Enrichment of comammox Nitrospira from three different seed sludges with addition of signaling molecules

Complete ammonia oxidation (comammox) bacteria have been detected in full-scale wastewater treatment plants. However, the slow growth rate limits the research and applications of comammox. Quorum sensing (QS) is an intercellular communication process to regulate microbial physiological metabolism, but its role in comammox Nitrospira has rarely been reported. In this study, denitrifying filter backwash, return and anoxic tank sludges were utilized as seeding inoculums to enrich comammox bacteria, with the addition of three types of signaling molecules (C6-HSL, C8-HSL and C12-HSL). Under ammonia- and dissolved oxygen (DO)-limited conditions, 12 lab-scale sequencing batch reactors (SBRs) were operated for 90 days. Quantitative polymerase chain reaction (qPCR) and 16S rRNA gene sequencing supported the enrichment of comammox Nitrospira in all SBRs. The highest absolute abundance of comammox Nitrospira amoA gene was detected in the anoxic tank sludge with exogenously added C8-HSL, reaching an average of 2.34×10⁶ copies/(g sludge). In this condition, comammox Nitrospira and ammonia-oxidizing archaea contributed up to 94 % of the total nitrification activity, with ammonia-oxidizing bacteria accounting for only 6 %. Overall, the role of QS in comammox Nitrospira enrichment was confirmed, with signaling molecules significantly accelerating the growth of comammox Nitrospira, promoting functional enzyme activity, and strengthening nitrifying bacterial competitiveness. Among the signaling molecules tested, C8-HSL exhibited the most pronounced promotional effect, followed by C6-HSL and C12-HSL. Using anoxic tank sludge as the seed sludge with the addition of C8-HSL provides a rapid and reliable enrichment strategy for comammox Nitrospira. These findings offer insights into the role of QS in comammox bacteria enrichment, and might facilitate the development of biotechnologies for wastewater treatment based on comammox Nitrospira.