Nitrososphaera viennensis was routinely grown as batch cultures in 30 mL Quickstart universal containers (STERILIN) at 42°C in freshwater medium (FWM) (De La Torre et al., 2008; Tourna et al., 2011) amended with non-chelated trace element solution (MTE) (Könneke et al., 2005), 7.5 μmol L⁻¹ FeNaEDTA, 2 mmol L⁻¹ NH₄Cl, 2 mmol L⁻¹ NaHCO₃, 1 mmol L⁻¹ sodium pyruvate, and 10 mmol L⁻¹ HEPES buffer (final pH 7.5). Cultures used as inoculum for bio reactors were inoculated with 1% (v/v), but for regular culture maintenance 1:10⁶ (v/v) inoculum was used. Long term storage works best by aliquoting late exponential cultures without any additives and storing them at −70°C. For reviving the culture 5% (v/v) inoculum should be used and a lag phase of 1–2 days need to be expected. Growth was followed by measuring NO₂⁻ production and NH₃ consumption as described before (Tourna et al., 2011).

To elucidate the effect of gassing on μ, N. viennensis was grown in 2 L all-glass bioreactors (Eppendorf AG, Hamburg, Germany) filled with 1.5 L. The medium composition and conditions were the same as for batch cultures, except for omitting HEPES buffer, and the inoculum was 5% (v/v). The pH was controlled at pH 7.50 (± 0.05 deadband) using 0.5 mol L⁻¹ HCl and 0.5 mol L⁻¹ NaOH for titration. Dissolved molecular oxygen concentration (dO₂) was measured with optodes (Hamilton) and recorded but not controlled. Cultures were continuously stirred at a rate of 400 rpm and depending on the experiment not gassed (in- and off-gas clamped off), gassed with 2 sL L⁻¹ h⁻¹ air, gassed with 1 sL L⁻¹ h⁻¹ air or 1 sL L⁻¹ h⁻¹ air/N₂ mix (12.6 Vol.-% O₂). In the last batch experiment cultures were gassed with 2 sL L⁻¹ h⁻¹ air for 63 h, in- and off-gas clamped off and NO-donor 2,2′-(2-Hydroxy-2-nitrosohydrazinylidene)bis-ethanamine (DETA NONOate) added via a septum in different concentrations (0.15, 0.6 and 2.4 μmol L⁻¹, sterile H₂O as control). After 25 h one reactor was gassed with 2 sL L⁻¹ h⁻¹ air, one with 2 sL L⁻¹ h⁻¹ 99% air 1% CO₂ mixture, one was kept clamped off and one clamped off and 2 mmol L⁻¹ NaHCO₃ were added via a septum.

Chemostat cultures were grown like batch cultures but without NaHCO₃ in the medium, instead the cultures were gassed with an air/CO₂ mix of 0.5, 1 or 2 Vol.-% CO₂ with 2 sL L⁻¹ h⁻¹ gassing rate. The culture volume was 1.5 L but had to be reduced to 1.1 L at the highest D of 0.07 h⁻¹ to not exceed the maximum pump rate of 100 mL h⁻¹. The chemostats were started in late exponential growth phase of the batch and due to the relatively low μ of the organism, the minimum volume exchanges for steady states, after every applied or observed change, were reduced from the usual five (99.3% (v/v) exchanged) to three exchanges (95.0% (v/v) exchanged) to speed up the experiment.

Peristaltic pumps were calibrated by weighing feed- and harvest bottles every day to avoid drifting over time and the reactor volume was corrected with every sampling. Deviations of the reactor volume were usually between 1 and 3%. Outliers of BR1 and BR2 at 1,271 h and 1,295 h were caused by technical issues of the outflow pumps after the reduction of the working volume to 1.1 L, causing a culture volume increase to about 2 L over night and thus a decrease in residual NH₄⁺. Outliers of BR2 at 1,343 h and 1,346 h were due to a ripped inflow pump tube, causing a temporary reduction of culture volume to about 0.9 L, a decrease in residual NH₄⁺ and a contamination with small rod-shaped microorganisms that was treated with kanamycin.

To estimate the activity of the biofilm on the reactor walls, the planktonic cells were removed by draining the reactors and refilling them with fresh and sterile medium twice. At a working volume of 1.5 L and 7.5 mL remaining in the bioreactors, a dilution of the planktonic culture of 1:40,000 was achieved (43 nmol L⁻¹ NO₂⁻ remaining). After that the continuous cultures were started again with a D of 0.065 h⁻¹. During the washing procedure, precipitated carbonate detached from the acid/base/medium inlet in BR2 and started to scrape off the biofilm from the reactor wall, thereby creating a constant loss of biofilm to the system.

