Anammox bacteria enable efficient removal of nitrogen from sewage in processes involving partial nitritation and anammox (PN/A) or nitrification, partial denitrification, and anammox (N-PdN/A). In mild climates, anammox bacteria must be adapted to ≤15 °C, typically by gradual temperature decrease; however, this takes months or years. To reduce the time necessary for the adaptation, an unconventional method of 'cold shocks' is promising, involving hours-long exposure of anammox biomass to extremely low temperatures. We compared the efficacies of gradual temperature decrease and cold shocks to increase the metabolic activity of anammox (fed batch reactor, planktonic "Ca. Kuenenia"). We assessed the cold shock mechanism on the level of protein expression (quantitative shot-gun proteomics, LCHRMS/MS) and the structure of membrane lipids (UPLCHRMS/MS). The shocked culture was more active (0.66±0.06 vs 0.48±0.06 kg-N/kg-VSS/d) and maintained the relative content of N-respiration proteins at levels consistent levels with the initial state, whereas the content of these proteins decreased in gradually acclimated culture. Cold shocks also induced a more efficient expression of potential cold shock proteins (e.g. ppiD, UspA, pqqC), while putative cold shock proteins CspB and TypA were upregulated in both cultures. Ladderane lipids characteristic for anammox evolved to a similar end-point in both cultures; this confirms their role in anammox bacteria adaptation to cold and indicates a three-pronged adaptation mechanism (ladderane alkyl length, introduction of shorter non-ladderane alkyls, polar headgroup). Overall, we show the outstanding potential of cold shocks for low-temperature adaptation of anammox bacteria and provide yet unreported detailed mechanisms of anammox adaptation to low temperatures.

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

Department of Biotechnology Delft University of Technology van der Maasweg 9 2629 HZ Delft the Netherlands

Department of Biotechnology Delft University of Technology van der Maasweg 9 2629 HZ Delft the Netherlands; Department of Water Technology and Environmental Engineering University of Chemistry and Technology Prague Technická 5 166 28 Prague Czechia

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

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

References provided by Crossref.org

Effect of temperature on the compositions of ladderane lipids in globally surveyed anammox populations

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