Adaptation of anammox bacteria to low temperature via gradual acclimation and cold shocks: distinctions in protein expression, membrane composition and activities

Anammox bacteria enable an 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 {degrees}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, LC-HRMS/MS) and structure of membrane lipids (UPLC-HRMS/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 up-regulation of cold shock proteins (e.g. CspB, TypA, ppiD). Ladderane lipids characteristic for anammox evolved to a similar end-point in both cultures which confirms their role in anammox bacteria adaptation to cold and indicates a three-pronged adaptation mechanism involving ladderane lipids (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. HighlightsO_LIAnammox bacteria were adapted to low T by gradual acclimation and cold shocks C_LIO_LIThe shocked culture was more active (0.66{+/-}0.06 vs 0.48{+/-}0.06 kg-N/kg-VSS/d) C_LIO_LIN-respiration proteins content decreased in gradually acclimated bacteria C_LIO_LISeveral cold shock proteins were upregulated more efficiently by cold shocks C_LIO_LIAt {downarrow}T, anammox adjusted ladderane membrane lipid composition in three aspects C_LI Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY