P33: Cottonseed meal derived soluble sugars as an economical feedstock for the fermentative production of C4 dicarboxylic acid using engineered E. coli

Bio-based production of succinic acid still faces the challenge of becoming cost competitive against petrochemical-based processes. As the use of an economical source of fermentable sugars is one of the effective approaches to reduce the cost of fermentation process, we therefore aimed to examine the use of soluble sugars derived from cottonseed as the biomass feedstock to produce succinate. Cottonseed meal, a protein rich by-product of oil extraction from cottonseed, contains soluble sugars such as stachyose, raffinose, sucrose, glucose, and galactose. We have developed a simple and efficient method to extract soluble sugars (85-90 mM hexose sugars) from cottonseed meal, and examined the production of succinate using engineered E. coli strains with or without heterologous expression of oligosaccharide catabolizing genes. Furthermore, we evaluated oligosaccharide hydrolysis pretreatments using dilute acid, ionic liquid catalyst, and alpha-galactosidase. The sulfonic acid functionalized ionic liquid modified silica catalyst was inefficient in hydrolyzing stachyose, raffinose, and cottonseed meal extract containing soluble sugars. Whereas, dilute H₂SO₄ and alpha-galactosidase were highly effective in hydrolyzing cottonseed meal extract oligosaccharides without forming inhibitors such as acetic acid and furfural. The HL27659(pKK313) consumed 73 mM hexose sugars from cottonseed meal extract hydrolysate and produced 64 mM succinate with a high molar succinate yield of 0.87 in 24h under aerobic conditions. This study demonstrated the use of inexpensive cottonseed soluble sugars to produce succinate aerobically with close to maximum theoretical succinate yield. Studies are being performed to integrate oligosaccharide catabolizing genes in the E. coli chromosome and optimize fermentation conditions in bioreactors.