Intensifying Nitrogen Removal Capacity of Membrane Biofilm Reactor Coupling n-DAMO and Anammox Processes by Dosing Signaling Molecules

Coupling nitrite- and nitrate-dependent anaerobic methane oxidation (n-DAMO) with anammox processes enables efficient biological nitrogen removal from wastewaters. However, the inherently slow growth and low activity of n-DAMO microorganisms have hindered their broader applications. Here, we investigated the impact of the exogenous quorum sensing signaling molecule dose on the performance of a lab-scale membrane biofilm reactor (MBfR) integrating n-DAMO and anammox. Long-term operation (∼380 days) demonstrated that the dosage of C6-HSL (N-hexanoyl-l-homoserine lactone, 0.2 μM) enhanced the total nitrogen removal rate of MBfR by 59.0%. In situ batch tests showed that the nitrate removal rate of n-DAMO archaea (Candidatus Methanoperedens) and the nitrite removal rate of anammox bacteria (Candidatus Brocadia) increased by 65.6 and 48.5%, respectively, while the nitrite removal rate of n-DAMO bacteria (Candidatus Methylomirabilis) exhibited a modest increase of 15.4%. qPCR and reverse transcription qPCR confirmed that C6-HSL promoted both growth and specific activity of n-DAMO archaea and anammox bacteria, while it had only manorial effects on n-DAMO bacteria. C6-HSL gradually accumulated in the biofilm and was sensed by these nitrogen-converting microorganisms, triggering gene regulatory responses associated with growth and metabolism. Notably, once the biofilm reached a threshold signaling molecule level, additional C6-HSL dosing became unnecessary. The findings reveal a quorum-sensing-mediated strategy to stimulate key microbial populations in n-DAMO–anammox systems, offering a practical approach for accelerating reactor start-up and intensifying its nitrogen removal capacity.