Breeding Of Mn²⁺-tolerant And Citirc Acid Producing Aspergillus Niger

Citric acid is an important intermediate product in the TCA cycle, which is widely usedin food, medicine, cosmetics and other industries, and it has great commercial value. Thelow-cost raw materials which generally contain Mn²⁺, such as starch, jade, rice flour, tapiocaflour, wheat starch, et al, are usually used in citric acid fermentation. However, Mn²⁺candecrease the citric acid production and substrate conversion rate. In order to solve this trouble,it was important to screen the Mn²⁺-tolerant and high yield citric acid-producing strain. In thisresearch, a citric acid-producing strain Aspergillus niger deposited in our laboratory wasmutagenized, and the mechanism of Mn²⁺-resistant by the mutant strain was investigated.Finally, according to the Mn²⁺-metabolic regulation, the optimization of citric acidfermentation was determined.Mn²⁺-tolerant and high yield citric acid strain was bred. Aspergillus niger7#was used asthe original strain to obtain mutants by ultraviolet and nitrosoguanidine mutagenesis.254bigger-colonies mutants with different concentrations of manganese were selected, and aMn²⁺-tolerant and high yield citric acid mutant strain UV-141was obtained after thefermentation screening, which had good genetic stability. The citric acid production of mutantstrain UV-141was increased by12.1%compared to original strain. The contents of oxalicacid and aconitic acid were both less than1g/L in the fermentation broth by the mutantUV-141. When the Mn²⁺concentration was up to600mg/L, the citric acid yield was able tomaintain constant by mutant UV-141. The citric acid production was increased by92.4%withadding300mg/L Mn²⁺, when it was cultured with glucose as the sole carbon source in shakeflask fermentation.The mechanism of Mn²⁺-tolerant by the mutant UV-141was studied. In order to revealthe causes of Mn²⁺-tolerant and high yield citric acid for the mutant UV-141, the trackinganalysis of key enzymes activities and the respiratory pathways throughout the entire period offermentation was carried out. It was observed that in the presence of Mn²⁺, all of the specificactivities level of aconitase, NAD⁺-isocitrate dehydrogenase and NAD⁺-isocitratedehydrogenase in the original strain increased, but both of the specific activities of aconitaseand NAD⁺-isocitrate dehydrogenase in the mutant UV-141were apparently no effects. In thepresence of high Mn²⁺concentration, the specific activity level of aconitase decreased, and thespecific activities level of NAD⁺-（NAD⁺-） isocitrate dehydrogenase were at a low level. Inaddition, Mn²⁺could weaken the decrease of citric acid yield, which was caused by theinhibition of alternative respiratory chain （AOX） in presence of SHAM （salicylhydroxamicacid）. Therefore, it is beneficial for the synthesis of citric acid to adding the suitable Mn²⁺concentration in mutant UV-141.According to the regulation of Mn²⁺-metabolic, the optimization of the citric acidfermentation was carried out. The fermentation composition in mutant strain was determinedand the single factor experimental results showed that the optimum fermentation mediumcomponents （g/L） were as follows: sugar concentration of fermentation medium150g/L, cornliquid ratio14%. pH5.0, Mn²⁺200mg/L. In shake flask level, the optimum culture conditions were10%inoculation quantity,240r/min, liquid volume30mL/250mL, culture temperature35°C, fermentation time4d, seed age30h. Under the fermentation conditions above, thefinal concentration of citric acid reached125.0g/L. When Mn²⁺concentration was adjusted to200mg/L in a5L fermentor, the citric acid production was up to140g/L and increased by7.4%compared with no Mn²⁺adjustment.