O2 versus N2O respiration in a continuous microbial enrichment

Despite its ecological importance, essential aspects of microbial N2O reduction—such as the effect of O2 availability on the N2O sink capacity of a community—remain unclear. We studied N2O vs. aerobic respiration in a chemostat culture to explore (i) the extent to which simultaneous respiration of N2O and O2 can occur, (ii) the mechanism governing the competition for N2O and O2, and (iii) how the N2O-reducing capacity of a community is affected by dynamic oxic/anoxic shifts such as those that may occur during nitrogen removal in wastewater treatment systems. Despite its prolonged growth and enrichment with N2O as the sole electron acceptor, the culture readily switched to aerobic respiration upon exposure to O2. When supplied simultaneously, N2O reduction to N2 was only detected when the O2 concentration was limiting the respiration rate. The biomass yields per electron accepted during growth on N2O are in agreement with our current knowledge of electron transport chain biochemistry in model denitrifiers like Paracoccus denitrificans. The culture’s affinity constant (KS) for O2 was found to be two orders of magnitude lower than the value for N2O, explaining the preferential use of O2 over N2O under most environmentally relevant conditions. Electronic supplementary material The online version of this article (10.1007/s00253-018-9247-3) contains supplementary material, which is available to authorized users.


Figure S1
Chemostat operation under N 2 O limitation (D = 0.026 h -1 , pH 7, 20°C) showing (a) the time points at which continuous operation was interrupted to perform batch experiments -marked with circles -together with the relative abundance of the main 16S rRNA gene OTUs making up the community, (b) incoming and outgoing acetate and NH 4 + concentrations and, (c) biomass concentration (in gVSS/L) and optical density (OD 660 ) of the culture. The corresponding values prior to day 45 and during the start-up of the enrichment can be found in Conthe et al. (2018b). The biomass yields and biomass specific conversion rates are presented in Tables 1 and 2, in the main text, and the taxonomic assignment of 16S rRNA OTUs is available in Table S1. Bacteria

Figure S2
Concentration of N 2 O, N 2 , CO 2 and Argon in the offgas (above) and incoming gas (below) of the experiment on day 106 -N 2 O only, presented in the main text as Figure 1a. The averaged data for each step -numbered in the graph -is presented in Table S2. Acetate was added manually to the culture during steps 5 and 7 to ensure that it was present in excess. NH 4 + was also in excess throughout the experiment. pH was kept constant at 7.0 ± 0.1 1 2 3 4 5 6 7 Table S2 Average concentration and rates of N 2 O, N 2 , CO 2 , O 2 and Argon supplied and produced during each of the steps (numbered 1 through 7) in the experiment with only N 2 O on day 106 -presented in Figure 1a and Figure S2.
Step 1 corresponds to steady state operation.

Figure S3
Concentration of N 2 O, N 2 , CO 2 and Argon in the offgas (above) as well as the incoming gas (below) of the experiment on day 132 (O 2 only) -presented in the main text as Figure 1b. The averaged data for each step -numbered in the graph -is presented in 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6

Table S3
Average concentration and rates of N 2 O, N 2 , CO 2 , O 2 and Argon supplied and produced during each of the steps (numbered 1 through 9) in the experiment with only O 2 on day 132 - Figure 1b in the main text and Figure S3.
Step 1 corresponds to steady state operation.  Amount of NH 4 + (above) and biomass (below) during the 9 steps of the experiment corrected for the broth volume in the chemostat. The average biomass valies for each step was calculated based on the initial VSS measurement (assuming a molar weight of 24,6 g/mol) corrected for biomass growth during each step. This growth was estimated based on the NH 4 + consumption shown above, assuming biomass contains 0,2 N -mole per C-mole. This estimation correlates with the VSS measurements performed at the end of steps 3 and 6 and with the OD 660 , as shown in the inset. These values were used to calculate the biomass specific rates (q) for each step.

Table S4
Average concentration and rates of N 2 O, N 2 , CO 2 , O 2 and Argon supplied and produced during each of the steps (numbered 1 through 9) in the experiment with simultaneous presence of O 2 and N 2 O on day 110 - Figure 3a in the main text and Figure S4.  Figure 3b in the main text and Figure S5.  Figure 3c in the main text. The averaged data for each step is presented in Table S5. pH was kept constant at 7.0 ± 0.1 1 2 3 4 5 6 7 8  Figure 3c in the main text and Figure S6. ! ! ! Table S7 Parameters and stoichiometry used to calculate the Gibbs free energy dissipation values during microbial growth following the methodology of Kleerebezem and Van Loosdrecht (2010). To obtain the overall stoichiometry of microbial metabolism (MET): first, the stoichiometry of the redox reactions describing the catabolism (CAT) and anabolism (AN) need to be established from the balanced redox half reactions (D for donor half reaction; A for acceptor half reaction and An* for the anabolic half reaction based on the C and N source -in this case acetate and ammonium). In a subsequent step, the stoichiometric coefficent -here λ cat , derived from the experimentally determined biomass yields on substrate (Y x/Acetate ) -is used to couple the CAT and AN equations into the overall metabolic growth equation. The Gibbs free energy dissipation during the growth reaction is calculated by multiplying the stoichiometric coefficients obtained with the Gibbs free energy of formation of these compounds (G f 0 ) corrected for non-ideality considering T = 20 °C and pH 7.