Hydrochloric Acid → Vanillin

Temperature

30 °C

A culture of Micromucor isabellinus (Zyl 849) grown on yeast malt agar was used to inoculate a 500 mL starter culture flask containing 100 mL medium (15 g glucose; 5 g (NH4)2SO4; 2 g K2HPO4; 0.2 g NaCl; 0.2 g MgSO4; 0.015 g CaCl2, trace element solution, 1 mL; vitamins solution 10 mL; made up to 1 liter with pH 6.0 phosphate buffer (0.2M)) containing 2 g/L vanillic acid which was incubated at 30° C. with shaking at 200 rpm for 24 hours. This starter culture was added to 5 liters of the same medium in a fermenter with the medium components made up to 1 liter with deionised water and vanillic acid added to a concentration of 1.5 gL-−1, immediately prior to inoculation. The fermenter contents were stirred at 30° C. for 18 hours with the pH of the medium being maintained at pH 5.2 by the addition of 5M sodium hydroxide solution. After this time the medium pH was gradually reduced to 3.7 over 45 minutes by the addition of 5M HCl and temperature was maintained at 30° C. Dissolved oxygen concentration was maintained at 70% of saturation throughout the process. Assay was by hplc as described above. Prior to the pH being allowed to drop at the 18 hour stage, the amount of vanillic acid in the system was measured at 1.48 gL-−1; vanillin was detected in solution at 0.02 gL-l. As the reaction proceeded various amounts of vanillic acid or glucose were added as follows: 19.25 hours, 50 g glucose; 20.25 hours, 3 g vanillic acid; 21.5 hours, 4 g vanillic acid; 23 hours, 4 g vanillic acid. Substrate and product concentrations were measured as being: 19.25 hours, vanillic acid 1.5 gL-−1, vanillin 0.1 gL-−1;: 20.25 hours, vanillic acid 1.16 gL-−1, vanillin 0.36 gL-−1;: 21.5 hours, vanillic acid 1.07 gL-−1, vanillin 0.74 gL-−1;: 23 hours, vanillic acid 1.06 gL−1, vanillin 1.22 gL-−1;: 26 hours, vanillic acid 0.83 gL-−1, vanillin 1.96 gL-−1.

1. customwas incubated at 30° C.
2. additionThis starter culture was added to 5 liters of the same medium in a fermenter with the medium components
3. additionadded to a concentration of 1.5 gL
4. stirringThe fermenter contents were stirred at 30° C. for 18 hours with the pH of the medium
5. concentrationDissolved oxygen concentration
6. temperaturewas maintained at 70% of saturation throughout the process
7. customto drop at the 18 hour stage
8. additionwere added
9. custom19.25 hours, 50 g glucose
10. custom20.25 hours, 3 g vanillic acid
11. custom21.5 hours, 4 g vanillic acid
12. custom23 hours, 4 g vanillic acid
13. concentrationSubstrate and product concentrations
14. custom19.25 hours, vanillic acid 1.5 gL
15. custom20.25 hours, vanillic acid 1.16 gL
16. custom21.5 hours, vanillic acid 1.07 gL
17. custom23 hours, vanillic acid 1.06 gL−1, vanillin 1.22 gL
18. custom26 hours, vanillic acid 0.83 gL

ORD record

ord-c972e99bd5c14f4abe925d21360b5ca2

Source dataset

uspto-grants-2005_01

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US06844019B1