Micro-aeration and low influent C/N are key environmental factors for achieving ANAMMOX in livestock farming wastewater treatment plants.

Anaerobic ammonium oxidation (ANAMMOX)-mediated system is a cost-effective green nitrogen removal process. However, there are few examples of successful application of this advanced wastewater denitrification process in wastewater treatment plants, and the understanding of how to implement anaerobic ammonia oxidation process in full-scale is still limited. In this study, it was found that the abundance of anaerobic ammonia-oxidizing bacteria (AnAOB) in the two livestock wastewater plants named J1 and J2, respectively, showed diametrically opposed trends of waxing and waning with time. The microbial communities of the activated sludge in the two plants at different time were sampled and analyzed by high-throughput sequencing of 16S rRNA genes. Structural equation models (SEMs) were used to reveal the key factors affecting the realization of the ANAMMOX. Changes in the concentration of dissolved oxygen and C/N had a significant effect on the relative abundance of anaerobic ammonia oxidation bacteria (AnAOB). The low concentration of DO (0.2∼0.5 mg/L) could inhibit the activity of nitrifying bacteria (NOB) to achieve partial oxidation of ammonia nitrogen and provide sufficient substrate for the growth of AnAOB, similar to the CANON (Completely Autotrophic Nitrogen removal Over Nitrite). Unlike CANON, heterotrophic denitrification is also a particularly critical part of the livestock wastewater treatment, and a suitable C/N of about 0.6 could reduce the competition risk of heterotrophic microorganisms to AnAOB and ensure a good ecological niche for AnAOB. Based on the results of 16S rRNA and microbial co-occurrence networks, it was discovered that microorganisms in the sludge not only had a richer network interaction, but also achieved a mutually beneficial symbiotic interaction network among denitrifying bacteria (Pseudomonas sp., Terrimonas sp., Dokdonella sp.), AnAOB (Candidatus Brocadia sp.) at DO of 0.2∼0.5 mg/L and C/N of 0.6. Among the top 20 in abundance of genus level, AnAOB had a high relative abundance of 27.66%, followed by denitrifying bacteria of 3.67%, AOB of 0.64% and NOB of 0.26%, which is an essential indicator for the emergence of an AnAOB-dominated nitrogen removal cycle. In conclusion, this study highlights the importance of dissolved oxygen and C/N regulation by analyzing the mechanism of ANAMMOX sludge extinction and growth in two plants under anthropogenic regulation of AnAOB in full-scale wastewater treatment systems.