Effect Of Sodium On Citric Acid Fermentation And Its Resistant Strains Screening In An Integrated Citric Acid- Methane Fermentation Process

To solve the problem of extraction wastewater pollution in citric acid production, an integrated citric acid- methane fermentation process was established by our laboratory. Cassava and corn starch are used as raw material in this process to citric acid production, while unused materials(fiber and pectin) and metabolites of A. niger in fermentation are converted to biogas through mesophilic anaerobic digestion. The biogas can be used to produce electricity and heat while the anaerobic digestion effluent(ADE) was further treated and recycled for the next citric acid fermentation, thereby avoiding the wastewater discharge and decreasing the water resource consumption. Metal ions contained in ADE could inhibit citric acid fermentation in recycling, therefore, the effect of metal ions on citric acid fermentation was studied and its influence machine was also investigated. Moreover, Genome Shuffling was used to screen strains with a resistance to high concentration of Na+. The main research results were shown as follows:Excessive ammonia and metal ions contained in ADE were the major inhibitors for citric acid fermentation in recycling. Air stripping was used to remove ammonia and part of precipitable metal ions and the treated ADE was recycled for the next batch of citric acid fermentation. Furthermore, excessive glucoamylase was added to medium after inoculation to improve the citric acid production and the recycling process performed for 10 batches. The average citric acid production was 145.9±3.4 g·L-1 and 2.5% lower than the control(149.6 g·L-1). Metal ions in ADE was detected by flame atomic absorption spectrometry and high concentration of Na+ was confirmed to be the major inhibitor for the proposed process.The activity of glucoamylase, isomaltase, citrate synthase, isocitrate dehydrogenase and the other endoenzyme in citric acid fermentation were investigated and the result indicated that 1000 mg·L-1 Na+ in fermentation medium could increase the activity of isocitrate dehydrogenase and promote the metabolism of ciric acid. Meanwhile, the activity of glucoamylase and isomaltase during the fermentation was inhibited, resulting in the incomplete degradation of residual total sugar which was approached to 30 g·L-1. The available sugar used to citric acid fermentation declined and the citric acid synthesis rate and citric acid production significant decreased. Excessive glucoamylase addition could significantly relieve the inhibition for citric acid fermentation caused by high concentration of Na+, but the citric acid production was still lower than the control.To completely relieve the inhibition for citric acid fermentation caused by high concentration of Na+, Na+-tolerant and high yield citric acid strain was bred. The original strain Aspergillus niger MZ-11 was mutated through ultraviolet and ARTP(Atmospheric and Room Temperature Plasma) mutagenesis to obtain the optimal positive mutations and establish the initial mutant library. After two round Genome Shuffling with selective medium contained 1000 mg·L-1 Na+, 441 positive mutations were selected. And a Na+-tolerant and high yield citric acid mutant strain G2-107 was obtained after the fermentation screening, which also had good genetic stability. The citric acid production of fusant G2-107 with 1000 mg·L-1 Na+ increased by 20.9% compared to original strain.and the resistant of Na+ increased to 400 mg·L-1.With glucoamylase addition, the resistant of Na+ concentration was up to 600 mg·L-1 which indicated that G2-107 exhibits substantial improvement in multiple stress tolerance to Na+.