermentation broth using ethyl acetate as an extractant. The result found that D-lactic acid was successfully purified from the fermentation broth with final concentration of 815.10 g/L and approximately 99% of lactic acid solution purity from preliminary test. The results obtained from these experiments revealed the optimum condition for D-lactic acid fermentation by L.plantarum D1X1. The low-cost inoculum medium and low-cost production medium for D-lactic acid production were cheaper than mMRS approximately 35 times. The production process was successfully substantiated in 1-L and 10-L scales. Moreover, the repeated batch fermentation process was also validated. All fermentation processes shows the stable of production efficiency and yield of lactic acid. In addition, D-lactic acid purification from fermentation broth was successfully achieved and obtained the high lactic acid concentration and solution purity. It can conclude that, the knowledge that achieved from this experiment can be applied for D-lactic acid production and purification for industrial scale.