Biosynthesis of citric acid from glycerol-containing waste by yeasts

The aim of the study is to investigate the possibility of using glycerol-containing substances such as biodiesel production wastes for different yeasts species culture and microbiological synthesis of citric acid (CA). 59 natural strains of different yeasts (Debaryomyces, Candida, Yarrowia, Pichia, Sacharomyces, Torulopsis) and 7 mutant strains of Yarrowia lipolytica (YL) have been used. All mutants were characterized as non growing on acetate-containing medium and were selected as producents of CA from glucose. Refined glycerol (RF) and glycerol-containing wastes were used. Yeast acid producing capacity was evaluated by chalk dissolved zones in nitrogen limited cell growth conditions. Selection of producents was made on media with RF. All strains were capable to grow on media with glycerol. Growth limitation led to acid excretion in 41 strains (40 st. of YL and 1 of Candida paludigena). The rest strains did not produce acids. Natural YL st. 704 and its mutants UF5, NG40, N11, N13 and N15 were selected as the most active acid producents. After identification of produced acids, biomass evaluation, CA and isocitric acid (ISA) content and their ratio determination the YL st. 704 (CA accumulation 21.16 g/l) and its mutant N15 (CA:ISA ratio 11.2:1) were selected for following examination. Conditions that stimulate CA synthesis were investigated and determined: media pH 5.0, high aeration level, glycerol concentration in media 20-70 g/l. The strain productivity on media with RF in 10 l-volume fermenter have been reached 110 g/l of CA with CA:ISA ratio 7.3:1. Under acid accumulation capacity examination on media with biodiesel wastes in 3 l-volume fermenter the YL 704 st. produced 61.7 g/l of CA and 46.6 g/l of ICA (CA:ISA=1.3:1). The ratio change in comparison with RF maybe connected with lipid acid admixture in biodiesel wastes. The mutant N15 on the medium with biodiesel wastes continued predominantly synthesis of CA (71 g/l with CA:ISA ratio 12.7:1). Its volume productivity reached 0.89 g/l in hour. It is concluded that biodiesel wastes are suitable as carbon source for biotransformation to CA