Lactic acid production by Furfural-Tolerant Lactic acid bacteria

Abstract

This research aimed to isolate the furfural tolerant lactic acid-producing bacterial strain which is capable of utilizing lignocellulosic material as substrate and producing optically pure L- or D-lactic acids. Among the 407 lactic acid bacterial isolates obtained from Miang, a traditional fermented tea from north Thailand, 25 isolates were selected according to their ability to grow in MRS broth containing both glucose and xylose as the sole carbon source. Identification of these isolates indicated that 20 isolates were Lactobacillus pentosus, 2 isolates were Lactobacillus fermentum, and 2 isolates were Lactobacillus lactis and the remaining was Pediococcus pentosaceus. All isolates were capable of furfural tolerance and utilizing both glucose and xylose as the carbon sources, however only 17 isolates produced high level of total acids (>10 g/L) from MRS containing 20 g/L glucose as a carbon source. Study on lactic acid production in MRS containing mixed glucose and xylose as the carbon sources found that only 4 isolates, L. pentosus Y23, L. pentosus CMY28, L. pentosus A1-1 and L. pentosus CMY46 showed the conversion of mixed sugar to lactic acid higher than 70%, growth in MRS broth supplemented with 6 g/L furfural. However, all 17 isolates produced mixed L- and D- lactic acid as the final products. The study on bacteria from the gut of Eri silkworm found totally 3.95 x 107 CFU/g of bacteria. From random selection of 790 isolates, 654 isolates were capable of growth on nutrient agar (NA) containing xylose as a sole carbon source but there were only 504 isolates showed acid producing ability. However, among 504 isolates, only 51 isolates were able to grow and produced acid in de Man Rogosa Sharpe (MRS) agar containing xylose as a carbon source. Thirty-nine from 51 isolates showed the ability in acid production within 12 h and there were only 12 isolates (WES1, WXI, WX3, WG1, WG2-3, WX1-4, WX2, UDX1, SC1, SX2, SCT2 and MJG2) were able to grow and produced optically L-lactic acid both at 45°C. The isolate WX1 was found to produce optically pure L-lactic acid from glucose, xylose and arabinose and also able to grow in MRS medium supplemented with 2 g/L furfural. The strain WX1 was identified to be Enterococcus mundtii based on 16S rRNA gene sequence analysis. The study of optically L-lactic acid production by E. mundti WX1 in the various ratios of mixed glucose and xylose at 37°C indicated an occurring of glucose repression effect on xylose consumption due to glucose was firstly utilized in bacterial growth and production of L-lactic acid while xylose was also utilized with the low consumption rate. To overcome the limitation of mixture sugar utilization, the co-culture of E. mundti WX1 and Lactobacillus rhamnosus SCJ-9, a homofermentative lactic acid bacterium capable of L-lactic acid production from only glucose, was performed in glucose/xylose mixture in a ratio of 60:40. L-Lactic acid production by co-culture was achieved up to 8.5 g/L from total sugar concentration of 10 g/L. To investigate the optimal medium for production of L-lactic acid by co-culture strategy, the statistical experimental Plackett-Burman design was applied to screen the significant influencing nutritional compositions on L-lactic acid production. Among eigh medium compositions based on MRS medium including Tween80, yeast extract, peptone, beef extract, MgS04-7H2O, K2HPO4 and ammonium sulfate, the result indicated that Tween80, MnSO4 and yeast extract were identified as the significant nutritional factors influence on L-lactic acid production (p<0.05). The optimal concentrations of these three variables revealed by central composite design (CCD) and response surface methodology (RSM) were 20.42 g/L yeast extract, 9.8 g/L Tween80 and 1.18 g/L MnSO4 with a predicted value of 18.8 g/L L-lactic acid from 20 g/L carbon source (glucose/xylose mixture in a ratio of 60:40). Furthermore, investigation on the effect of sugar mixture concentration in the rage of 20-100 g/L on L-lactic acid production by co-culture found that high L-lactic acid yield 0.98 g/g at 24 h when cultivated using 20 g/L of mixed sugar and high yield of 0.8 g/g was also observed at 40 g/L of mixed sugars. Corn stover was used as substrate for L-lactic acid production by co-culture of E. mundtii WX1 and L. rhamnosus SCJ-9. The chemical composition of corn stover was analyzed and found 27.16% hemicellulose, 31.39% cellulose, and 4.14% lignin. Pretreatment process with 1-4% H2SO4 and formic acid were investigated. The dilute acid pretreatment using 1% H2SO4 and formic acid followed by heating with steam at 121°C for 30 min showed the highest remaining of hemicellulose and cellulose in the substrate. In order to investigate changes in sugar released from substrate, the enzymatic hydrolysis of pretreated corn stover by using Cellic CTec2 was applied at the rage of 10-40 FPU/g DM followed by incubation at 50°C for 48 h. The maximum released sugar was 35 g/L from corn stover hydrolysis by 30 and 40 FPU/g DM. The 250 g of corn stover pretreated with 1% H2SO4 at 121°C, 30 min followed by enzymatic hydrolysis with Cellic CTec2 30 FPU/g DM at 50°C, for 48 h were investigated and fermenting sugar released was glucose 67.18 g and xylose 15.75 g. Then, L-lactic acid fermentation by co-culture strategy using pretreated corn stover as carbon source supplemented with optimum medium was investigated at 37°C for 48 h in 2.5 L jar fermenter. The L-lactic acid of 66.25 g was obtained at 48 h of fermentation, which was calculated to the yield of 0.8 g/g and the maximum productivity was 1.1 g/L/h at 24 h of fermentation time. The purification of L-lactic acid from 7 L fermentation broth was performed using ion- exchange and electrodialysis, the purified L-lactic acid was achieved with 50.18% recovery.