Citric acid production from yeasts : comparison of a parent and a mutant strain of Candida guilliermondii, and subsequent reversion of the mutant : a thesis presented in fulfilment of the requirements for the degree of Master of Philosophy in Food Technology at Massey University

Abstract

Citric acid production from yeasts has been studied widely owing to the short duration of fermentation, the broad choice of carbon source and the better yields obtained when compared to the currently used submerged or surface fermentation with Aspergillus niger. In this work two strains of Candida guilliermondii were compared for their citric acid-producing capabilities, these being parent strain Candida guilliermondii NRRL Y-448, and mutant strain Candida guilliermondii IMK1. The mutant was previously selected for its ability to produce much higher concentrations of citric acid than the parent. These strains were grown under various nutrient limitations to determine if nutrient limitation had an effect on the amount of citric acid produced. Several differences were observed between the non-citric acid-producing parent and the citric acid-producing mutant. The mutant generally consumed less glucose (g.g-1), produced less biomass (g.L-1) and produced much higher levels of citric acid – the best production (7.34 g.g-1) seen from the culture grown under phosphorus-limited (0.15 mM) conditions. Upon assessment of enzyme activities it was found that the mutant also exhibited reduced activity of the enzyme NAD-ICDH (NAD-dependent isocitrate dehydrogenase), a recognised control point for the over-production of citric acid. NAD-ICDH is inhibited by increased concentrations of ATP - these are associated with the accumulation of citric acid in the cell in the stationary phase of growth. This reduction in NAD-ICDH activity correlated with a dramatic increase in the activity of NADP-ICDH (NADP-specific isocitrate dehydrogenase), the activity of which was thought to compensate for the loss of activity of NAD-ICDH. However, in a subsequent experiment, the mutant was found to have reverted - losing its ability to produce citric acid. This loss of productivity occurred before the levels of adenine nucleotides in the cell could be assessed, meaning that the suggested inhibition of NAD-ICDH by elevated levels of ATP could not be confirmed. Upon analysis of the revertant, it was found that glucose consumption (grams per gram of cells) had increased, as had the production of biomass (g.L-1). Even though the revertant failed to consume as much glucose as the parent, in many instances it produced higher levels of biomass. Upon analysis of enzyme activity, it was found that the activity of NAD-ICDH had increased, so reducing the accumulation of citric and isocitric acids. The activity of NADP-ICDH had decreased somewhat, but activity of this enzyme remained at significant levels. It is proposed that the activity of NADP-ICDH in the revertant was responsible for the increased efficiency of biomass production. In conclusion, it is suggested that overproduction of citric acid in Candida guilliermondii IMK1 was due to the consumption of lowered levels of glucose combined with the reduced activity of the enzyme NAD-ICDH, which it is speculated was due to elevated concentrations of ATP in the cell.