Study On The L-lactic Acid Production From Jerusalem Artichoke

Recently, the sharply enlarged manufacture of the bio-degradable polylactide polymer has increased the global interest in the production of L-lactic acid by using fermentative route. Jerusalem artichoke can grow well in poor land and shows a high tolerance to frost and various plant diseases. However, there have been few studies on the L-lactic acid production from Jerusalem artichoke, which was not fermentable for Lactobacillus sp. In this dissertation, Aspergillus niger SL-09, an active inulinase producer was used to form the inulinase, and Lactobacillus sp. G-01 as a parent L-lactic acid production strain were used to investigate the production of L-lactic acid from Jerusalem artichoke. Its contents and results are as follows:⑴The optimization of medium contents and fermentation conditions for Aspergillus niger SL-09 to form inulinase was conducted. The optimum medium contains glucose 40g/L, peptone 30g/L, sucrose ester 4g/L. Under the optimum fermentation conditions, inulinase activity of 230U/mL was obtained at 30℃on reciprocating shaker with 140 rpm in 96 h. Further studies found that sucrose ester was a more effective inducer than inulin for inulinase production, which can promote the synthesis of mRNA for the formation of inulinase. Meanwhile, as sugar ester was more resistant to hydrolysis than inulin, so that sucrose ester acts as a persistent signal to initiate a response in the cell to provoke the transcription of mRNA available for fructanohydrolase synthesis. Moreover, it was found that the medium with glucose and sucrose ester as carbon source and inducer respectively, were more favorable for the growth and enzymes synthesis of this strain. The strain SL-09 was identified as Aspergillus niger by genetic and morphological analysis.⑵In the process of inulinase purification, salt out with 30% and 70% of ammonium sulfate were used respectively. Through the salting out by dialysis and concentrate, the enzymes were obtained. Further studies found that the enzyme form by Aspergillus niger was composed by inulinase and invertase, and the difference of molecular weight between the both enzyme was not significant. The molecular weight of the inulinase was around 165～210 kDa,and the invertase was 160～170 kDa. The Michaelis-Menten constant of the inulinase and invertase at 30℃were Km=34.9 mmol/L, and vm=909μmol/min for invertase, and Km=68.3 mmol/L, vm=476.2μmol/min for inulinase, respectively. Those results signified that the maximum reaction rate of invertase was near 2-fold higher than that of the inulinase, however, the substrate concentration required for maximum rate was only half of the inulinase.⑶The parent strain of Lactobacillus sp. G-01 was treated by diethyl sulfate (DES), N-methyl-N'-nitro-N- nitrosoguanidine (NTG), and domesticated with high concentration of fructose and lactic acid. A strain G-02 which could accumulate 92.8 g/L L-lactic acid using glucose as carbon source was obtained. The strains of G-02 also presented favorable genetics stability after several times of transfers. Strains G-02 was identified as Lactobacillus casei, homofermentation, and can produce L(+)-lactic acid with an optical purity of over 95%. Therefore, this strain was a favorable strain for L-lactic acid production.⑷To avoid the product repress in the inulinase production system and decrease the biomass of Aspergillus niger in the enzyme production medium, and to remove the contamination in the simultaneous saccharification and fermentation process for production of lactic acid from Jerusalem artichoke, the Lactobacillus sp. G-02 was inoculated to the enzyme production medium to form superiority bacterium before lactic acid fermentation and the compose of the medium was optimized. The results signified that when the Lactobacillus sp. strain was inoculated at 12 h of the culture, the enzyme activity was enhanced significantly. After 60 h of co-culture, inulinase activity reached 250 U/mL, which was 4-fold higher than that of the control (50 U/mL) with single strain. Further studies found that the inoculation of Lactobacillus sp. decreased the fermentable sugar in the medium dramatically, and the biomass of Aspergillus niger was decreased to half of the value in the control. The mycelial configuration of the Aspergillus niger changed obviously, which was more favorable to survive in the competitive environment.⑸The simultaneous saccharification and fermentation process for L-lactic acid production from Jerusalem artichoke was investigated. It was found that the optimized condition was inulinase activity 50 U/mL, inoculation volume 35%, Jerusalem artichoke concentration 230 g/L. It was also found that the citrate metabolism endowed cells with extra ability to counteract the acid toxicity, and then enhance the lactic acid productivity. The optimized citrate concentration was 10 g/L. Under the optimized condition in the fed-batch culture, the L-lactic acid with the concentration of 141.5 g/L was obtained at 40℃in 30 h, with a yield of the 52.4g lactic acid/100g Jerusalem artichoke flour.