Research On Microbial Production Of Vanillin

Vanillin(4-hydroxy-3-methoxybenzaldehyde or vanillic aldehyde) is the most important flavoring material and aromatic compound in the world. Because of its unique flavor and structure, it is extensively used not only in food industry but also in medical, pharmaceutical, electroplating, agricultural, manufacturing and chemical analysis industry.The demand for vanillin is huge. Even though chemically synthesized vanillin occupies most of the market share, its safety is always questioned by consumers. Since then, increasing emphasis has been put on health and food safety, which results in buoyant demand for natural vanillin. On the other hand, the worldwide demand of natural vanillin cannot be met by the limit supply from the time consuming, labor intensive extraction from plants like Vanilla orchid, it is necessary to develop alternative routes of producing natural vanillin through biotechnology at an economic price.This paper has focused on microbial production of vanillin from isoeugenol, utilizing Bacillus fusiforms CGMCC1347 as strain, aiming at solving the following problems: byproduct formation, low solubility of substrate, toxicity of substrate and product to bacteria, and poor reusability of bacteria.1. Medium optimization The fermentation medium was optimized in terms of the indegridents used as carbon and nitrogen sources, and their individual concentrations, in the fermentation medium. The results had shown that the choice of carbon sources had no difference on the conversion, and that the transformation was even more productive if without addition of any carbon source. On the other hand, nitrogen sources exhibited a significant influence on the conversion. Organic nitrogen source(with peptone the best) is superior to inorganic one. The optimal fermentation medium was determined to be: 10 g/L peptone, 5.2 g/L KH2PO4, 14 g/L K2HPO4·3H2O, 1 g/L Mg SO4·7H2O, p H 7.0.2. Ionic liquids(IL) as additives in the reaction system Ionic liquids were used as additives to explore their effects on vanillin production. Among the 35 ionic liquids tested, 11 of them can slightly improve the conversion: four imidazolium([MMIm][Me SO4], [EMIm][Me SO4], [BMIm][Me SO4], [Me(OEt)3EIm][Tf2N]); six ammonium([Choline][Cl], [Choline][H2PO4], [NHMe3][Me SO3], [NBu4][Me SO3], [NHMe3][H2PO4], [ETM][Tf2N]); and one phosphonium([PBu4][Ac]). The influence of ionic liquids on vanillin production is associated with both the substrate solubility in the ILcontaining reaction system and the toxicity of ionic liquids to Bacillus fusiforms CGMCC1347.3. Deep eutectic solvents(DES) as additives in the reaction system Deep eutectic solvents were used as additives to explore their effects on vanillin production. Among the 45 DESs tested(24 conventional DES and 21 NADES(natural deep eutectic solvents)), 36 of them(23 conventional ones and 13 NADES) can obviously improve the conversion. The components of deep eutectic solvents(like, choline chloride, choline acetate, urea, ethylene glycol) can also improve the conversion. Deep eutectic solvents can increase substrate solubility, which accounts for the effect of DES on conversion.4. Toxicity of deep eutectic solvents to Bacillus fusiforms CGMCC1347 Toxicity of deep eutectic solvents to Bacillus fusiforms CGMCC1347 was investigated by the use of laser scanning confocal microscopy, flow cytometry and ultraviolet spectrophotometer. Results had demonstrated that the presence of DESs did result in the damage of the cell membrane integrity, presumably accompanied by spills of nucleic acid and protein. Choline chloride-based DESs presented higher toxicity than Choline acetate-based ones, and the toxicity increased with an increase in the DES concentration.5. Cell immobilization Bacillus fusiforms CGMCC1347 was immobilized by entrapment into calcium alginate gels. The spherical beads that were produced had a diameter of about 2-4 mm. After 13 cycles of reaction, the conversion showed no trend of decrease, maintaining at 1.2 g/L. Beads were proved to be very durable, showing no sign of break, agglomeration or disintegration. After being used for 13 cycles, the bacterial cells in the beads looked identical to those original ones, as can be seen from their electron scanning micrographs. All these demonstrate that the immobilization method is effective in maintaining the activity of the bacterial cells and faciliting their reusuability.In conclusion, this paper has provided valuable information regarding microbial production of vanillin and also the application of using ILs and DESs as additives in whole-cell biocatalysis.