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

Abstract

Highlights 19• Ladderane size and cold exposure affected anammox activation energy (Ea). 20• Ea improved with more C18 [3]-ladderanes over C20 and larger polar headgroup. 21• Long-term cold exposure reduced Ea at 10-15 °C, not activity per se. 22 Graphical abstract 26 27 28 Abstract 29 The application of partial nitritation-anammox (PN/A) under mainstream conditions can enable 30 substantial cost savings at wastewater treatment plants (WWTPs), but how process conditions 31 and cell physiology affect anammox performance at psychrophilic temperatures below 15 °C 32 remains poorly understood. We tested 14 anammox communities, including 8 from globally-33 installed PN/A processes, for (i) specific activity at 10-30 °C (batch assays), (ii) composition of 34 membrane lipids (U-HPLC-HRMS/MS), and (iii) microbial community structure (16S rRNA gene 35 amplicon sequencing). Crucially, the key parameters impacting anammox activity were the 36 membrane lipid composition and cultivation temperature. The size of ladderane lipids and the 37 content of bacteriohopanoids were key physiological drivers of anammox performance at low 38 temperatures. Higher contents of (i) short C18 [3]-ladderane alkyl and (ii) large 39 phosphatidylcholine headgroup were determined in anammox more active at 15-30 °C and 10-15 40 °C, respectively. At below 15 °C, the activation energies of most mesophilic cultures severely 41 increased while those of the psychrophilic cultures remained stable; this indicates that the 42 adaptation of mesophilic cultures to psychrophilic regime necessitates months, but in some cases 43can take up to 5 years. Interestingly, biomass enriched in the marine genus "Candidatus 44Scalindua" displayed exceptionally highest activity at 10-20 °C (0.50 kg-N.kg-VSS -1 .d -1 at 10 °C, 45Ea10-30 °C = 51±16 kJ.mol -1 ), indicating outstanding potential for nitrogen removal from cold 46 streams. Collectively, our comprehensive study provides essential knowledge of cold adaptation 47 mechanism, will enable more accurate modelling and suggests highly promising target anammox 48 genera for inoculation and set-up of anammox reactors, in particular for mainstream WWTPs. 49 Keywords: 50 anaerobic ammonium oxidation; adaptation to low temperature; ladderane phospholipid; 51 Scalindua; Kuenenia; Brocadia 52 53 2 Materials and methods 127 2.1 Anammox biomasses 128 The mesophilic biomasses sampled from full-scale installations (Landshut / DE,

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

“…Compared to the unadapted “ Ca . Kuenenia” (Kouba et al, 2019), cold shocks (5 °C, 1 h) and gradual adaptation (1 °C/day) increased the anammox activity by 130% and 40%, respectively (shocked once per 3 days 0.74±0.15 kg-N/kg-VSS/d; gradually acclimated 0.45±0.06 kg-N/kg-VSS/d; unadapted culture 0.32±0.019 kg-N/kg-VSS/d; 15 °C), suggesting cold shocks to be the superior strategy for low temperature adaptation. In the literature, gradual adaptation (De Cocker et al, 2018) along with the enrichment of cold-adapted species (Hendrickx et al, 2014) are the established strategies for achieving high-performance anammox at low temperatures (10-20 °C), while the cold shocks have only very recently emerged as a promising alternative.…”

“…Brocadia (2 %), and “ Ca . Scalindua (1 %) (Kouba et al, nonpublished data). The inoculum was obtained from a membrane bioreactor (MBR) maintained at 30 °C and a pH of 7, under which the doubling time was 3 days.…”

“…Compared to the unadapted "Ca. Kuenenia" (Kouba et al, 2019), cold shocks (5 °C, 1 h) and On a practical note, increasing the process resiliency against low temperature would require the application of the present cold shock regime once every 6 weeks, which means that a 6-month winter period would require the application of 4-5 such regimes. The shock applications could be made less frequent by optimizing cold shock parameters such as shock duration and temperature which have the potential to extend the time-span of the increased resiliency post-shock.…”

“…Scalindua”, showed that the length of a single ladderane lipid alkyl (C18/C20 [5]-ladderane ester) increased as the cultivation temperature increased from 10 to 20 °C. Recently, membrane composition was correlated to anammox temperature coefficients; specifically, higher relative contents of short C18 [3]-ladderane alkyls and large phosphatidylcholine headgroups were detected in cultures more active at 15-30 °C and 10-15 °C, respectively (Kouba et al.). This shows that other lipid structures are also a key part of the mechanism involved in anammox adaptation to low temperatures.…”

“…Anammox bacteria can cost-effectively remove nitrogen from the mainstream of WWTP via processes such as partial nitritation and anammox (PN/A) as well as nitrification, partial denitrification and anammox (N-PdN/A). However, the mainstream of WWTP is much colder (10-20 °C) than most current anammox reactors used to treat mesophilic centrates (30-37 °C), making the adaptation of anammox bacteria to low temperatures a crucial issue(Cao et al, 2017; Kouba et al. ).…”