| Multi-stage control strategy for the hyperproduction of ganoderic acid and Ganoderma polysaccharide during submerged fermentation of medicinal mushroom Ganoderma lucidum was developed. The response of the cell growth and metabolites accumulation to environment factor was quite different.A novel three-stage light irradiation control strategy in the submerged fermentation of medicinal mushroom Ganoderma lucidum for the efficient production of bioactive metabolites ganoderic acid (GA) and Ganoderma polysaccharides was developed. Significance of light quality, i.e. blue light (390-500 nm,λmax=470 nm), red light (560-700 nm,λmax=625 nm), and white light (400-740 nm,λmax=550 nm), was studied at first. Interesting, there was a gradual decrease trend of GA content after the culture of day 2 when the maximal GA content was obtained, while GA content decreased slowly under white light irradiation after day 6. The dark environment was favorable to the specific GA biosynthesis (i.e., GA content) before day 6, and after that the optimum was white light irradiation culture. General speaking, white light irradiation was the best for the cell growth and total GA accumulation. The irradiation intensity of white light significantly affected the cell growth and GA biosynthesis. A relatively lower irradiation density of white light (i.e., 100 and 300 lux) was beneficial for the specific GA biosynthesis before day 6, while GA content was higher under higher irradiation density of white light (i.e., 500 and 1000 lux) at the later-stage of cultivation. 500 lux white light irradiation culture was the best from the viewpoint of GA accumulation. Therefore, a two-stage light irradiation control strategy by combing the first 2 days dark culture with the following 500 lux white light irradiation culture was developed. The highest GA production in the two-stage culture process was 276.0±12.5 mg/L, which was increased by 19% compared to 500 lux white light irradiation culture (232.4±15.8 mg/L) and by 178% compared to the dark culture (99.4±1.0 mg/L). While there still existed a gradual decrease trend of GA content after day 2 when the maximal GA content was obtained in the two-stage culture process. A following three-stage light irradiation control strategy was further demonstrated in order to turn around the sharply decrease of GA content after day 2. The first-stage culture was the two-day dark culture; the second-stage culture was the following six-day 100 lux white light irradiation culture, and the third-stage was 500 lux white light irradiation culture until the end of fermentation. During the three-stage culture process of G. lucidum, the gradual decrease trend of GA content after day 2 was turned around, which suggested that 100 lux white light irradiation was beneficial for the metabolic flux towards the GA biosynthesis. The maximal GA content of 3.1±0.1 mg/100 mg DW was obtained, which was higher by 41% compared to the two-stage culture process. The maximal GA production (i.e., 466.3±24.1 mg/L) and productivity (i.e., 38.9 mg/L per day) in the three-stage culture process were 69% and 101% higher than those obtained in the two-stage culture process.A pH-shift and DOT-shift integrated fed-batch fermentation process for the efficient production of ganoderic acid and Ganoderma polysaccharides by medicinal mushroom Ganoderma lucidum was developed for the first time. Investigations on the impact of pH and dissolved oxygen (DOT) on the submerged fermentation of G. lucidum suggested that the response of the cell growth and metabolites accumulation to pH and DOT was quite different. A high GA production of 321.6 mg/L was obtained through a 4-day culture at pH 3.0 followed by 6-day culture at pH 4.5, which was 2.9-folds of the control experiment which was carried out at an initial pH of 5.5. Simultaneously, a high GA production of 487.1 mg/L was achieved by combining a 6-day culture at DOT 25% with a following 6-day culture at DOT 10%, which was higher by 43% and 230 % compared with the culture at DOT 25% and 10%, respectively. A multi-stage integrated fed-batch fermentation process by combining pH-shift from 3.0 to 4.5 on day 4, DOT-shift from 25% to 10% on day 6 with carbon source feeding on day 9 resulted in a significant synergistic enhancement of GA accumulation up to 754.6 mg/L, which are the highest reported in the submerged fermentation of G. lucidum in stirred-tank bioreactors. Besides, in the integration of DOT-shift on day 6 and carbon source feeding on day 9, the highest EPS (Extracellular polysaccharides) and IPS (Intracellular polysaccharides) production was 3.54 and 4.86 g/L, which are also the highest reported in the submerged fermentation of G. lucidum in stirred-tank bioreactors.By investigating the shear stress on a pH-shift and DOT-shift integrated fed-batch fermentation process of G. lucidum, a relatively higher biomass (22.62 g/L), GA content (3.53 mg/100 mg DW) and GA production (798.0 mg/L) was obtained in bioreactor at lower impeller tip speed (0.154 m/s-1.234 m/s), which was 104%, 85% and 276% higher than higher impeller tip speed (0.154 m/s-2.161 m/s), respectively. So, the integrated strategy cultivation of G. lucidum was under lower impeller tip speed from the view point of GA production.Process scale-up of the G. lucidum cultivation for the production of ganoderic acid and Ganoderma polysaccharides was developed under the integrated approaches of pH, DOT and shear stress. When pH shifted from 3.0 to 4.5 on day 4, DOT shifted from 25% to 10% on day 6 and impeller tip speed was controlled from 0.154 m/s to 1.234 m/s, the maximum GA content (3.7 mg/100 mg DW) and production (673.1 mg/L) was attained, which was near to that of 7.5 L STR.This is the first report investigating the significance of light irradiation control strategy, performance analyses of a multi-stage integrated fed-batch fermentation process and scale-up for the cell growth and metabolites accumulation on the medicinal mushroom fermentation. Such work is very helpful to other mushroom fermentations for useful metabolites production on the medicinal mushroom fermentation.
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