| Citric acid?2-hydroxy-1, 2, 3-propanetricarboxylic acid, CA?, an intermediate of the tricarboxylic acid cycle, is a significantly important platform-compound. Currently, CA has become the world largest edible organic acid in terms of production and consumption. Fermenting starchy-material using Aspergillus niger for the CA accumulation was presently main production mode. Though CA has become the one of the most important bulk fermentation products, its production process still faces representative problems in the fermentation industry, such as starchy-liquefaction lack of scientific evaluation criteria, seed-culture with cumbersome and instability process, material utilization with incompletness and fermentation mode with low energyefficiency. In the paper, after analyzing the relationship between catalytic efficiency of A. niger and the molecular weight?Mw? characteristics of the dextrin, we established the evaluation method of starchy-material liquefaction on basis of the the dextrin Mw. Based on the physiological and morphological characteristics of A. niger, we studied ultrasonic wave pretreating the spores and pellet-dispersion technology, and established seed recycling cultivation process. Integration strategies of segmentation fermentation and pellet dispersion, the segmentation recycling fermentation process was further constructed. By staged-addition enzyme strategy, the glucose supply and consumption rate was guaranteed to be matching, along with the CA yield and material utilizationratio enhanced. Main results achieved in this work were highlighted below.?1? Method evaluating the starchy-material liquefied process was established on basis of the Mw characteristics of the dextrin. Dextrin sample with the molecular weight narrow distribution was prepared by alcohol fractional precipitation method. Pure A.niger glucoamylase?GM? with three isozymes was obtained after the A. niger culture was separated and purified. The Mw of subunit GM I, GM II, GM III was 30 kDa, 50 kDa, 100 kDa, respectively. Analysis of the relationship between catalytic efficiency of A. niger and the molecular weight?Mw? characteristics of the dextrin, the evaluation method of starchy-material liquefaction was established on basis of the the dextrin Mw. Kinetic parameter Km attained the minimum when Mw of the dextrin was 1.9 kDa. The Mw of more liquefied components was aggregated in the range of 1.41.9 kDa, which was favorable to the saccharification efficiency. Furthermore, the starchy-material was fine regulated and the liquefied component was easy to be utilized by glucoamylase compared with the traditional method of dextrose equivalent value?DE value?, and then were used for CA fermentation. CA production rate and total sugar uptake rate were evidently improved with residual total sugar decreasing by 10.8% and fermentation efficiency enhancing by 21.1% in 9 h shorter fermentation time.?2? Spores were rapidly germinated after ultrasonic wave pretreatment with inner water promotion permeation. On basis of the physiological properties of the A.niger spores, microwave pretreated the spores and promoted the water infiltration in the spores and the spores were fast-germination with shortening the time of seed cultivation. The seed viability was also improved with fermentation productivity enhancing by 8.13%. Based on the physiological characteristics and growth kinetics parameters in the whole life cycle of the A.niger in the fermenter, their own glucoamylase could directly reflect the cell viability. Strain viability-fluctuation was thereby effectively avoided in comparison with traditional seed-transferring on basis of seed-age. Integrated strategies of the ultrasonic pretreating the spores and glucoamylase representing seed-transferring, high activity seed was obtained and lag-phase was shortened, along with total fermentation time shortening by 7.9 h and fermentation efficiency enhancing by 11.76%.?3? Seed recycling cultivation was established on basis of the pellet-dispersion strategy. Mycelium-pellets inoculation substituted for the traditional spores inoculation mode, effectively avoiding the longtime?spore preparation 30 days? of the traditional seed culture. On this basis, using pellet-dispersion strategy, the bottleneck caused by the mycelium structure was overcome and the seed recycling culture process was established. The seed recycled for eight batches and can still restore high cell-viability. The dispersed fermentations were evidently superior to the pelleted fermentations. CA yield in the eight-recycling batches on average with the dispersed pattern was enhanced by 26.5% in comparison with the pelleted pattern, along with 2.5% increasement in comparison with the initial control.?4? CA recycling production process in the segmented fermentation was established by integrating the strategies of pellet dispersion and segmentation fermentation. Segmented fermentation strategy effectively realized the separation of the cell-growth and CA synthesis. In the pelleted-recycled segmentation fermentations, mycelium morphology restricted the cell-growth and CA accumulation. Application of pelletdispersion strategy could effectively control the mycelium morphology characteristics. The fermentation pellets morphology was stable in the six-successive batches. CA yield increased from 70.3 g·L–1 to 97.0 g·L–1 while still lower than the control. Furthermore, under the optimized conditions(segmentation level 2/10, limited-nitrogen addition 15 g·L–1, segmentation time 24 h), fermentations recycling ten batches in 5 L fiveconjoined-fermenters were stable and CA yield even increasing by 1.26% compared with the control. Simultaneously, the total sugar uptake was decreased by 10.01% with significantly enhancing the production efficiency.?5? Staged-addition glucoamylase strategy guaranteed the glucose supply rate in the CA synthesis phase and enhanced the CA synthetic efficiency. Against high total sugar in the broth, p H stability of the commercial glucoamylase and A.niger glucoamylase was analyzed. Commercial glucoamylase was found to be more stable in the low-pH environment. Pre-saccharification and fermentation strategy?commercial glucoamylase, 60 °C for 2 h? was firstly proposed with the residual total sugar decreasing 10.4% and fermentation efficiency enhanced 0.11 g·L–1·h–1. However, there existed other problems such as higher initial glucose inhibiting the cell-growth and glucoamylase activity greatly lost in the middle and later periods of the fermentation. Furthermore, glucoamylase addition in the fermentation process, on basis of stagedaddtion mode, effectively compensated the enzyme activity loss resulted from the p H sharp-decrease, insuring the glucose supply rate. The fermentation efficiency was evidently enhanced by 13.3% with residual total sugar decreasing by 31.3%, simplifing the subsequent product separation and extraction process. |
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