Characterization of microbial growth in lignin-based residues and biodegradation of vanillin: : Optimizing factors for maximizing the extraction of a biodegradation compound of vanillin and investigating the potential for lipid accumulation.

Abstract: Vanillin (4-hydroxy-3methoxybenzaldehyde) is one of the most employed aromatic and flavoring additives in food and cosmetic industry. The industrial interest in vanillin could also apply to its biodegradation products. The microbial transformation of vanillin can open the possibility of new products with new areas of application for products related to vanillin. For example, vanillyl alcohol, vanillic acid and ferulic acid are currently used in the pharmaceutical or food industry. Some species reported to biodegrade vanillin into the related products vanillyl alcohol and vanillic acid, are: Brettanomyces anomalus and Saccharomyces cerevisiae. Moreover, certain microorganisms possess the ability to accumulate lipids when cultivated on different carbon sources, opening the possibility of microbial lipid production as another industrial application. The present investigation focuses on the optimization of extraction methods for vanillin biodegradation products, as well as identifying the isolates of a collection of microorganisms originating from the Faroe Islands that are amenable to being cultivated on a lignin-based media. Finally, the potential for microbial lipid accumulation was also studied. Two analytical methods, Thin-Layer Chromatography (TLC) and Gas Chromatography (GC) were employed for characterizing the biodegradation products obtained after 24 hours and 72 hours of culture in growth medium supplemented with 1 mM of vanillin. The results showed that after 24 hours of incubation, the model microorganism, strain FMYD002, had consumed some of the vanillin and transformed it into biodegradation products. TLC retention factors and GC chromatograms revealed that the main biodegradation product after 24 hours - when compared to a standard – is likely to be to vanillyl alcohol. Furthermore, vanillin and its biodegradation products were relatively temperature-stable based on a temperature test of supernatant from a 24-hour culture, however, when the 72-hour culture had been subjected to the highest temperature (60 °C) some spontaneous decomposition occurred. The biodegradation pattern of the 72-hour culture evidenced by TLC revealed two additional biodegradation products, one of which migrates in a similar fashion to vanillic acid. After 72 hours of incubation, the biodegradation product presumed to be vanillyl alcohol was no longer observed. Acidification tests showed that the best route for extraction of the product believed to be vanillyl alcohol is to adjust the extracted sample to a pH of 9. The cultivation test of the isolates in media prepared from different lignin-based residual products showed that 26 out of 60 initial strains grew regardless of the concentration of lignosulfonates and vanillin. Moreover, 17 strains grew in nitrogen-limited medium. Eight of the strains accumulated lipids. A preliminary categorization of isolates based on their colony morphology and capacity of growth on different substrates showed that to some extent, their morphology can predict the ability to grow on lignin- and vanillin-based media. This could help future scientists to easily screen for and select isolates with interesting activity for the ligno-cellulose industry.