Abstract: Citric acid is the most mature platform compound that is mainly produced by submerged fermentation of Aspergillus niger.Traditionally,A.niger spores are directly inoculated in the fermentation,which results in delayed cell growth and fluctuant production level.In this study,a spore pre-culture strategy was proposed to shorten the seed culture cycle.Particulate matter and shaking were applied to control the morphology uniformity of mycelium pellets.Firstly,the effect of pre-culture conditions on spore germination was investigated.The results showed that starch as a carbon source could hardly promote the spore germination of A.niger.Glucose could significantly promote spore germination(P<0.05),with the highest spore germination rate and bud tube length.Nitrogen sources also promoted spore germination.Compared with inorganic nitrogen sources,organic nitrogen sources with rich nutrients had more significant effects on spore germination(P<0.05),and the germination rate and bud tube length were the highest when adding corn pulp.The germination rate and the length of bud tube gradually increased with the increase of Ca2+ concentration in the range of 0-10 mmol/L.However,when Ca2+ concentration increased to 100 mmol/L,spore germination was inhibited,germination rate and bud tube length were also decreased.A.niger spores had wide adaptability to pH and could germinate within the range of pH 3-8,and the optimal pH was 5-7.The length of bud tube was the highest at pH 5.The germination of spores was slow at 25 ℃.The spores stopped germination and stayed dormant at 40 ℃.At 35 ℃,the spore germination rate and the length of bud tube were the highest.In stationary culture,the germination rate of spores was low and most spores were still dormant.However,the germination rates of spores were both higher in the 50-150 r/min shaking table culture,and the difference of spore germination was not significant,indicating that spore germination required a small amount of oxygen supply.The germination rate was almost 0 when the spore concentration was higher than 0.8×108 CFU/mL.The germination rate and the length of bud tube increased with the decrease of spore concentration when the spore concentration was lower than 0.8×108 CFU/mL.The results showed that low spore concentration was beneficial to germination,while high spore concentration inhibited spore germination.With a prolonged pre-culture time,the germination rate and the length of bud tube increased gradually.After 10 h of culture,the germination rate almost did not increase,but the length of bud tube continued to increase,indicating that spores had completed germination and entered the stage of mycelium growth.Based on the results,the optimal spore pre-culture conditions were:glucose and corn steep liquor as carbon and nitrogen source,10 mmol/L Ca2+,pH 5.0,35 ℃,and the initial spore concentration was 0.8×107 CFU/mL,the aerobic pre-culture time was 10 h.Subsequently,the effects of particulate matter and oscillating dispersion treatment on spore aggregation and mycelium pellet morphology were investigated,to control the formation of uniform micro mycelium pellets from spores.The results showed that particulate matter had a significant effect on the morphology of mycelium pellets when the resting spores were directly cultivated in seed culture.In spore pre-culture,scattered spores were formed when the particulate matter was added.Without particulate matter,clumps of spores formed.The results showed that adding particulate matter before spore germination had a significant effect on spore aggregation.At the same time,no matter whether the seed medium contains particles or not,the scattered spore can form a large number of micro mycelium pellets,while the aggregated germinating spores all formed a smaller number of large mycelium pellets.The results showed that after spore germination,the particulate matter had little effect on the morphology of mycelium pellets,and the morphology of mycelium pellets mainly depended on whether the germinating spores condensed or not.In addition,a large number of micro mycelium pellets can also be formed after the aggregation of germinating spores is dispersed,which further indicated that agglomeration of germinating spores played a decisive role in the morphology of mycelium pellets.Therefore,the final morphology of mycelium pellets can be controlled by adding particulate matter before spores germination and oscillating dispersion after conidial agglomeration.Furthermore,the size of mycelium pellets had a significant impact on the fermentation results,and the corresponding fermentation strength and acid production rate per unit cell of small mycelium pellets were significantly higher than those of large mycelium pellets(P<0.05).Finally,the new process was scaled-up and verified on the 500-5 000 L fermenters.In seed culture,the original process required a lag time of about 8 h,while the pre-cultured group rapidly entered the growth stage with no obvious lag time,which shortened the cycle by more than 12 h.Compared with the control group,the rate of sugar consumption and acid production increased in the pre-cultured group.The final fermentation results showed that the mycelium pellets concentration and the pellet formation ratio increased significantly(P<0.01) by 0.4×104 CFU/mL and 18.3%,respectively.The mean diameter of mycelium pellets decreased significantly(P<0.05) by 28 μm.The maximum acid production rate was increased by 7.5%,the fermentation intensity increased by 0.14 g/(L·h),and the fermentation cycle was shortened by 2 h.The final citric acid concentration and the conversion of sugar and acid increased by 2 g/L and 1.2%,respectively.This study has important reference significance for spore germination,morphology control,and production efficiency improvement of filamentous fungi.
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