Handbook of Vanilla Science and Technology

Basic understanding of the metabolic pathways is important for a successful commercial scale-up in a most cost effective way. All process parameters are equally important. Besides making a decision on the production organism, we need to consider reactor and fermentation type. Cost of the media and substrate, its availability, toxicity, and solubility are all critical parameters. Also, product inhibition, product down-stream metabolism, product toxicity to cells, and product recovery need to be evaluated. Several approaches have been attempted to increase the productivity. Table 19.1 summarizes the highest yields of vanillin by different microorganisms produced from ferulic acid, eugenol, and isoeugenol, respectively.

Table 19.1 The highest microbial bioconversion yields of vanillin from various precursors

Substrate | Microorganisms | Yield (g/l) | Molar yield (%) | Variable parameters | Reference

Ferulic acid | S. setonii ATCC39116 | 13.9 | 75 | pH 8.5, glucose 17 hr | Muller et al. 1998

Ferulic acid | Amycolatopsis sp. HR167 | 11.5 | 77.8 | 32 hr | Rabenhorst and Hopp 2000

Ferulic acid | A. niger I-147 P. cinnabarius MUCL 384672 | 3.6 | 82 | continuous, 360 hrs phospholipids cellobiose | Lessage-Messen et al. 1999, 2002

Ferulic acid | P. putida Zyl581 | 2.2 | 73 | pH 8.5 43 hr | Cheetham et al. 2005

Ferulic acid from sugar beet | P. putida Zyl581 | 1.7 | 54 | pH 8.5 40 hr | Cheetham et al. 2005

Ferulic acid | S. setonii ATCC 39116 | 9.3 | 90 | pH 8.2, glucose pH 7.0 | Gunnarsson and Palmkvist 2006

Ferulic acid from rice bran | A. niger CGMCC 0774 P. cinnabarius CGMCC 1115 | 2.8 | 61.6 | 72 hr, resin HD-8 | Sun et al. 2008

Vanillic acid | Micrococcus isabellinus Zyl849 | 1.5 | 80 | pH 3.8; 20 hr continuous process; oil extraction; 18.9 g product | Cheetham et al. 2005

Glucose | E. coli ATCC 98859/Neurospora crassa SY7A | 0.3 | ― | 20 g/l glucose 0.4 g/l methionine 48 hr, pH 7.0 | Frost 2002

Eugenol | Pseudomonas sp. HR199 | 0.3 | 89 | ― | Overhage et al. 2003

Eugenol | lipoxygenase | 0.14 | 1.1 | 36 hr, charcoal | Wu et al. 2008

Isoeugenol | Pseudomona putida IE27 | 16.1 | 71 | pH20 C, 10% DMSO | Yamada et al. 2007

Isoeugenol | Pseudomona putida IE27 | 29.1 | 5.1 | in 60% isoeugenol 24 hr | Yamada et al. 2007

Isoeugenol | Pseudomonas chlororaphis | 1.2 | 12.9 | ― | Kasana et al. 2007

Isoeugenol | Bacillus pumillus S-1 | 3.8 | 40.5 | ― | Hua et al. 2007

Isoeugenol | Bacillus fusiformis CGMCC1347 | 8.1 | 17.5 | pH 7.072 hr, resin H103 | Zhao et al. 2006

Isoeugenol | Bacillus fusiformis SW-B9 | 32.5 | 5.8 | 60% isoeugenol 72 hr | Zhao et al. 2005

Isoeugenol | lipoxygenase | 2.46 | 13.3 | H₂O₂, charcoal | Li et al. 2005

19.4.1 Direct bioconversion process

In this process, the substrate is directly biotransformed into vanillin, which is recovered by subsequent organic solvent extraction or by hydrophobic adsorption. Economically feasible yields are achieved with purified ferulic acid as a substrate. Its conversion yields about 6 to 13 g/l vanillin, with a molar yield of about 75% within 40 to 50 hours of fermentation. This was reported for a biotransformation with actinomycetes, such as Amycolatopsis sp. HR167 (Rabenhorts and Hopp 1997) and Streptomyces setonii ATCC 39116 (Muller 1998). Ferulic acid (22.5 g/l) added in a NaOH solution was converted by S. setonii to vanillin (9-13 g/l) at 40 to 75% molar rate (Muheim et al. 2001). To achieve this high bioconversion rate, resting cells of S. setonii were used. Glucose was depleted. The pH of the bioconversion was maintained at pH 8.5.

Amycolatopsis sp. was given as precursor 19.9 g/l of ferulic acid in a stepwise fed-batch mode. After 47 hours, the culture produced 11.5 g/l vanillin with a molar conversion rate of 77% (Rabenhorst and Hopp 1997). On the other hand, S. setonii started to accumulate vanillin after glucose was exhausted and ferulic acid had been added in a stepwise fed-batch manner. Productivity varied, based on the fermentation volume, between 51% and 75%. The best yield of 13.9 g/l was achieved in a 10-liter fermentor from 22.5 g/l of ferulic acid (Muller et al. 1998). Besides vanillin, a number of other phenolic metabolites, such as vanillyl alcohol, vanillic acid, guaiacol, p-vinyl guaiacol, and 2-methyl-4-ethylphenol were produced during this bioconversion. This may present a challenge for the purification of vanillin.