Routine culture-based assay for screening microbial lignin deconstruction

Evaluating microbial lignin deconstruction is very challenging because of the absence of validated routine screening methods. Lignin is the most recalcitrant component of lignocellulose and a potential source of high value replacements for petro-chemicals. Lignin primarily consists of three main aromatic subunits:  p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) subunits linked by complex array of C-C and C-O bonds. Previous studies have focused primarily on lignin fungal breakdown that employ free radical mechanisms involving enzymatic mechanisms (white-rot) and/or small molecule (brown rot) catalysts. Bacterial lignin deconstruction has been documented but mechanistic models are lacking. We have developed a culture-based assay for screening lignin deconstructing species that was based on the clearance of the dark brown color from minimal media supplemented with colloidal lignin-rich substrates within 3 – 5 days and the appearance of large cell-lignin aggregates. As a proof of concept, we demonstrated clearance of the assay broth by Streptomyces viridosporus T7A, a model lignin depolymerization species, compared to an uninoculated control, while no change was observed with Escherichia coli.  Using this assay, we identified potential lignin degrading species, including Lysinbacillus, a previously unidentified lignin deconstruction species. Lysinibacillus, rapidly cleared cultures with lignin and forming cell-lignin aggregates characterized by microscopy. This lignin-clearing assay has been demonstrated with lignin from Ionic-liquid pre-treated and saccharified eucalyptus, as well as commercially available forms of insoluble lignin.  Efforts are currently focused on understanding the detailed mechanisms of the assay and validating lignin deconstructing capabilities of species that were identified using this assay.