Handbook of Vanilla Science and Technology

Fermentations using filamentous type of organisms such as actinomycetes or fungi can present some challenges, such as highly viscous broths, unfavorable pellet formation, and uncontrolled fragmentation and lysis of the mycelium, which can result in a reduced productivity and more complex downstream processing.

When isoeugenol was used as a vanillin precursor with a growing culture of B. pumillus S-1, isoeugenol, at a concentration of 10 g/l, was converted into 3.75 g/l vanillin in 150hours, with a molar yield of 40.5% (Hua et al. 2007). This was the highest reported yield for isoeugenol so far.

Solid state fermentation by Phanerochaete chrysosporium, using autoclaved green coconut husk waste as a substrate, yielded vanillin in the range between 44 to 52 ug/g of solid support. Sun dried husks were found suitable for vanillin production. Vanillin was extracted by organic solvent from the solid support (Barbosa et al. 2008).

19.4.2 Bi-phasic fermentation

The use of an additional phase, whether solid or liquid, provides a three-fold benefit: protects the cells against the substrate toxicity, prevents downstream product degradation, and speeds up product recovery. A typical choice of second liquid phases would be either oil or a nonpolar solvent. On the other hand, solid phase can be selected from active charcoal, or a variety of hydrophobic inert resins such as XAD2, XAD4, FPX66, etc.

For example, a biphasic bioconversion of isoeugenol using Bacillus fusiformis led to a yield of vanillin up to 32.5 g/l, when isoeugenol (60%) was used as a substrate and as a non-polar solvent (Zhao et al. 2005). The disadvantage of this system is very high viscosity and difficulty in recovering vanillin from the isoeugenol phase.

A biphasic water/hexane system was employed in the production of 4-vinylguaiacol from ferulic acid by Bacillus pumillus. The yield of 9.6 g/l of vinylguaiacol from 25 g/l of ferulic acid indicates 38% conversion yield, or 49.7% molar yield (Lee et al. 1998).

Use of a biphasic water/vegetable oil process with Micromucor isabellinus Zyl849 produced 18.9 g/l vanillin from vanillic acid after 20 hours (Cheetham et al. 2005). Vanillic acid concentration was maintained at 1.5 g/l throughout the experiment. Culture medium was continuously pumped through an extraction vessel and extracted by warm oil at pH 6.0. The aqueous portion containing vanillic acid, which was not extracted into the oil phase, was returned to the fermentor. A similar experiment was done with A. fumigatus Zyl747, with the yield of 16.26 g of vanillin after 665 hours (Cheetham et al. 2005).

19.4.3 Mixed culture fermentation

This type of fermentation has received the most attention in the recent years (Lesage-Meessen et al. 1996,2002; Cheetham et al. 2005; Sun et al. 2008). The approach depends on the substrate and selected microorganisms.

If the precursor is plant material containing ferulic acid, the fermentation process consists of three steps:

I ferulic acid is released from the plant materials either by microbial or enzymatic hydrolysis;

II transformation of ferulic acid into vanillic acid; and III reduction of vanillic acid into vanillin.

Once ferulic acid is unbound, it is biotransformed to vanillic acid in the second step performed, for example by Ascomycetes such as Aspergillus niger, Basidiomycetes such as Bjerkandera adusta, and Actinomycetes such as S. setonii. In the third step, vanillic acid is reduced to vanillin and vanillyl alcohol, for example by Pycnoporus cinnabarinus.

As an example of such a mixed culture bioconversion, is a process using autoclaved maize bran and two fungi A. niger CNCMI-1472 and P. cinnabarinus MUCL 38467. First, A. niger released ferulic acid that was concomitantly deacetylated into vanillic acid. Afterwards, P. cinnabarinus reduced vanillic acid into vanillin (Lesage-Meessen et al. 2002) with the yield of 767 mg/l vanillin. In another experiment, A. niger was fed ferulic acid continuously at 430 mg/l per 24 hours to a final concentration of 5.05 g/l. After culturing for 15 days, the final concentration of vanillic acid in the culture was 3.60 g/l with a molecular conversion rate of 82%. Cellobiose was used as an elicitor and XAD2 resin was used to isolate vanillin from the broth. When only A.niger’s extracellular enzymes were used in a combination with P. cinnabarinus, 584 mg/l of vanillin was obtained (Lesage-Meessen et al. 1999, 2000). The whole process is quite long, because it requires also preculture of both fungi and purification of the product.