The influences of different nitrogen and carbon concentrations on the growth of nitrifiers in the cultures were assessed by qPCR of the amoA and nxrA genes, representing the ammonia and nitrite oxidizers, respectively. cuatro + concentrations and no substantial growth for the Nitrobacter-like NOB under NH4 + concentrations of 1,000 and 1,500 ?M when 1,000 ?M HCOstep step three – was supplied (Figure 4). A HCO3 – concentration of 1,000 ?M increased the growth of AOB in Culture0dos at NH4 + concentration ?500 ?M and NOB in Culture03 at NO2 – concentrations ?200 ?M, but this growth became steady when nitrogen concentration increased (Figure 4). By contrast, an excess of HCO3 – (3,000 ?M) promoted higher growth of AOB and NOB in all cultures (Student’s t-test, p < 0.01) when compared to cultures with 1,000 ?M HCO3 – (Figure 4).
Underneath the exact same substrate (HCO
FIGURE 4. Increases in the (A) amoA and (B) nxrA gene copy numbers at different NH4 + or NO2 – concentrations. Culture01 and Culture02 were fed NH4 + , while Culture03 was fed NO2 – . The amoA gene copy numbers at time zero for Culture01 and Culture02 were 2.2 ? 10 4 ± 7.9 ? 10 2 and 4.0 ? 10 4 ± 1.3 ? 10 3 per ml, respectively. The nxrA gene copy number at time zero for Culture01 and Culture03 was 2.0 ? 10 3 ± 2.5 ? 10 2 per ml. Error bars represent one standard deviation from biological triplicate experiments.
3 – and NH4 + ) concentrations, the growth of AOB was significantly higher (Student’s t-test, p < 0.01) in Culture02 than in Culture01, with the amoA gene showing increases from 4.8 ? 10 4 ± 1.9 ? 10 4 to 3.2 ? 10 5 ± 6.3 ? 10 4 per ml for Culture01 and increases from 2.6 ? 10 5 ± 2.5 ? 10 4 to 6.1 ? 10 6 ± 7.5 ? 10 5 per ml for Culture02 (Figure 4A). Growth of the Nitrobacter-like NOB was similar between Culture01 and Culture03 when the HCO3 – was in excess, with increases in the aplikacje randkowe amino nxrA gene ranging from 5.3 ? 10 3 ± 5.0 ? 10 2 to 8.0 ? 10 4 ± 3.8 ? 10 4 per ml culture for Culture01 and from 6.8 ? 10 3 ± 3.0 ? 10 3 to 1.6 ? 10 5 ± 7.7 ? 10 4 per ml for Culture03 (Figure 4B).
Dialogue
In this study, a biofilter from an aquarium was used to culture AOB and NOB, which have functional roles in removing NH3 and NO2 – that are toxic to aquatic life (Keuter et al., 2011; French et al., 2012; Wu et al., 2013). A culture-dependent approach was adopted to allow testing of the bacterial physiology. In particular, nitrification activities were examined with and without the synergistic interactions between the AOB and NOB. A 15-month cultivation period (over 60 transfers) with NH4 + as the sole energy source for the nitrifying community successfully cultured a number of AOB and NOB, as confirmed by both 16S rRNA gene amplicon and metagenomic sequencing. Culture02, cultured using chlorate, contained a few Nitrobacter 16S rRNA gene sequences (Supplementary Figure S1), but the NOB were unlikely to be functional as stoichiometric conversion of NH4 + to NO2 – was obtained and the nxrA gene was not amplified via PCR (Supplementary Table S1). The AOA belonging to Nitrososphaera were initially found in the biofilter, but no archaeal amoA gene or 16S rRNA gene was subsequently detected in the cultures. The relatively low culturing temperature (25°C as opposed to the 37°C preferred by AOA) (Wu et al., 2013), short retention time (Xia et al., 2011), high NH4 + concentration () and high dissolved oxygen levels (Yan et al., 2012) likely favored AOB over AOA. We attempted to use ampicillin to isolate AOA with 500 ?M NH4 + in synthetic medium (Mosier and Francis, 2008), but we detected no nitrification activity after incubating for more than one month.