Study On Vanillin Preparation By Bioconversion With A PDMS Membrane Bioreactor

Vanillin is one of the most widely used agents in foods, pharmacy, flavors, andother synthetic chemicals. The special vanillin produced by bioprocess is called"biovanillin", which usually means bioconversion processes with microorganismsand enzymes.However, vanillin is not so easily extracted and purified from the broth in mostcommon bioconversion processes, due to its further oxidation and product inhibition.A practical process is usually complex or low in efficiency, and intruding impuritiesin its isolation and purification deteriorate the final product and disagree withrequirements for its natural features.In this study, we established a system of silicone rubber membrane bioreactor(abbreviated SRMBR) with a composite membrane PDMS prepared in our Lab.,which could selectively transfer vanillin into a solvent from its broth, withoutnegative effects on bioprocess in the bioreactor. Using this system and an enzyme asthe biocatalyst, we investigated vanillin preparation by bioconversion.The ferment volume of this system was 1L, with an external-circulating moduleof 0.024m² membrane area. The performance tests showed that the system couldeffectively separate vanillin from the broth by perstraction. When the concentrationof the feed solution was 1.2%(w/w) at the temperature of 30℃, and the flow rate ofupstream and downstream are 120L/h and 80L/h, respectively, the overall masstransfer coefficient was 1.4027×10^-3m/h.The enzyme was lipoxygenase (SLO) isolated from soybean, and the substrate bio-catalyzing performances of SLO were verified by shaking test, and apre-optimization for bioconversion conditions was achieved. SLO isolationconditions: temperature 4℃and no adjustment of pH. The specific activity of SLOreached about 6.00×10⁵U/mL.We found that temperature was the most important factors affecting SLOactivity, which means that the SLO is thermosensitive and must work undertemperate environment. Furthermore, SLO must be operated at alkali environment tokeep its activity. The pre-optimization results for operating conditions of the systemwere temperature 28℃and pH8.0.We used activated charcoal as an enhancer to further optimizing bioconversionprocess in the system. Although Tween20 can emulsify Clove oil and enhance thedispersion in water, its encapsulation decreases the touch oportunity of Clove.oil andthe enzyme. Activated charcoal can absorb the Clove oil on its surface, so it.is betterthan tween20 in enhancing dispersion and touch opportunity. H₂O₂ can not enhancethe bio-catalysis but initialize the bio-reaction as an activity inducer of SLO. MuchH₂O₂ could destroy SLO structure and then deteriorate its activity.Compared with shaking flask experiment, the SRMBR system effectivelyprevented the product inhibition of the enzyme and vanillin oxidation loss, thebioconversion rate of clove oil increased to 1.04% from 0.45%, and the maximumvanillin concentration in receiving solution was 130.10mg/L which was much higherthan 11.13 mg/L in the flask. By adding activated charcoal into the bioreactor, theconversion rate was improved to 1.09%, and the maximum vanillin concentrationwas 136.48 mg/L in receiving solution, vanillin purity was quantified to 96.77% byHPLC analysis.The experiment carried out in the PDMS membrane bioreactor showed thesystem can make a good coupling of vanillin bioconversion and separation. Theconversion rate of Clove oil was enhanced, and the enzyme inhibition was reduced.Furthermore, being selectively transferred and dissolved into the downstreamsolution, high purity of vanillin could easily extracted from the downstream solution.However, there were still some problems, for example, SLO activity not continuing longer, lower extraction rate of vanillin, etc. It seems that these problemscan be overcome by improving conditions in SRMBR, optimizing SLO, modifyingthe module and increasing membrane area.In this work, we have brought forward a novel process technology that couldprepare vanillin from Clove by bioconversion with a SRMBR bioreactor and SLOenzyme. The process technology was characterized in continuously feeding substrateinto the fermentor and continuously extracting product vanillin by the perstractivePDMS membrane. This process could elevate significantly substrate utilization andreduced the residual broth discharge. More experiments are being conducted.