Extended Data Fig. 3: N. inopinata growth experiments.
(a) Ammonium consumption over time in treatments containing either ammonium and guanidine, or ammonium only. 150 µM N for both guanidine and ammonium were added to a washed culture of N. inopinata (after pre-incubation with guanidine and ammonium for several months) and incubated for 12 days. On days 6, 8 and 10, additional spikes of around 150 μM of ammonium were added to the incubations (dashed grey lines). (b) Nitrite and nitrate production (combined). (c) 16 S rRNA gene copy numbers measured by qPCR. (d) Concomitant utilization of guanidine and ammonium in treatments containing both substrates. (e, f) N balance for treatments receiving guanidine and ammonium, and ammonium only (e), and for guanidine, no guanidine (starved) and guanidine dead control (f). N-balances include guanidine (where added), ammonium, nitrite and nitrate. Urea concentrations were not included as they were always <2.5 µM. (g) Nitrite and nitrate (combined) production rate per cell and day in treatments receiving guanidine and ammonium, or ammonium only. Rates were calculated for time intervals before complete ammonium depletion (incl. day 4), by normalizing the difference in NOx concentration to average cell numbers between time points and the duration of the time interval. * N. inopinata NOx production when incubated with guanidine and ammonium versus ammonium only, Welch two sample t-test: t = −2.4714, d.f. = 14.174, P = 0.02673. (h) Guanidine utilization rate per cell and day in treatments receiving only guanidine, or guanidine and ammonium. Rates were calculated across all time intervals as guanidine concentrations never dropped to 0, by normalizing the difference in guanidine concentration to average cell numbers between time points and the duration of the time interval. Colour gradient indicates the average guanidine concentration between time intervals. Higher utilization rates coincided with higher guanidine concentrations. No significant differences were found between treatments (Welch two sample t-test). (i) Cell yield per mol nitrite and nitrate produced across treatments, calculated between beginning and end of the incubation (12 days for treatments receiving guanidine and ammonium, or ammonium only; 126 days for the treatment receiving guanidine only). Note that all treatments contained residual nitrite (given as %), which may affect the overall energy conserved, and thus the cell yield per mol nitrite and nitrate produced. No significant differences were found between treatments (anova). All experiments were done in five biological replicates, datapoints show means, error bars standard deviations. Boxplots in (g-i) depict the 25–75% quantile range, with the centre line depicting the median (50% quantile); whiskers encompass data points within 1.5x the interquartile range.
