Size matters: Anaerobic granules exhibit distinct ecological and physico-chemical gradients across biofilm size.

Anaerobic biological decomposition of organic matter is ubiquitous in Nature wherever anaerobic environments prevail, and is catalysed by hydrolytic, fermentative, acetogenic, methanogenic, and various other groups. It is also harnessed in innovative ways in engineered systems that may rely on small (0.1-4.0 mm), spherical, anaerobic granules. These biofilms are crucial to the operational success of a range of widely applied engineered-ecosystems designed for wastewater treatment. The structure and function of granule microbiomes underpin their utility. Here, granules were separated into ten size fractions (proxies for age), hypothesizing that small granules are 'young' and larger ones are 'old'. Gradients were observed across size in terms of volatile solids, density, settleability, biofilm morphology, methanogenic activity, and profiles of extracellular polymeric substances, suggesting ongoing development of physico-chemical characteristics as granules develop. Short-read amplicon sequencing indicated a negative relationship between granule size and community diversity. Furthermore, as size increased, the methanogenic archaea dominated the microbiome. Small granules were found to harbour a sub-group of highly specific taxa, and the identification of generalists and specialists may point to substantial resilience of the microbiome. The findings of this study indicate opportunities for precision management of wastewater treatment systems. They suggest that size is an important indicator for aggregate utility - size may, indeed, determine many of the characteristics of both the individual-granule microbiomes and the overall function of a wastewater treatment system.