Continuous production of butyric acid using cell-recycle mixed culture system

Abstract

Butyric acid has been widely used

as food additives and fiber modifiers to resist heat and sunlight

ingredient to prepare various chemicals including butanol and biodegradable polymers, and medicines for treating hemoglobinopathies, cancer, and gastrointestinal diseases. To this day, industrial production of butyric acid is dominated by chemical synthesis using crude oil as starting materials. However, oil price and other environmental issues cause price increasing of butyric acid and needs of food and health industries shift to reliable biocompatibility. Therefore, it is getting important to produce bio butyric acid through biological processes. For biological process of butyric acid, we can use two kinds of the butyric acid fermentation bacteria. First bacteria produce butyric acid as final product and the other bacteria produce butanol as final product. The second one is Clostridium species and that fermentation called acetone-butanol-ethanol (ABE) fermentation. This is one of the oldest industrial fermentation and at the same time, one of the largest biotechnological processes. In several researches, butyric acid was biologically produced by Clostridium pure culture system. Pure culture system is easy to maintain fermentation condition and shows high production yield. However, pure culture system requires high processing cost for construction and operation, since several disadvantages, such as vulnerable to contamination, needs easily degradable substrate, and low product selectivity. On the other hand, mixed culture system, using two or more microorganisms in fermentation, is more required for industrial mass production. Mixed culture system doesn？t require sterilization, showing high adaptive capacity owing to microbial diversity, could be used for continuous process, and has high capacity to use complex substrates. However, mixed culture system still has limitations like low production yield, low selectivity, and difficulty to keep stabile microbial community

therefore it is more important to determine the optimum fermentation conditions and advanced fermentation techniques.The objectives of this study are: 1) to construct the continuous stirred tank reactor with cell recycle for butyric acid production from molasses, waste in sugar production industry, and 2) to determine optimum conditions in the continuous bioreactor for high production of butyric acid via mixed culture fermentation.To achieve these objectives: first, heat treatment and accumulation was progressed for screening butyric acid producing microorganisms

second, batch tests under different pH and temperature were analyzed to determine optimum conditions through statistical methods such as response surface methodology (RSM) and carbon material balance analysis

third, optimum conditions determined from batch experiments were applied to continuous fermentor and also optimization study was progressed in continuous condition

and finally, we developed the high-performance butyric acid production system.The batch test showed that the fermentation at pH 4.5 and 40？C enabled the highest butyrate production of 5.93 g/l with a yield of 0.118 g/g (39.33% of theoretical yield, 0.3 g/g). The RSM analysis predicted the optimum condition as pH 4.80 and temperature 39.79？C with maximum production of 5.73 g/L. At continuous condition, optimum pH was 4.5 showing the same as the batch test result, and the highest butyric acid production was 4.3-4.8 g/L/day at pH 4.5 and temperature 35？C. Optimum cell density condition for butyric acid production was 20-30 g/L with 50 g/L molasses and the maximum butyric acid production was 6.3-7.7 g/L/day and average butyric acid production was 5.03 g/L at pH 4.5 at temperature 40？C, and optimum F/M ratio was 1.24 g glucose/g cell dw&#8729

day. In this study, we finally got three main results

1) pH is the most important operating factor to decide composition of organic acids and key factor to control the selectivity of targeted butyric acid

2) we need to keep high cell density under optimum F/M ratio, since high cell density under optimum F/M ratio can increase the butyric acid production and yield

and 3) continuous fermentation process would be preferred due to higher selectivity of targeted product than batch culture.We constructed the continuous stirred tank reactor with cell recycle showing high productivity and high selectivity for the production of butyric acid from molasses, through applying optimum condition determined from batch experiments. This study provides the strategy to optimize operating factors of the mixed culture bioreactor for stable and economical production of butyric acid in a large scale, and also suggests a useful guideline to enhanced understanding the organic acid production process for industrial application.