The fed-batch experiment started with two batch cultures of 1.1 L gassed with an air/0.5 Vol.-% CO₂ mix of 2 sL L⁻¹ h⁻¹. The 400 mL feed medium contained 32 mmol L⁻¹ NH₄Cl (10 mmol L⁻¹ final), 4.75 mmol L⁻¹ pyruvate (2 mmol L⁻¹ final), 4.75x MTE (2x final), 4.75x FeNaEDTA (2x final) and was exponentially fed in the beginning to maintain a μ of 0.02 h⁻¹. Due to unreliable pump rates the feed mode was changed to linear with a feed rate of 2 mL h⁻¹. The pyruvate concentration in the feed medium for the continuous culture was increased to 5 mmol L⁻¹ to avoid pyruvate depletion which resulted in stagnating cell concentration during the fed-batch.

Samples were taken from the bioreactors after the first 2 mL were discarded to remove old culture from the tubing. One micro liter aliquots were pipetted into 1.5 mL reaction tubes and centrifuged at 4°C for 30 min at 23,000 g in a benchtop centrifuge (Eppendorf Centrifuge 5424 R). Supernatant was removed and used directly for NO₂⁻ and NH₃ measurements while the cell pellets were stored at −20°C for DNA extraction.

Basic FWM was autoclaved in 5 L Schott bottles and supplements were added in a laminar flow hood after room temperature was reached. The GL45 caps with tubing and 0.2 μm filter were always transferred from one bottle to the next, while keeping the inlet and outlet tubes clamped off. After attaching the bottles back to the reactor tubes via luer locks, the connections were put into beakers with boiling water for at least 10 min to reduce the contamination risk, before opening the clamps and restarting the pumps. While BR2 was slightly contaminated due to ripped feed pump tubing, BR1 remained clean over the whole run (103 days with 36 bottle exchanges). The contamination of BR2 occurred at 1,343 h and lasted at most up to 2,041 h.

DNA from cell pellets was extracted by bead beating using SDS-buffer, phenol/chloroform/isoamylalcohol [25:24:1 (v/v/v), Fisher BioReagents] and chloroform/isoamylalcohol [24:1 (v/v)] as already described elsewhere (Zhou et al., 1996; Griffiths et al., 2000; Rittmann and Holubar, 2014; Abby et al., 2018).

DNA samples were checked for bacterial contamination using the 16S rRNA primers Eubac27F 5′-AGA GTT TGA TCC TGG CTC AG-3′ and Eubac1492R 5′-GGT TAC CTT GTT ACG ACT T-3′. PCR conditions were 95°C for 5 min as initialization, followed by 35 cycles of 30 s denaturing at 94°C, 30 s primer annealing at 55°C, 2 min elongation at 72°C, and finishing with final elongation at 72°C for 10 min. Reactions were done in 25 μL containing 2 μL sample, 0.15 μL GoTaq Polymerase, 5 μL 5x Flexi Buffer, 2 μL MgCl₂ 25 mmol L⁻¹, 0.5 μL dNTP 10 mmol L⁻¹, 0.25 μL BSA 20 mg mL⁻¹, 1.25 μL Primer F 10 μmol L⁻¹, 1.25 μL Primer R 10 μmol L⁻¹, 12.6 μL nuclease free water.

The archaeal 16S rRNA gene was quantified to estimate the cell number using the primers Arch931F 5′-AGG AAT TGG CGG GGG AGC A-3′ (Jackson et al., 2001) and Arch1100R 5‘-BGG GTC TCG CTC GTT RCC-3′ (Øvreås et al., 1997) in triplicate 20 μL reactions containing 10 μL qPCR Master Mix 2x (Luna Universal qPCR Master Mix, NEW ENGLAND BioLabs Inc.), 1 μmol L⁻¹ of each primer, nuclease free water and 4 μL DNA sample (diluted to have approximately 10 ng per reaction) or standard. Reactions were performed in a qPCR (BIO-RAD CFX Connect Real-Time System) with the following conditions: initialization at 95°C for 2 min, 40 cycles of 30 s denaturing at 95°C, 30 s joint annealing-extension at 60°C, and extension with fluorescence measurement at 60°C for 30 s. The specificity of qPCR products was confirmed by melting curve analysis. Standards were prepared by amplifying the 16S rRNA gene of N. viennensis under the conditions as described above in a 50 μL reaction using 2 μL DNA template and the archaeal 16S rRNA primers A109F 5‘-ACK GCT CAG TAA CAC GT-3′ (Großkopf et al., 1998) and A1492R GYY ACC TTG TTA CGA CTT-3′ (Nicol et al., 2008). PCR product was cleaned up using the Machery-Nagel DNA cleanup kit and DNA concentration was measured with Qubit™ dsDNA BR Assay Kit (Thermo Fisher Scientific) before preparing serial dilutions with nuclease free water. The standard curve had an efficiency of 93.38% and an R² of 0.999.

