Climate change is a current crisis that requires reduced reliance on non-renewable fuels, and lignocellulose offers an abundant and undervalued renewable fuel alternative. Lignin is a strong barrier to the conversion of cellulose to biofuels, and lignin valorization is necessary for lignocellulosic biofuels to be market competitive. As the largest natural source of aromatics, lignin is potentially a valuable source of flavors, fragrances, dyes, and biofuels. A mechanistic understanding of anaerobic microbial lignin modification is required for lignin valorization.
Tolumonas lignolytica BRL6-1 is a novel, facultative anaerobic bacterium that was isolated from tropical forest soils on lignin as sole carbon source. In concordance with the "Molecular Transformation" theme of EMSL and the mission of the Department of Energy Biological and Environmental Research (DOE BER), this proposal aims to determine the molecular mechanism of Tolumonas lignolytica BRL6-1 in modifying and depolymerizing lignin under anoxic conditions. Our hypotheses are that (H1) BRL6-1 produces a small molecule that acts as both a chelator and redox agent in the presence of lignin in anoxic conditions, and, (H2) lignin is being modified due to electron exchange between iron, lignin, and redox agent.
To test these hypotheses, BRL6-1 was grown in lignin-amended media and un-amended media controls and we have been able to make some insights into the mechanism of BRL6-1 interaction with lignin. In the presence of lignin, BRL6-1 has a greater biomass and shorter lag phase compared to un-amended conditions, indicating lignin is beneficiary to BRL6-1 metabolism. Proteomics showed that during early exponential phase, up-regulated proteins (based on 2-fold change or greater) were related to iron transport when lignin was present. Supernatant fractions were tested for chelating ability via Arnow assays, and chelating compounds <10kDa were detected in the lignin-amended supernatant. These preliminary results support our working model of BRL6-1 lignin modification by small molecules.
With EMSL support to further this project, we aim to (i) determine the structure of these redox- or chelator-active molecules, (ii) identify and quantify the free radicals produced by BRL6-1 in the presence of lignin, and (iii) define which linkages and subunits of lignin are modified in the presence of BRL6-1. If our hypothesis is correct, this would be the first demonstration of anaerobic microbial free radical generation for lignin modification to our knowledge.