Citric acid production by immobilized cells of the yeast Candida guilliermondii : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Biotechnology and Bioprocess Engineering at Massey University
The feasibility of using cells of Candida guilliermondii immobilized onto sawdust particles for production of citric acid was investigated. C. guilliermondii IMK1 from a stock culture (Department of Process and Environmental Technology, Massey University, Palmerston North, New Zealand) was reisolated for further study including strain improvement work by induced mutation using UV light. A mutant strain DT2 was isolated which produced a citric acid concentration of 9.2 g/l (yield 25 % (w/w)) in shake flask culture, using a defined medium containing 36 (g/l) glucose, compared with 4.9 (g/l) citric acid produced (yield 14 % (w/w)) by the parent strain. Experiments in a laboratory scale batch fermenter, in which a higher concentration of citric acid (11.7 g/1) was achieved, proved that citric acid production using the mutant strain C. guilliermondii DT2, could be scaled up successfully from shake flask to a 2 1 fermenter. This mutant was used throughout subsequent experiments. Sawdust was selected, as the most appropriate support material to immobilize the mutant strain C. guilliermondii DT2 via the adsorption method. Experiments using different concentrations of nitrogen nutrient in defined medium using cells of C. guilliermondii DT2 immobilized onto sawdust particles, in repeated batch shake flask culture, demonstrated a marked effect of the nitrogen concentration on citric acid production. Thus, an overall productivity of 0.11 (g/l.h) was obtained using a defined medium containing 0.53 (g/l) ammonium chloride, compared to overall productivities of 0.04 (g/l.h) and 0.01 (g/l.h) using defined media containing 0.1 (g/l) and no ammonium chloride, respectively. No significant effect of nitrogen concentration on citric acid yield was observed in this investigation. In contrast, similar experiments, in repeated batch shake flask culture, for the effect of phosphate concentration on citric acid production showed no effect of phosphate on either the production rate or yield of citric acid. In bubble column culture experiments, using cells of C.guilliermondii DT2 immobilized onto sawdust, the importance of pH control in citric acid production was demonstrated. In addition, it was demonstrated that the activity of immobilized cells which have lost the ability to produce citric acid can be revived by supplying medium containing sufficient concentrations of nitrogen and phosphate. Reduction of the nitrogen concentration in the medium from 0.53 (g/l) to 0.05 (g/l), provided that the reactor was well established, showed no significant influence on citric acid productivity, but significantly improved the citric acid yield. The highest productivity of around 0.21 - 0.24 (g/l.h) at a dilution rate of 0.21 h-1, accompanied by a citric acid yield of about 10 - 11% (w/w), was reached and maintained for more than 140 hours of stable operation. Overall, it was concludcd that cells of C. guilliermondii were succesfully immobilized onto sawdust particles, and the immobilized cell reactor produced citric acid at a higher rate compared to a free cell system. In particular, a high rate of citric acid production in a bubble column reactor, operated in continuous mode, was achieved.