The adaptation of bacteria involved in anaerobic ammonium oxidation (anammox) to low temperatures will enable more efficient removal of nitrogen from sewage across seasons. At lower temperatures, bacteria typically tune the synthesis of their membrane lipids to promote membrane fluidity. However, such adaptation of anammox bacteria lipids, including unique ladderane phospholipids and especially shorter ladderanes with absent phosphatidyl headgroup, is yet to be described in detail. We investigated the membrane lipids composition (UPLC-HRMS/MS) and dominant anammox populations (16S rRNA gene amplicon sequencing, Fluorescence in situ hybridization) in 14 anammox enrichments cultivated at 10-37 °C. "Candidatus Brocadia" appeared to be the dominant organism in all but two laboratory enrichments of "Ca. Scalindua" and "Ca. Kuenenia". At lower temperatures, the membranes of all anammox populations were composed of shorter [5]-ladderane ester (reduced chain length demonstrated by decreased fraction of C20/(C18 + C20)). This confirmed the previous preliminary evidence on the prominent role of this ladderane fatty acid in low-temperature adaptation. "Ca. Scalindua" and "Ca. Kuenenia" had distinct profile of ladderane lipids compared to "Ca. Brocadia" biomasses with potential implications for adaptability to low temperatures. "Ca. Brocadia" membranes contained a much lower amount of C18 [5]-ladderane esters than reported in the literature for "Ca. Scalindua" at similar temperature and measured here, suggesting that this could be one of the reasons for the dominance of "Ca. Scalindua" in cold marine environments. Furthermore, we propose additional and yet unreported mechanisms for low-temperature adaptation of anammox bacteria, one of which involves ladderanes with absent phosphatidyl headgroup. In sum, we deepen the understanding of cold anammox physiology by providing for the first time a consistent comparison of anammox-based communities across multiple environments.

Radboud University Department of Microbiology Institute for Water and Wetland Research 1Heyendaalseweg 135 6525 ED AJ Nijmegen the Netherlands

TU Delft Department of Biotechnology Building 58 Van der Maasweg 9 2629 HZ Delft the Netherlands

TU Delft Department of Biotechnology Building 58 Van der Maasweg 9 2629 HZ Delft the Netherlands; TU Delft Department of Water Management Building 23 Stevinweg 1 2628 CN Delft the Netherlands

University of Chemistry and Technology Prague Department of Biochemistry and Microbiology Technická 5 166 28 Prague Czechia

University of Chemistry and Technology Prague Department of Food Analysis and Nutrition Technická 5 166 28 Prague Czechia

University of Chemistry and Technology Prague Department of Water Technology and Environmental Engineering Technická 5 166 28 Prague Czechia

University of Chemistry and Technology Prague Department of Water Technology and Environmental Engineering Technická 5 166 28 Prague Czechia; University of Chemistry and Technology Prague Department of Biochemistry and Microbiology Technická 5 166 28 Prague Czechia

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On anammox activity at low temperature: effect of ladderane composition and process conditions