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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 °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.HighlightsAnammox bacteria were adapted to low T by gradual acclimation and cold shocksThe shocked culture was more active (0.66±0.06 vs 0.48±0.06 kg-N/kg-VSS/d)N-respiration proteins content decreased in gradually acclimated bacteriaSeveral cold shock proteins were upregulated more efficiently by cold shocksAt ↓T, anammox adjusted ladderane membrane lipid composition in three aspectsGraphical abstract

The application of partial nitritation-anammox (PN/A) under mainstream conditions can enable substantial cost savings at wastewater treatment plants (WWTPs), but how process conditions and cell physiology affect anammox performance at psychrophilic temperatures below 15 °C remains poorly understood. We tested 14 anammox communities, including 8 from globally-installed PN/A processes, for (i) specific activity at 10-30 °C (batch assays), (ii) composition of membrane lipids (U-HPLC-HRMS/MS), and (iii) microbial community structure (16S rRNA gene amplicon sequencing). Crucially, the key parameters impacting anammox activity were the membrane lipid composition and cultivation temperature. The size of ladderane lipids and the content of bacteriohopanoids were key physiological drivers of anammox performance at low temperatures. Higher contents of (i) short C18 [3]-ladderane alkyl and (ii) large phosphatidylcholine headgroup were determined in anammox more active at 15-30 °C and 10-15 °C, respectively. At below 15 °C, the activation energies of most mesophilic cultures severely increased while those of the psychrophilic cultures remained stable; this indicates that the adaptation of mesophilic cultures to psychrophilic regime necessitates months, but in some cases can take up to 5 years. Interestingly, biomass enriched in the marine genus “Candidatus Scalindua” displayed exceptionally highest activity at 10-20 °C (0.50 kg-N.kg-VSS−1.d−1 at 10 °C, Ea10-30 °C = 51±16 kJ.mol−1), indicating outstanding potential for nitrogen removal from cold streams. Collectively, our comprehensive study provides essential knowledge of cold adaptation mechanism, will enable more accurate modelling and suggests highly promising target anammox genera for inoculation and set-up of anammox reactors, in particular for mainstream WWTPs.HighlightsLadderane size and cold exposure affected anammox activation energy (Ea).Ea improved with more C18 [3]-ladderanes over C20 and larger polar headgroup.Long-term cold exposure reduced Ea at 10-15 °C, not activity per se.Marine “Ca. Scalindua” was exceptionally suitable for cold streams.Anammox Ea at 15-30 °C was 79±18 kJ.mol−1.Graphical abstract