DNA concentration of extracted samples was measured with Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific) and several samples with concentrations ranging from 3.58 to 35.5 ng μL⁻¹ were quantified by qPCR as described above. Using a linear regression model the mass of DNA was correlated to the cell number, assuming that each cell contained a single copy of the 16S rRNA gene. Using this model one copy of 16S rRNA correlates to 3.42 fg which is 25.6% more than the calculated mass of a N. viennensis genome of 2.72 fg (2.52 Mbp genome size). Assuming a cell diameter of 0.75 μm (0.6–0.9 μm cell diameter) and a density of 1.1 g cm⁻³ a theoretical N. viennensis cell would weight 2.43·10⁻¹³ g or 243 fg.

The outflow of the continuous cultures was collected in sterile 5 L bottles and usually harvested once a week by concentrating the biomass using a tubular centrifuge (CEPA) at 40,000 rpm and subsequent centrifugation of the cell concentrate in a bucket centrifuge (Thermo SCIENTIFIC Sorvall LYNX 4000 Centrifuge) at 4°C, 24,470 g for 40 min. The pellets were resuspended in a small amount of supernatant and split up into pre-weighted 2 mL reaction tubes which were centrifuged in a benchtop centrifuge (Eppendorf Centrifuge 5424 R) at 4°C for 30 min at 23,000 g. Supernatant was removed, reaction tubes weighed and stored at −70°C.

Initial experiments of N. viennensis grown as batch cultures in 2 L bioreactors showed that gassing with air had a detrimental effect on μ even at low gassing rates such as 2 sL L⁻¹ h⁻¹ (0.0276 h⁻¹) when compared to cultures without gassing (0.0445 h⁻¹, Supplementary Figure S1). In non-gassed cultures dO₂ decreased to about 50% of its initial concentration. Therefore, growth under decreased gassing rate and reduced dO₂ was tested. As shown in Supplementary Figure S2 reducing the gassing rate to 1 sL L⁻¹ h⁻¹ did not increase μ (0.0267 h⁻¹) and reducing dO₂ to about 60% did decrease μ to 0.0205 h⁻¹, which contradicted the results of the first experiment. Due to the hypothesized importance of NO for the archaeal ammonia oxidation pathway (Kozlowski et al., 2016) we decided to test if the reduction of μ is a result of stripping NO from the system by gassing. As shown in Figure 1, addition of the NO-donor 2,2′-(2-Hydroxy-2-nitrosohydrazinylidene)bis-ethanamine (DETA NONOate) to early exponential phase cultures (300 μmol L⁻¹ NO₂⁻ produced) did not show any effect on recovering μ. However, adding 1 Vol.-% CO₂ to the in-gas or 2 mmol L⁻¹ NaHCO₃ to the culture medium, and keeping the bioreactor closed did increase μ from 0.0259 ± 0.0002 h⁻¹ to 0.0338 h⁻¹ and 0.0395 h⁻¹, respectively. Gassing with air or keeping the reactor closed did not change μ (0.0260 h⁻¹) or even reduced it (0.0234 h⁻¹), respectively. Hence, the above results showed that the organism was carbon limited likely due to the stripping of CO₂ from the supplied gassing.

To determine μ_max in closed batch systems, N. viennensis was grown in serum flasks with 0.5 Vol.-% CO₂ and different dO₂ concentrations (Supplementary Figure S3). A slight increase of μ was observed with decreasing dO₂ from 0.0484 ± 0.0004 h⁻¹ to 0.0508 ± 0.0004 h⁻¹at 21 Vol.-% to 5 Vol.-% O₂, respectively (224.7 to 53.5 μmol L⁻¹ dO₂ considering a pressure of 1,106 hPa and an incubation temperature of 42°C). Due to the minor effect of dO₂ on μ, 21 Vol.-% O₂ was used to gas the following cultures and a μ_max of 0.0484 h⁻¹ is therefore considered. Moreover, by using an inoculum of 1:10⁶ (v/v), μ was increased from 0.024 h⁻¹ (Stieglmeier et al., 2014) to 0.048 h⁻¹ (data not shown).

