The implementation of anaerobic ammonia removal has potential for reducing the operating costs in wastewater treatment plants. However, the application of this process in mainstream treatment presents various challenges, particularly due to the sensitivity of anammox bacteria to low temperatures. To address these challenges, changes in the bacterial community were investigated under decreasing temperatures (from 30 to 12 °C) within a deammonification process conducted in a 10-L sequencing batch reactor (SBR) over a period of 114 days. The aim of the study was to determine which groups of microorganisms are most strongly linked by ecological relationships with anammox bacteria and how these relationships change in response to decreasing temperature. Community dynamics were analyzed by combining metagenomics, network analysis, and specific process rates. The nitrification process was most efficient at 25 °C, as indicated by high values of the ammonia and nitrite utilization rate, and nitrate production rate, all of which declined as the temperature decreased. In contrast, the activity and abundance of anammox bacteria remained stable throughout all phases of the experiment. The decreasing temperature negatively impacted Nitrospira bacteria, the main competitors of anammox in the deammonification system. Despite the reduced process efficiency, the SBR maintained stable functionality even at temperatures as low as 12 °C. Network analysis identified which bacterial groups most frequently co-occurred and interacted, as well as those most sensitive to temperature fluctuations. Among the bacterial nodes, Nitrospira, Phycisphaerae, and Prosthecobacter exhibited the highest degree of connectivity, suggesting that they participate in multiple interactions, such as metabolite exchange, cooperation, or competition. Nevertheless, the temperature node had a greater number of connections than any bacterial node, indicating that this environmental factor had a more important effect on microbial community formation than direct ecological relationships. These results contribute to a comprehensive understanding of the role of anammox bacteria in deammonification systems, their ecological interactions within microbial networks, and their activity under suboptimal temperature conditions, which is crucial for future practical applications.
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