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.
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