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South African edible oil processing plants produce approximately 3 x 105 tonnes of oil annually with up to 3 tonnes of water for every tonne of oil produced. Wastewater that contains oil extracts varies in organic loading from 30,000 to 60,000 mg.l-1 COD. This wastewater can be used to grow oleophilic fungi to produce valuable industrial products. The global vitamin B market is approximately R25.5 billion with 4500 metric tonnes being produced. A large proportion of this is produced using the fungus Eremothecium gossypii using oil substrates. The aim of this study was to to develop a novel method to produce riboflavin with the aid of fungi, using edible oil effluent (EOE) as substrate, and to optimize the production thereof by statistical experimental design. Four fungi were surveyed for their growth potential on EOE and two, E. gossypii (CBS109.51) and C. famata (ATCC 208.50) were found to produce sufficient riboflavin for further study. Mutation of these organisms using ethylmethane sulphonate (EMS) increased riboflavin production from 3.52 mg.l-1 to 38.98 mg.l-1, an 11-fold increase. An enzyme pathway responsible for this was found to involve isocitrate lyase and comparison of this enzyme’s activity in the mutant against the wild-type using Michaelis-Menten kinetics showed a higher reaction velocity (Vmax) with a reduced substrate affinity (Km) indicating that the mutation was associated with this enzyme. Biomass comparisons were fitted to the sigmoid Gompertz model which was used to compare the wild-type to the mutant and increased specific growth rates and doubling times were observed in mutated cultures of E. gossypi. A strategy of statistical experimental design was pursued to optimize media components and iterative fractional factorial experiments culminating in a central composite optimization experiment were conducted. Statistically verified mathematical models were developed at each stage to identify important media components, predict media interactions, show directions for improvement and finally, predict maximum riboflavin production. An eight-factor resolution IV fractional factorial increased riboflavin production to 112 mg.l-1 followed by a four-factor resolution V experimental design which increased riboflavin production to 123 mg.l-1. A two-factor (yeast extract and NaCl) central composite experimental design predicted a maximum riboflavin production of 136 mg.l-1 which was a 3.5-fold increase from the mutant, and 38.6-fold higher than the E. gossypii wild-type. The optimized value was achieved within predicted confidence intervals in confirmatory experiments. Cost implications for production of riboflavin on EOE were calculated and a 10% technology uptake by the edible oil industry could yield a riboflavin industry with a 63.65 million rand turnover and a potential 24.96 million rand gross profit margin.

Optimization of riboflavin production by fungi on edible oil effluent