Different acetonitrile degraders and degrading genes between anaerobic ammonium oxidation and sequencing batch reactor as revealed by stable isotope probing and magnetic-nanoparticle mediated isolation

Microbial degraders play crucial roles in wastewater treatment processes, but their use is limited as most microbes are yet unculturable. Stable isotope probing (SIP) is a cultivation-independent technique identifying functional-yet-uncultivable microbes in ambient environment, but is unsatisfactory for substrates with low assimilation rate owing to the low isotope incorporation into DNA. In this study, we used acetonitrile as the target low-assimilation chemical in many wastewater treatment plants and attempted to identify the active acetonitrile degraders in the activated sludge, via DNA-SIP and magnetic-nanoparticle mediated isolation (MMI) which is another cultivation-independent approach without the requirement of substrate labeling. The two approaches identified different active acetonitrile degraders in a 3-day short-term anaerobic ammonium oxidation (ANAMMOX). MMI enriched significantly more acetonitrile-degraders than SIP, showing the advantages in identifying the active degraders for low-assimilation substrates. Sequencing batch reactor (SBR, 30-day degradation) helped in more incorporation of ¹⁵N-labeled acetonitrile into the active degraders, thus the same acetonitrile-degraders and acetonitrile-degrading genes were identified by SIP and MMI. Different acetonitrile degraders between ANAMMOX and SBR were attributed to the distinct hydrological conditions. Our study for the first time explored the succession of acetonitrile-degraders in wastewater and identified the active acetonitrile-degraders which could be further enriched for enhancing acetonitrile degradation performance. These findings provide new insights into the acetonitrile metabolic process in wastewater treatment plants and offer suggestive conclusions for selecting appropriate treatment strategy in wastewater management.