Leveraging Microscopy to Characterize the Morphology and Autofluorescence of Lignocellulose Degrading Microbes

Lignocellulose, the woody complex responsible for the strength of plant cell walls, is the most abundant renewable biomass resourceavailable. Its valorization promises to enable sustainable production of value-added products from biofuels to pharmaceuticals; however,lignocellulose is recalcitrant to chemical and biological degradation. Rumenous anaerobic microbial consortia, found in the primary stomach of ruminants(cows, sheep, etc.), naturally evolved to degrade lignocellulose as an energysource. Within the rumen, anaerobic fungi are the primary lignocellulose degraders. Methanogenic archaea consume byproducts of fungal metabolism andare critical to consortium function. Biomass breakdown is spatially dependent in these systems, as fungi bind to biomass and each other, and methanogens associate within the fungal network. To mechanistically understand lignocellulose degradation in anaerobic consortia, we aim to characterize spatial organizationsof the constituent microbes. The morphology ofa representative anaerobic fungus, Neocallimastix sp. (S3), was characterized via brightfield microscopy; morphology of a representative methanogen, Methanobacterium bryantii, is already well characterized.To distinguish between constituentmicrobes in a mixed culture, non-overlapping fluorescence in both species is necessary. Autofluorescence in the fungus was observed with excitationand emission channels spanning virtually the entire visible light spectrum, with limitedUVactivity.Autofluorescence in methanogens is well-characterized, with UV excitation and blue light emission. Therefore, we developed methods to resolve fungi and methanogens in co-culture without the use of stains, probes, or dyes. Following optimization of cultivation of imageable consortia, the spatial organization of rumenousconsortia will be quantified to further our understanding of consortium function.