Fermentative Process Optimization And Mechanisms Of Butyric Acid Production From Rice Straw With Undefined Mixed Culture

Lignocellulosic biomass is a renewable bioresource in plentiful supply, and its utilization for the production of fuels and chemicals could meet the energy needs and sustainable development. However, the utilization of this low-cost bioresource is very limited. The cost of cellulolytic enzymes is one of the major technological and economical hindrances in the production of fuels and other products from lignocellulosic biomass. Applying mixed culture fermentation technology to anaerobically convert lignocellulosics into valuable products is a promising strategy to overcome the impediment. In a mixed culture fermentation process, there is no necessary supplement of cellulase. Butyric acid is a valuable chemical with wide applications in chemical, food, and pharmaceutical industries and potential applications in production of biobutanol and biodegradable plastics. However, the butyric acid production from lignocellulosic biomass is hindered by the cost of cellulolytic enzymes, low concentrations in the fermentation broth, low yield and high recovery cost. This study describes an alternative mixed culture fermentation technology to anaerobically convert lignocellulosic biomass into butyric acid without supplementary cellulolytic enzymes. The objectives of this study were to evaluate the NaOH pretreatment conditions of rice straw, to obtain a stable undefined mixed community for butyrate production from the pretreated rice straw, to structure and control the fermentation systems for butyric acid production, and finally to explore the mechanisms of butyric acid fermentation from rice straw with undefined mixed culture.Rice straw was soaked in1%NaOH solution to increase digestibility. By soaking rice straw at50°C for72h removed66%of the lignin, but retained84%of the cellulose and71%of the hemicellulose. At the same time, eight inocula sampled from various natural sources were used to start the enrichment procedure. After8months of continuously transferring, an anaerobic microbial community possessing stable activity of both cellulose and hemicellulose utilization and high selectivity of butyric acid production was obtained. The PCR-DGGE and sequence analysis revealed that microbial community included cellulolytic and xylanolytic bacteria, butyrate-producing bacteria and other acidogenic bacteria. Using this cellulose-degrading butyrate-producing microbial community as butyric acid producer in batch fermentation, about6g/L of butyric acid was produced from the pretreated rice straw, which accounted for76%of the total volatile fatty acids (VFAs). This percentage of butyric acid was equivalent to those from glucose fermentation by pure butyrate-producing bacteria species like Clostridium butyricum and Clostridium tyrobutyricum.To enhancing the rice straw conversion and butyric acid production, the effects of pH, inhibitors of methanogenesis and fermentation models were studied. The pH range of6.0to6.5was found favorable for butyric acid production. CaCO3combined with NaHCO3was believed as an effective buffer system for maintaining pH at that desired range and7.6g/L of butyric acid was obtained. Applying0.08g/L chloroform into the fermentation system, methanogenesis was totally inhibited without negative influence on butyric acid production. In this study, due to the decline of butyric acid production batch by batch, repeated batch operation was not suitable for butyric acid fermentation with undefined mixed culture. Comparing to batch operation, fed-batch operation was more suitable for butyric acid concentration. In the fed-batch operation, the inhibition of butyric acid production by acid products and Na+and the aging of mixed culture were relieved by the discharging of the fermentation broth.A stirred-tank reactor with20L effective volume was operated in fed-batch fermentation mode with fermentation broth discharging of3L/d followed by fresh fermentation medium feeding of3L/d, and the pH was controlled at6.0~6.5.28.5g/L of total VFAs was obtained. The butyric acid concentration reached16.1g/L, which was higher than many of those produced by pure cultures from glucose or sucrose. The butyric acid production corresponded to35%of the maximum theoretical yield, and total VFAs production corresponded to64%of the maximum theoretical yield based on the acetic acid equivalent. The PCR-DGGE analysis showed that the microbial community structure was assumed stable in the fed-batch fermentation process.It is concluded that two mechanisms are responsible for the butyric acid production in the undefined mixed cultured-based processes. The first one based on the balance theory of butyric acid production and acetic acid production. In the metabolic pathway of butyric acid fermentation, four ATPs are produced in acetate branch, and three ATPs in butyrate branch. At the beginning of fermentation, more acetic acid was produced for more ATP to meet the higher energy demand. As time went on, excreted acetic acid was taken up and converted into butyric acid, and the metabolism shifted to butyric acid production to response the decreasing of pH and the excess of NADH+H+. The second one based on the secondary fermentation in undefined mixed cultures. Butyric acid can be converted from the products of primary fermentation by some species within undefined mixed culture through several secondary fermentation reactions: chain elongation of acetic acid with ethanol, lactic acid oxidation with acetic acid and H+as electron acceptor.