Handbook of Vanilla Science and Technology

A similar process using pre-purified ferulic acid as the starting substrate, with a combination of yeast R. rubra with bacterium Nocardia sp., was mentioned as a possible combination. First, R. rubra IFO 889 produces vanillic acid by β-oxidation from ferulic acid. Vanillic acid is then converted by Nocardia sp. NRRL 5646 carboxylic acid reductase to vanillin (Li and Rosazza 2000).

A bacterial fermentation coupled with an enzymatic reduction serves as another example. A recombinant bacteria E. coli, which contains a part of the plant shikimate pathway, produces vanillic acid from glucose. Subsequently, vanillic acid is reduced to vanillin by a purified aryl aldehyde dehydrogenase (Frost 2002).

19.4.4 Continuous fermentation with immobilized cells

Since the bioconversion of ferulic acid requires cells in their resting growth phase, it is possible to engineer a continuous system. Cells, in particularly unicellular micro-organisms such as bacteria and yeasts, are easily immobilized on highly inert and porous polymeric materials. Ferulic acid or another substrate is then fed at a calculated rate to allow for the bioconversion to take place during one pass. The product is recovered either by a solvent extraction or by a hydrophobic adsorption. Physically immobilized C. glutamicum was fed 1 g/l/hr of ferulic acid and it produced continuously 0.024 g/l/hr of vanillin, which was continuously removed by XAD resin over 164-hour period (Labuda, unpublished data). Even though the productivity was quite low, improvement of this system could result in a commercially viable process.

19.4.5 Enzymes

Vanillin production from eugenol and isoeugenol by enzymatic biotransformation is another way to produce vanillin. The use of styrene dioxygenase, vanillyl alcohol oxidase (dehydrogenase), and lipoxygenase have been reported (van den Heuvel et al. 2004; Li et al. 2005). Creosol and vanillylamine were transformed to vanillyl alcohol and then to vanillin via a two-step process under the catalysis of vanillyl alcohol oxidase (van den Heuvel et al. 2004).

A crude enzyme preparation of lipoxygenase extracted from soybean converted isoeugenol to vanillin. With the addition of powdered activated carbon and hydrogen peroxide, 2.46 g/l vanillin was obtained with a molar yield of 13.3% (Li et al. 2005). Similar to the observation with microbial biotransformations, the vanillin yields from isoeugenol were higher than those from eugenol.

Soybean lipoxygenase was employed in a bioconversion of clove oil into vanillin in a silicon rubber membrane bioreactor. It reached 0.12 g/l vanillin after 36 hours. The conversion rate was 1.01%. Upon addition of charcoal into the conversion broth, vanillin concentration in the receiving solution reached 0.14 g/L, and the conversion rate of clove oil increased to 1.14% (Wu et al. 2008).

19.4.6 Cofactors

Sulfhydro compounds, for example, dithiothreitol (DTT), gluthathione were reported as important for bioconversion of ferulic acid to vanillin by various microorganisms (Labuda et al. 1992b). However, the mechanism has never been elucidated. The effect of sulhydro compounds is very broad. Recent patents on formation of vanillin used compounds such as thioacetic acid, as a part of their vitamin solutions, without elaborating on their importance (Cheetham et al. 2005).

DMSO (10%) was used to dissolve isoeugenol in the water phase. It was effective in promoting vanillin producing activities in P. putida when isoeugenol was a precursor. The addition of DMSO also inhibited the formation of vanillic acid (Yamada et al. 2007).

Addition of phospholipids accelerated bioconversion of ferulic acid to vanillic acid. Phospholipids, such as soy phospholipids (phosphatidylcholine, lysophosphatidylcholine, phosphatidyl-ethanolamine, acylphosphatidylethanolamine, phosphatidylinositol, phospha-tidic acid, etc.) were suggested to be used at a concentration range of 0.1 to 20 g/l (Lesage-Meessen et al. 1999).

Peroxidase, laccase, and some lyases, which require hydrogen peroxide as an electron donor, were reported to improve the conversion of isoeugenol into vanillin by crude enzyme extracts from soybeans. The maximum concentration of vanillin of 2.46 g/l after 36 hours was reached with the aid of 0.1% H₂O₂ and 10 g/l of charcoal. It corresponds to a molar yield of 13.3% (Li et al. 2005).

19.5 CONCLUSION