De Novo Growth Of Methanogenic Granules Indicates A Biofilm Life-Cycle With Complex Ecology

Methanogenic sludge granules are densely packed, small (diameter, approx. 0.5-2.0 mm) spherical biofilms found in anaerobic digesters used to treat industrial wastewaters, where they underpin efficient organic waste conversion and biogas production. A single digester contains millions of individual granules, each of which is a highly-organised biofilm comprised of a complex consortium of likely billions of cells from across thousands of species - but not all granules are identical. Whilst each granule theoretically houses representative microorganisms from all of the trophic groups implicated in the successive and interdependent reactions of the anaerobic digestion process, parallel granules function side-by-side in digesters to provide a meta-organism of sorts. Granules from a full-scale bioreactor were size-separated into small, medium and large granules. Laboratory-scale bioreactors were operated using only small (0.6-1 mm), medium (1-1.4 mm) or large (1.4-1.8 mm) granules, or unfractionated (naturally distributed) sludge. After >50 days of operation, the granule size distribution in each of the small, medium and large bioreactor types had diversified beyond - to both bigger and smaller than - the size fraction used for inoculation. New granules were analysed by studying community structure based on high-throughput 16S rRNA gene sequencing. Methanobacterium, Aminobacterium, Propionibacteriaceae and Desulfovibrio represented the majority of the community in new granules. H2-using, and not acetoclastic, methanogens appeared more important, and were associated with abundant syntrophic bacteria. Multivariate integration analyses identified distinct discriminant taxa responsible for shaping the microbial communities in different-sized granules, and along with alpha diversity data, indicated a possible biofilm life cycle.\n\nImportanceMethanogenic granules are spherical biofilms found in the built environment, where despite their importance for anaerobic digestion of wastewater in bioreactors, little is understood about the fate of granules across their entire life. Information on exactly how, and at what rates, methanogenic granules develop will be important for more precise and innovative management of environmental biotechnologies. Microbial aggregates also spark interest as subjects in which to study fundamental concepts from microbial ecology, including immigration and species sorting affecting the assembly of microbial communities. This experiment is the first, of which we are aware, to compartmentalise methanogenic granules into discrete, size-resolved fractions, which were then used to separately start up bioreactors to investigate the granule life cycle. The evidence, and extent, of de novo granule growth, and the identification of key microorganisms shaping new granules at different life-cycle stages, is important for environmental engineering and microbial ecology.