To determine the K_S and D_crit of N. viennensis eight different D (0.035, 0.038, 0.042, 0.046, 0.050, 0.060, 0.065, and 0.070 h⁻¹) were applied in two bioreactors. As shown in Figure 2, at D 0.035 h⁻¹ S stabilized at an unexpectedly high level around 750 μmol L⁻¹ NH₄⁺ and increasing D further only had a marginal effect on S. In section A (D 0.035 to 0.050 h⁻¹, 382 to 990 h) S stabilized at 801.9 ± 43.2 μmol L⁻¹ NH₄⁺. Activity of both cultures spontaneously increased 58 h after D was set to 0.050 h⁻¹ (94.5% of medium exchanged) and S decreased to 575.2 ± 21.2 μmol L⁻¹ NH₄⁺. The increase of D to 0.060 h⁻¹ was responsible for the abrupt increase of S to 690.1 ± 29.9 μmol L⁻¹ NH₄⁺ which gradually decreased, despite negative outliers, to 456.2 ± 7.9 μmol L⁻¹ NH₄⁺. Overall S stabilized at 541.6 ± 68.6 μmol L⁻¹ NH₄⁺ in section B (D = 0.050 to 0.070 h⁻¹, 1,032 to 1,654 h) and negative outliers at 1,271, 1,295, 1,343, and 1,346 h were due to technical issues (see Materials and Methods). Another spontaneous increase of activity occurred in both cultures 11 days after D was set to 0.070 h⁻¹ (18 working volume exchanges), which first led to a complete consumption of NH₄⁺ in both cultures but subsequently two different S stabilized. As shown in section C of Figure 2 (D = 0.070 and 0.065 h⁻¹, 1,796 to 2,035 h), S of the BR1 culture stabilized at 262.2 ± 19.9 μmol L⁻¹ NH₄⁺ and at 7.4 ± 11.1 μmol L⁻¹ NH₄⁺ for the BR2 culture. From section C to section D (D 0.065 h⁻¹, 2,062 to 2,402 h) the reactor volume was increased to minimize the effect of sampling larger volumes which caused S of BR1 to increase from 258 to 356 μmol L⁻¹ NH₄⁺ (ratio of concentrations matches the ratio of volumes). S of BR2 gradually increased from 4.1 μmol L⁻¹ NH₄⁺ to 375.0 μmol L⁻¹ NH₄⁺ probably as a result of the disturbances caused by the repeated sampling process.

The cell concentrations declined gradually with D from 7.67·10⁷ ± 2.11·10⁷ cells mL⁻¹ at D = 0.035 h⁻¹ to 4.36·10⁷ ± 2.18·10⁶ cells mL⁻¹ at D = 0.046 h⁻¹ (549 to 932 h). μ calculated from measured cell concentrations as shown in Supplementary Figure S4 gradually increased with increasing D from 0.0362 ± 0.0011 h⁻¹ at D = 0.035 h⁻¹ to 0.0437 ± 0.0007 h⁻¹ at D = 0.046 and after the activity increase at D 0.050 h⁻¹ μ increased further to 0.0505 ± 0.0003 h⁻¹ (1,107 h). Cell concentrations remained surprisingly stable from D = 0.050 to 0.060 h⁻¹ at 4.13·10⁷ ± 5.30·10⁶ cells mL⁻¹ (1,032 to 1,419 h) and declined to 3.08·10⁷ ± 7.06·10⁵ cells mL⁻¹ at D = 0.070 h⁻¹ (1,654 h) before the second activity increase, after which cell concentrations started to alternate in both reactors. Average NH₄⁺ and cell concentrations of sections A to D are shown in Supplementary Figure S5. K_S and D_crit could not be determined due to the unusual growth behavior and biofilm formation, respectively.

