Research On The Production Of Fumaric Acid By Fermentation From Corn Cobs Hydrolysate

Fumaric acid, as an important four carbon platform compounds, is widely used in material, chemical, food, medicine, feed additives and other fields. However, with the depletion of oil and increasing price of fossil resource, producing fumaric acid in industry from benzene will be eliminated gradually. To produce fumaric acid with natural renewable resources by microorganism fermentation, can not only achieve resource substitution, but also provide a broad space for the development of fumaric acid industry. In this paper, dilute acid pretreatment and cellulose enzyme was used to hydrolysis corn cobs to reducing sugar, and detoxification treatment was done subsequently to make it more beneficial for microbial growth and metabolish. Meanwhile, Rhizopus oryzae was domesticated and its capacity of fermentation by using different carbon source was analyzed. Research on metabolic mechanism of Rhizopus oryzae to produce fumaric acid with five and six carbon sugars was carried out, and the metabolic network was built. On this basis, the production of fumaric acid was improved by optimizing the process parameters. The main research results were as follows:(1) With the orthogonal experiment, the optimal conditions of dilute acid pre-treatment were:sulfuric acid concentration 1.5%, reaction time 60 min, reaction temperature 120℃ and solid-liquid ratio 1:9, the concentration of reducing sugar in hydrolysate was 46.28±0.45 g/L. The optimal conditions of cellulase hydrolysis were:amount of enzyme 1000 U/g, reaction pH 4.8, reaction temperature 50℃, solid- liquid ratio 1:10 and reaction time 48 h, the concentration was 40.38±0.76 g/L. The concentration of total reducing sugar in corn cobs hydrolysate obtained through the dilute acid pre-treatment and cellulase was 43.33±0.62 g/L, including glucose 30.84±0.38 g/L and xylose 7.46±0.72 g/L, the concentration of glucose is about 4 times of xylose, and the yield of reducing sugar from corn cobs is 0.35 g/g.(2) The results of investigation on the detoxification of corn cobs hydrolysate indicated that the excessive alkali neutralization combined with activated carbon adsorption was better detoxification method. The optimal adsorption parameters were:activated carbon amount 6%, adsorption time 60 min, adsorption temperature 40℃. Under this condition, the removal rate of furfural was 70.71±0.25% and the loss rate of reducing sugar was 14.62±0.38%.(3) A furfural-resistant strain WHT5 with good genetic stability was gained through high-concentration furfural gradient medium cultivation, and the strain has better tolerance to fermentation inhibitors in corn cobs hydrolysate. Fermentation with the strain, the biomass was 23.07±0.53 g/L, about 3 times of the original strain. And the titer of expected product, fumaric acid, was reached 23.71±0.38 g/L, which was increased by 94.66% compared with the original strain.(4) Response surface methodology was used to obtain the optimal medium compositions of fumaric acid fermentation by Rhizopus oryzae from glucose and xylose, the outputs of fumaric acid were 37.18±0.56 g/L and 13.28±0.24 g/L, respectively. Single factor experiment was done to gain the optimal results from mixed sugar and corn cobs hydrolysate, the titers of fumaric acid were 42.18±0.42 g/L and 41.20±0.46 g/L, successively. Through metabolic products and key enzyme activity analysis, metabolic pathways and networks of Rhizopus oryzae to produce fumaric acid by five and six carbon sugar co-fermentation were confirmed. Then metabolic flux equations were built and metabolic flux was calculated. Single factor experiment was investigated to ensure the optimal pH 2.5 of pre-culture medium for Rhizopus oryzae, and mycelia morphology which was suitable for fermentation was obtained. The better process conditions for producing fumaric acid by fermentation was tempreture 32℃, agitation speed 500 r/min and ventilation 0.40 L/min·L. On these conditions, the accumulation of biomass in the broth was 8.6±0.24 g/L, the consumption of glucose and xylose were 92.72±0.86 g/L and 13.26±0.47 g/L, respectively. And the concentrations of organic acid were as follows:fumaric acid 49.54±0.62 g/L, malic acid 0.62±0.38 g/L, succinic acid 1.97±0.15 g/L, lactic acid 1.40±0.18 g/L and citric acid 0.50±0.06 g/L. The titer of fumaric acid was improved significantly.(5) Fermentation experiment with corn cobs hydrolysate in stirred tank was carried out on the conditions acquired above. The results were as follows:glucose consumption 89.66±0.64 g/L, xylose consumption 11.20±0.47 g/L, fumaric acid output 44.82±0.35 g/L and biomass accumulation 9.20±0.11 g/L. The conversion ratio of fumaric acid relative to the reducing sugars in the hydrolysate was 44.44%, and it relative to the corn cobs was 15.55%.