Methanogenic sludge granules are densely packed, small, spherical biofilms found in anaerobic digesters used to treat industrial wastewaters, where they underpin efficient organic waste conversion and biogas production. Each granule theoretically houses representative microorganisms from all of the trophic groups implicated in the successive and interdependent reactions of the anaerobic digestion (AD) process. Information on exactly how methanogenic granules develop, and their eventual fate will be important for precision management of environmental biotechnologies. Granules from a full-scale bioreactor were size-separated into small (0.6-1 mm), medium (1-1.4 mm), and large (1.4-1.8 mm) size fractions. Twelve laboratory-scale bioreactors were operated using either small, medium, or large granules, or unfractionated sludge. After >50 days of operation, the granule size distribution in each of the small, medium, and large bioreactor sets had diversified beyond-to both bigger and smaller than-the size fraction used for inoculation. Interestingly, extra-small (XS; <0.6 mm) granules were observed, and retained in all of the bioreactors, suggesting the continuous nature of granulation, and/or the breakage of larger granules into XS bits. Moreover, evidence suggested that even granules with small diameters could break. "New" granules from each emerging size were analyzed 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 (MINT) analyses identified distinct discriminant taxa responsible for shaping the microbial communities in different-sized granules.

395 words (249 in 'Abstract' + 146 in 'Importance') 8 Text: 3,980 words (excl. references, table footnotes, figure legends) 9Abstract. Methanogenic sludge granules are densely packed, small (diameter, 1 approx. 0.5-2.0 mm) spherical biofilms found in anaerobic digesters used to treat 2 industrial wastewaters, where they underpin efficient organic waste conversion and 3 biogas production. A single digester contains millions of individual granules, each of 4 which is a highly-organised biofilm comprised of a complex consortium of likely 5 billions of cells from across thousands of speciesbut not all granules are identical. 6Whilst each granule theoretically houses representative microorganisms from all of 7 the trophic groups implicated in the successive and interdependent reactions of the 8 anaerobic digestion process, parallel granules function side-by-side in digesters to 9 provide a 'meta-organism' of sorts. Granules from a full-scale bioreactor were size-10 separated into small, medium and large granules. Laboratory-scale bioreactors were 11 operated using only small (0.6-1 mm), medium (1-1.4 mm) or large (1.4-1.8 mm) 12 granules, or unfractionated (naturally distributed) sludge. After >50 days of 13 operation, the granule size distribution in each of the small, medium and large 14 bioreactor types had diversified beyondto both bigger and smaller thanthe size 15 fraction used for inoculation. 'New' granules were analysed by studying community 16 structure based on high-throughput 16S rRNA gene sequencing. Methanobacterium, 17Aminobacterium, Propionibacteriaceae and Desulfovibrio represented the majority of 18 the community in new granules. H2-using, and not acetoclastic, methanogens 19 appeared more important, and were associated with abundant syntrophic bacteria. 20Multivariate integration analyses identified distinct discriminant taxa responsible for 21 shaping the microbial communities in different-sized granules, and along with alpha 22 diversity data, indicated a possible biofilm life cycle. 23Importance. Methanogenic granules are spherical biofilms found in the built 24 environment, where despite their importance for anaerobic digestion of wastewater 25