To get the full picture of how D effects S, a new continuous culture run was set up with 0.5 Vol.-% CO₂ enriched air to reduce the precipitation of CO₃²⁻ both at the inlets and in the medium. Initial D was set at 0.01 h⁻¹ and all substrate was consumed. As shown in Figure 4 an increase in D to 0.020 h⁻¹ resulted in a temporary increase of S to 11 and 44 μmol L⁻¹ for BR3 and BR4 respectively, after which S returned to 0 μmol L⁻¹. Then CO₂ was increased to 2 Vol.-% at 96 h after D was increased, which surprisingly induced an increase of S in BR3 with a 55.5 h delay. S in BR3 slowly started to increase from 17 to finally 192 μmol L⁻¹ whereas in BR4 S concentrations increased up to 95 μmol L⁻¹ but only 216 h after the CO₂ was increased. The reduction of CO₂ to 0.5 Vol.-% caused an immediate consumption of all NH₄⁺ in both reactors (553.5 to 577 h). Increasing D to 0.030 h⁻¹ increased S in both reactors, but at this time BR4 showed higher S than BR3. A further increase of D to 0.040 h⁻¹ induced an extended increase of S similar to the behavior seen at D 0.035 h⁻¹.

Cell concentrations remained rather stable over the whole run from D of 0.010 h⁻¹ to 0.030 h⁻¹ at 6.94·10⁷ ± 1.02·10⁷ cells mL⁻¹. Only one strong increase from 7.24·10⁷ cells mL⁻¹ to 1.23·10⁸ cells mL⁻¹ was observed in BR4 directly after decreasing the CO₂ concentration of the in-gas from 2 Vol.-% to 0.5 Vol.-%. However, the cell concentration decreased again to 7.77·10⁷ cells mL⁻¹ before increasing D from 0.020 h⁻¹ to 0.030 h⁻¹ (Figure 4). The amount of CO₂ in the gas phase seems to affect S. Depending on the CO₂ concentration of 0.5 Vol.-% and 2 Vol.-% in the gas phase S starts to accumulate at D of 0.030 h⁻¹ or 0.020 h⁻¹, respectively.

Before starting a continuous culture with higher substrate concentrations, the inhibitory effect of NO₂⁻ was determined by batch cultures in 30 mL polystyrene tubes with varying NO₂⁻ starting concentrations of 0 to 19 mmol L⁻¹ and 1 mmol L⁻¹ NH₄⁺. NO₂⁻ showed only a weak linear inhibitory effect on μ with 0.0342 to 0.0411 h⁻¹ for 19 and 0 mmol L⁻¹ NO₂⁻ starting concentrations, respectively (Supplementary Figure S6).

The bioreactor experiment commenced with a 2 mmol L⁻¹ NH₄⁺ batch, followed by a fed-batch to increase NO₂⁻ up to 10 mmol L⁻¹ and then switching into continuous mode to see the effect of D on cell number and S. The NH₄⁺ to pyruvate ratio was changed in the feed from 2 to 5 to avoid excessive pyruvate concentrations that would promote bacterial growth in case of a contamination. For up to 6 mmol L⁻¹ NO₂⁻ the cell number correlated very well with the NO₂⁻ concentration but then the cell number stagnated in both reactors even though NH₃ was still oxidized. Pyruvate was added to the reactors at the end of the fed-batch to increase the concentration from 2 mmol L⁻¹ to 5 mmol L⁻¹ and the feed media for the continuous cultures were also adjusted to 10 mmol L⁻¹ NH₄Cl and 5 mmol L⁻¹ pyruvate. Initial D was 0.006 h⁻¹ to allow the cultures to recover but was further decreased to 0.005 h⁻¹ to reduce the increase of S. In both cultures S appeared to be drifting, which means that μ (based on NO₂⁻ concentration) was always lower than D but still increased with increasing D (Figure 5). With such low D the residence time would have been far too long to wait for three volume exchanges to establish steady states (25 days at D = 0.005 h⁻¹). D was increased incrementally after at least 200 h from 0.005 to 0.006, 0.008, 0.010 and 0.012 h⁻¹. S eventually stabilized around 5 mmol L⁻¹ at D = 0.01 h⁻¹ and an increase of D to 0.012 h⁻¹ did not further increase S. The cell number increased in both reactors from 1.76·10⁸ cells mL⁻¹ and 1.88·10⁸ cells mL⁻¹ at the beginning of the continuous phase up to 3.22·10⁸ cells mL⁻¹ and 2.62·10⁸ cells mL⁻¹ for BR5 and BR6, respectively, after D was increased to 0.06 h⁻¹ and from there gradually decreased to about 2.00·10⁸ at D = 0.01 h⁻¹ (Figure 5).

