Characterizing the microbial degradation of kraft lignin and lignin-derived compounds

Abstract

Analytical methods for characterizing the microbial degradation of Kraft lignin and lignin-derived compounds were utilized with the goal of biologically generating demethylated lignin for subsequent industrial applications. Selected ion flow tube mass spectrometry (SIFT-MS) technology was used for the first time with both bacterial and fungal cultures growing on lignin as a sole carbon source. Methanol and other volatile compounds were evaluated using this method and lignin-derived compounds were identified. Methanol oxidation products were found in the headspace of seven microbial cultures, as well as several unknown products not present in the SIFT-MS compound library. An assay was then developed to both confirm the results obtained by the SIFTMS and help to understand the nature of the microbial demethylation reactions. The Ti(III)-NTA assay was found to be an economical method for rapidly determining the relative degree of lignin demethylation by cultures of microorganisms and their enzymes. Using the Ti(III)-NTA assay, some fungal cultures were found to degrade lignin monomers completely and others to metabolize methanol. Four cultures were then selected for growth optimization; to both maximize vicinal diol generation and methanol formation. By altering variables such as induction day, incubation length, culture agitation, hydrogen peroxide concentration and micronutrient concentrations (known to promote enzyme production), the effect on four fungal species was investigated. Induction with vanillin after 1 week of growth on glucose resulted in the highest demethylation activity. In the final study, culture media from the fungus Absidia cylindrospora and the bacterium Sphingobium sp. SYK-6 were used to partially purify demethylating activity. The fungal enzyme had higher specific activity than the bacterial enzyme, but was much less abundant. Further research is needed to purify these enzymes responsible for demethylation.