## 3 Results and Discussion
The PN and PS concentrations in the three isolated EPS fractions are presented in Figure 6. In granules from R1, content of PN and PS in Sol- and LB-EPS remained at a similar level in the GBSR cycle, but increased about 2 times at the end of the cycle in case of TB-EPS (Figure 6). In R2 a significant decrease in PN concentration in Sol- and LB-EPS was observed at the end of the cycle that was accompanied by a slight increase in PN and PS concentrations in TB-EPS (Figure 6). PN and PS are the main components of EPS in aerobic granules. For granulation, the ratio of PN to PS in EPS is more important than their quantity in a given microbiological structure [44]. A high PN/PS ratio in EPS was observed in aerobic granules with very good settling properties [45]. Decreasing the amount of PN relative to PS decreases the consistency of granules, causes problems with biomass sedimentation and increase the risk of filamentous bacteria growth and sludge bulking [46]. It has been shown that the PN/PS ratio depends on the C/N ratio in the influent. For example, [PERSON] et al. [47], conducted research with C/N ratios of 5 and 15 in the wastewater fed to reactors. At the 15 C/N ratio, which was lower than C/N ratio used in our study (about 24), the PN/PS ratio in EPS was significantly lower than at the 5 C/N ratio, and granules with poorer settling and pollutant removal capacity were cultivated. Figure 6: Content and composition of EPS in granules at the beginning and end of the reactor cycle in (**A**) R1 and (**B**) R2. In the present study, the PN/PS ratio was low and remained at around 1 in both reactors at the beginning of the cycle. The largest changes in EPS concentration took place in the TB-EPS fraction. The PN and PS concentrations in TB-EPS at the beginning of the cycle was similar in both reactors but increased more than 1.5-time in R1 in comparison with R2 at the end of the cycle, which explains much better settling properties of granules from R1. Higher concentration of TB-EPS at the end of R1 cycle can be caused by the fact that in continuously aerated reactors, microorganisms use available carbon sources mainly for biomass and EPS synthesis. In intermittently aerated reactors, in contrast, they use more of the available carbon for denitrification and phosphorus removal. Up to 50% of the EPS in aerobic granular sludge can be used in the starvation phase and anaerobic conditions [48]. The high increase in PN in TB-EPS fraction at the end of the reactor cycle was advantageous for granules stability. Both TB-EPS and LB-EPS can support the aggregation of neighboring cells in AGS formation; however, LB-EPS does not bind cells together as strongly as TB-EPS and it can be more easily destroyed, which leads to the destruction of the granule structure [49]. Real-time PCR was used to estimate the total number of bacteria (_16S rRNA_ gene copy number), the number of gene for _amoA_ (ammonium monooxygenase gene) enzyme of AOB and full denitrifiers (nitrous oxide reductase gene _nosZ_) in granules during reactor operation (Figure 7). Standard curves were made showing the relationship between the Ct threshold value and logarithm of the number of gene copies in the tested biomass. The curves were linear in the studied range of gene copy numbers, and the reactors determination coefficient was 0.99, 0.98 and 0.99 for the 16S rRNA, _amoA_, and _nosZ_ genes, respectively. The number of gene copies in the above studies was referred to 5 ng DNA (unit of sample). Figure 7: The absolute number of copies of (**A**) bacterial _16 SrRNA_, (**B**) _amoA_ and (**C**) _nosZ_ genes in the reactors. The abundance of the _165 rRNA_ genes in the biomass of both reactors gradually decreased as the experiment was carried out. The largest number of 16S rRNA genes was recorded in the inoculum (\(1.4\times 10^{7}\) copies/5 ng DNA). The number of copies of the 16S rRNA in mature granules at 164 th cycle was at a level of \(9.52\times 10^{6}\)/5 ng DNA and \(6.75\times 10^{6}\) copies/5 ng DNA in R\({}_{1}\) and R\({}_{2}\), respectively.