Even though very low D were used in this continuous culture experiments, surprisingly high NH₄⁺ concentrations were measured. Once S stabilized, increases of D did not result in an increase of S as observed before with 2 mmol L⁻¹ cultures. The NH₄Cl to pyruvate ratio should remain 2:1 to ensure ROS detoxification.

For the following analysis only steady states below D = 0.050 h⁻¹ were considered, as we expect the results at and beyond this D to be heavily influenced by biofilm formation. Data points at D = 0.0124 h⁻¹ and 0.0146 h⁻¹ are from three different bioreactors that were used for lab scale biomass production (data not shown).

As shown in Figure 6A the cell number of N. viennensis increased slightly with D from 5.84·10⁷ ± 1.19·10⁷ cells mL⁻¹ at D = 0.010 h⁻¹ up to 7.67·10⁷ ± 2.11·10⁷ cells mL⁻¹ at D = 0.035 h⁻¹ from where it decreased linearly with D to 4.36·10⁷ ± 2.18·10⁶ cells mL⁻¹ at D = 0.046 h⁻¹.

NH₄⁺ was completely consumed up to D = 0.020 h⁻¹ by cultures gassed with 0.5 Vol.-% CO₂. For 2 Vol.-% CO₂ gassed cultures S started to accumulate at D = 0.020 h⁻¹ to 138 ± 58.9 μmol L⁻¹ and increased steeply up to 745.5 ± 27.6 μmol L⁻¹ NH₄⁺ at D = 0.035 h⁻¹ where it began to stagnate and reach a maximum of 850.0 ± 11.6 μmol L⁻¹ at D = 0.046 h⁻¹. Despite the early onset of increasing S, the cell concentration remained surprisingly stable up to D = 0.035 h⁻¹. Conversely, while the cell number was decreasing with D from 0.035 to 0.046 h⁻¹, S increased only marginally, thus changes in S appeared to be not reflected in the cell number and vice versa.

NO₂⁻ and cell productivities (P_NO2, P_C) are shown in Figure 6B. P_NO2 increased logarithmically from D of 0.010 to 0.020 h⁻¹ and from there on in a linear way up to 49.6 ± 0.3 μmol L⁻¹ h⁻¹ at D = 0.046 h⁻¹. P_C increases linearly from 5.84·10⁵ ± 1.19·10⁵ cells mL⁻¹ h⁻¹ at D = 0.010 h⁻¹ up to 2.69·10⁶ ± 7.39·10⁵ cells mL⁻¹ h⁻¹ at D = 0.035 h⁻¹ from which point it started to decline to 2.00·10⁶ ± 1.00·10⁵ cells mL⁻¹ h⁻¹ at D = 0.046 h⁻¹. As a result of a variable Y_(X/NH3), P_NO2 and P_C do not correlate in a fixed 1:1 ratio as would be expected. For comparison P_NO2 and P_C of 10 mmol L⁻¹ cultures at D = 0.012 h⁻¹ are also shown in Figure 6C with 53.6 ± 0.9 μmol L⁻¹ h⁻¹ and 2.61·10⁶ ± 2.30·10⁵ cells mL⁻¹ h⁻¹, respectively. The highest P_C was reached at D = 0.035 h⁻¹ which is therefore the optimal D for biomass production for N. viennensis.

As shown in Figure 6C an average Y_(X/NH3) of 3.36·10⁷ ± 5.77·10⁶ cells μmol⁻¹ L⁻¹ was determined from the batch cultures before changing into continuous mode. Y_(X/NH3) from steady states at D of 0.010 to 0.030 h⁻¹ or 10 mmol L⁻¹ NH₄Cl cultures (D from 0.005 to 0.012 h⁻¹) were well within this range, but from D = 0.035 h⁻¹, Y_(X/NH3) were highly elevated with 6.12·10⁷ ± 1.68·10⁷ cells μmol⁻¹ L⁻¹ and decreased to 3.79·10⁷ ± 1.90·10⁶ cells μmol⁻¹ L⁻¹ at D = 0.046 h⁻¹. Cultures at D of 0.020 h⁻¹ gassed with 0.5 Vol.-% and 2 Vol.-% CO₂ had very much the same Y_(X/NH3) with 3.82·10⁷ ± 1.96·10⁶ cells μmol⁻¹ L⁻¹ and 3.86·10⁷ ± 3.34·10⁵ cells μmol⁻¹ L⁻¹, respectively. While Y_(X/NH3) of steady states at low D are comparable to batch cultures, at higher D, Y_(X/NH3) increased up to 81.7% (D = 0.035 h⁻¹) compared to batch cultures.