Rhizopus oryzae for fumaric acid production : continuous feed strategies to manipulate the metabolism

Abstract

The four-carbon dicarboxylic acid, fumaric acid, of the tricarboxylic acid cycle remains a promising bio-based platform chemical. To date the most promising organism for pro- ducing fumaric acid is Rhizopus oryzae (ATCC 20344) that naturally excretes fumaric acid under nitrogen limited conditions. In order to investigate the fumaric acid produc- tion with R. oryzae, a novel immobilised biomass reactor was developed. Fumaric acid excretion in R. oryzae is always associated with the co-excretion of ethanol, an unwanted metabolic product from the fermentation. The cause of ethanol production was suspected to be a result of R. oryzae being a Crabtree-positive organism. For Crabtree-positive or- ganisms like Saccharomyces cerevisiae, ethanol overflow is negated by controlling the glucose input to the fermentation. The same strategy was employed for R. oryzae during a continuous production fermentation. It was shown that ethanol could be eliminated entirely during fumaric acid production, achieving a yield of 0.802 g g−1 fumaric acid on glucose [1]. The medium pH was identified as a key parameter affecting fumaric acid excretion. It was found that the selectivity for fumaric acid production increased at high glucose consumption rates for a pH of 4, different from the trend for pH 5 and 6, achieving a yield of 0.93 g g−1 [2]. This yield is higher than previously reported in the literature.

The use of lignocellulosic hydrolysate, predominantly comprised of glucose and xylose, for the production of fumaric acid would greatly improve the industrial viability of the process. A synthetic lignocellulosic hydrolysate (glucose-xylose mixture) was used in batch and continuous fermentations to investigate the feasibility of this substrate. The batch fermentation of the synthetic hydrolysate at the optimal conditions (urea feed rate 0.625 mg L−1 h−1 and pH 4) produced a fumaric acid yield of 0.439 g g−1. A spe- cific substrate feed rate (0.164 g L−1 h−1) which negated ethanol production and selected for fumaric acid was determined. Using this feed rate in a continuous fermentation a fumaric acid yield of 0.735 g g−1 was achieved; a 67.4 % improvement [3]. Metabolic analysis helped to identify a continuous synthetic lignocellulosic hydrolysate feed rate that selected for fumaric acid production, while achieving co-fermentation of glucose and xylose, avoiding the undesirable carbon catabolite repression.

Because this work demonstrates the viability of fumaric acid production from lignocellu- losic hydrolysate, the process developments discovered will pave the way for an industri- ally viable process.