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.
Finding optimal reaction conditions is usually complex, requires many experiments and is therefore demanding in terms of human, financial and environmental resources. This work provides a simple workflow for easier design of popular palladium catalyzed C-H functionalization reactions, where the active palladium catalysts contain carboxylate ligands. The key factor for optimizing reaction conditions is to find a balance between two opposing effects of the carboxylic acid in the reaction mixture; generation of more reactive palladium catalyst vs. deactivation of substrate by its protonation.
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