17Autotrophic nitrogen removal by anaerobic ammonium oxidizing (anammox) bacteria is an 18 energy-efficient nitrogen removal process in wastewater treatment. However, full-scale 19 deployment under mainstream conditions remains challenging for practitioners due to the high 20 stress susceptibility of anammox bacteria towards fluctuations in dissolved oxygen and 21 temperature. Here, we investigated the response of microbial biofilms with verified anammox 22 activity to oxygen shocks under favorable and cold temperature regimes. Genome-centric 23 metagenomics and metatranscriptomics were used to investigate the stress response on various 24 biological levels. We show that temperature regime and strength of oxygen perturbations 25 induced divergent responses from the process level down to the transcriptional profile of 26 individual taxa. Temperature induced distinct transcriptional states in compositionally identical 27 communities and transient pulses of dissolved oxygen resulted in the upregulation of stress-28 response only under favorable temperatures. Anammox species and other key biofilm taxa 29 display different transcriptional responses to the induced stress regimes.30 31Autotrophic nitrogen removal by biological anaerobic ammonium oxidation (anammox) is 33 increasingly implemented as an energy-efficient mechanism of fixed nitrogen elimination 34 during wastewater treatment. Deployed for mainstream wastewater treatment plants (WWTPs), 35 it may even permit operation under energy autarky 1 . In contrast to conventional nitrification- 36 denitrification system, external carbon sources are not required to reach very low effluent 37 nitrogen concentration, less aeration is needed and most of the organic load can be diverted for 38 valorization e.g. biogas or bio-plastics production [2][3][4] . Autotrophic N removal with anammox 39 involves the simultaneous oxidation of ammonium (NH4 + ) and reduction of nitrite (NO2 -) under 40 oxygen-limiting conditions 5 . In engineered systems aerobic ammonia-oxidizing bacteria (AOB) 41 oxidize a fraction of the available NH4 + to NO2 -(nitritation), which is subsequently used as a 42 terminal electron acceptor by anammox bacteria to oxidize the remaining NH4 + to N2 6 . The 43 process can be realized in single stage 7 or two-stage bioreactor systems 8 . Currently, autotrophic 44 N removal with anammox coupled to nitritation is already widely applied and represents a 45 robust method for the treatment of wastewaters with high N concentrations under mesophilic 46 conditions, e.g. effluents from anaerobic sludge digestion 9 . However, development of stable 47 anammox processes for mainstream municipal WWTPs suffers from unexplained process 48 instabilities due to unexpected fluctuation of environmental temperature and dissolved 49 oxygen 10-12 . 50 Anammox bacteria are characterized by very slow growth rates, low cell yields 13 , and a high 51 sensitivity to changing environmental conditions 14 . For their application in wastewater 52 treatment, they are grow...