Construction of acyl-homoserine lactone-producing engineered bacteria for activating low-temperature anammox process

Acyl-homoserine lactone (AHL)-mediated quorum sensing plays a crucial role in enabling anammox bacteria to adapt to environmental conditions. However, its practical application is constrained by the high cost of signaling molecules. In this study, we constructed a genetically engineered bacterial strain, designated ES-P1, by inserting the 3OC₆-HSL (typical signalings in AHLs) synthase gene into the pET28b (+) plasmid, followed by transformation of the recombinant plasmid into Escherichia coli. Through an optimized fed-batch fermentation process designed to maintain optimal growth and prevent substrate inhibition, we successfully produced 3OC₆-HSL, achieving a 53-fold increase in yield compared to previously reported AHL-producing bacteria, while reducing production costs by 94 % relative to chemically synthesized signaling molecules. Subsequently, the fermentation supernatant of ES-P1 (FSE) was introduced into a three-compartment anaerobic baffled reactor (ABR) operated at 10 °C to evaluate its biological effects. Due to the sequential configuration of the ABR, a concentration gradient of 3OC₆-HSL was established from the inlet compartment (C1) to the downstream compartments (C2, C3). The nitrogen removal rate in compartment C2 increased significantly by 43.3 %, whereas that in compartment C1 decreased notably by 13.2 %. In contrast, compartment C3 exhibited no statistically significant change in nitrogen removal performance. In C2, the secretion of endogenous signaling molecules was enhanced, accompanied by a marked increase in microbial interaction capacity and extracellular polymeric substances content. This study presents a strategy for enhancing the microbial adaptation of anammox bacteria to low-temperature environments through the use of cost-effective signaling molecules.