| Butyric acid is an important organic acid which has various applications It can be produced with chemical synthesis or microbial fermentation. Fermentation method has some disadvantages such as low product concentration, complexity of post-processing and high cost for potential industrial application. However, fermentation method offers a production process with mild conditions, low energy consumption, low pollution and wide source of raw materials. Besides, the commercial price of butyric acid from microbial fermentation is high due to a strong preference by consumers and manufacturers for using bio-based natural ingredients in foods and pharmaceuticals. Therefore, much attention has been paid on fermentation method in recent years, and as a key step of this method, separation of butyric acid from fermentation broth with high efficiency is strongly demanded. In this study, extraction and esterification was adopted to isolate butyric acid, and several conditions involved in the separation process were investigated.In order to separate butyric acid with extraction method efficiently, trioctylamine (TOA) and n-octanol were adopted as extractant and diluent respectively. Effects of TO A concentration, pH of butyric acid solution, phase ratio and temperature on distribution coefficient were studied. The optimal conditions were:TOA concentration 40% (v/v), butyric acid solution pH 2-3, phase ratio 0.8, temperature 25℃. According to researches on extraction and back-extraction of butyric acid from fermentation broth, high distribution coefficient (13.91) was obtained with much less use of extractant at fermentation broth pH 2-3 and phase ratio 0.3; Extraction yield could be improved to 96.39% by adopting a two-stage extraction process. Further study showed that 0.8 mol/L NaOH was suitable as the stripping solution with a high recovery ratio (86.4%). The selected extractant can be regenerated well under the optimal conditions.To explore the performance of immobilized lipase in esterification method for butyric acid separation, a novel method for lipase immobilization was developed. Candida rugosa lipase was immobilized on fibrous material (cotton cloth) following a three-step procedure involving polyethylenimine (PEI) pretreating cotton cloth, adsorption of enzyme to PEI coated cotton cloth and glutaraldehyde cross-linking. Effects of amounts of PEI, support and enzyme, pH of PEI aqueous solution, concentrations of glutaraldehyde and stabilizing agent PEG 2000 on immobilization were investigated. The obtained optimal conditions were:0.1 g enzyme,10-20 mg PEI, PEI aqueous solution pH 8-10, mass ratio of support to enzyme 12-15, glutaraldehyde concentration 0.1-0.5%, PEG 2000 concentration 15 mg/mL.Catalytic performance of immobilized lipase in ethyl butyrate synthesis from butyric acid was evaluated. The time-course curves of esterification reaction were determined at various substrate concentrations (0.5 M or 0.8 M). Effects of reaction medium, water activity of reaction system, temperature, amount of immobilized lipase, substrate concentration and molar ratio of alcohol to acid on initial reaction rate and conversion yield were studied intensively. The optimal reaction conditions were:reaction medium cyclohexane, high reaction water activity (0.8-1.0), temperature 25℃, lipase amount 0.6 g, butyric acid concentration 0.6 M and ethanol concentration 0.25 M. A kinetic model of Ping Pong Bi-Bi mode with inhibition of both substrates was proposed and validated by experimental data. Furthermore, ethanol was a stronger inhibitor than butyric acid according to the fitting data of the model. Good operational and storage stability of the immobilized lipase were observed. Finally, a recycle batch reactor using immobilized lipase was developed for ethyl butyrate production. The achieved ethyl butyrat production revealed the promising potential of this immobilized lipase in practical applications. |
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