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

Abstract

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.

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

“…Brocadia" in cultivations under 16, 25 and 35 °C as well as on ladderane lipids composition from anammox biomasses collected from various environments at distinct temperatures. This was initially corroborated by our previous study cultivating anammox enrichment at reduced temperatures and analyzing ladderanes after adaptation in comparison to control (Kouba et al, 2022b). The present study was able to further corroborate this by analyzing ladderanes in anammox biomasses collected from reactors under various cultivation temperatures.…”

“…To implement anammox process for the treatment of sewage, one of the issues is the adaptation of anammox populations to low temperatures (Cao et al, 2017). This has stimulated inquiry into the physiology that anammox bacteria use to adapt to the cold such as via cold-shock protein expression or changes in the phospholipid composition in their cell membranes (Kouba et al, 2022a;Kouba et al, 2022b).…”

“…Specifically, the ladderane alkyl chain length has been highlighted as one of the adaptation mechanisms. Increased amounts of C20 compared to C18 [5]-ladderanes have been microbially synthesized at elevated temperatures (Kouba et al, 2022b;Rattray et al, 2010). Other suggested ladderane adaptations include increased content of non-ladderane straight/branched (C14-16) alkyls and PE as polar headgroup (Kouba et al, 2022b).…”

“…Increased amounts of C20 compared to C18 [5]-ladderanes have been microbially synthesized at elevated temperatures (Kouba et al, 2022b;Rattray et al, 2010). Other suggested ladderane adaptations include increased content of non-ladderane straight/branched (C14-16) alkyls and PE as polar headgroup (Kouba et al, 2022b). However, the impact of microbial composition and process conditions on this C20:C18 ratio and the other components of anammox membranes such as the number of concatenated cyclobutane rings, length of more common alkyl chains, ether/ester alkyl bonds, and triterpenoids remain to be uncovered.…”

A Comprehensive Study of Gradient Conditions for Deep Proteome Discovery in a Complex Protein Matrix

“…Untargeted proteomics is also called discovery proteomics, and deep proteome profiling is one of challenges it faces [ 5 ]. Common nano-LC gradients used in discovery proteomics are single-stage linear or multi-stage linear [ 18 , 19 , 20 , 21 ]. It is worthwhile to explore various gradient conditions to optimize the proteome coverage for tims-ToF-based proteomics studies.…”