Micron-scale biogeography reveals conservative intra anammox bacteria spatial co-associations.

Micron-scale resolution can help to reliably identify true taxon-taxon interactions in complex microbial communities. Despite widespread recognition of the critical role of metabolic interactions in anaerobic ammonium oxidation (anammox) system performance, no studies have examined microbial interactions at the micron-scale in anammox consortia. To fill this gap, we extensively sampled (totally 242 samples) the consortia of a lab-scale anammox reactor at different length scales, including bulk-scale (∼cm), macro-scale (300-500 µm) and micron-scale (70-100 µm). We firstly observed evident micron-scale heterogeneity in anammox consortia, with the relative abundance of anammox bacteria fluctuated greatly across individual clusters (2.0%-79.3%), indicating that the biotic interactions play a significant role in the assembly of anammox communities under well-controlled and well-mixed condition. Importantly, by mapping the spatial associations in anammox consortia at micron-scale, we demonstrated that the conserved co-associations for anammox bacteria were restricted to three different Brocadia species over time, and their co-associations with heterotrophs were random, implying that there was no statistically significant symbiotic interaction between anammox bacteria and other heterotrophic populations. Further metagenomic binning revealed that the quorum sensing with secondary messenger c-di-GMP potentially holding on the conservative metabolic cooperation among Brocadia species. These results shed new light on the social behavior of the anammox community. Overall, delineating of biological structures at micron-scale opens a new way of monitoring the microbial spatial structure and interactions, paving the way for improved community engineering of biotreatment systems.