A twilight for the complete nitrogen removal via synergistic partial-denitrification, anammox, and DNRA process
Start-up phaseTo avoid the toxic effect of higher nitrite concentration on microorganisms30, the influent NH4+–N and NO2−–N during the first 66 days was maintained at 50 and 60 mg/L, respectively. The NH4+–N removal efficiency in each reactor reached up to 60% quickly with a certain amount of NO3−–N in the effluent. On the 5th day, the NH4+–N removal from reactors R1–R4 was 32.1, 36.9, 40.4, and 39.1 mg/L (Fig. 1a), respectively cumulative concentration of NO3−–N was 23.7, 33.3, 39.9, and 31.1 mg/L in respective reactors (Fig. 1c). The higher concentration of NO3−–N might be due to the proliferation of nitrifying bacteria or lower denitrifying bacteria activity. It has also been reported that other bacteria, despite nitrite-oxidizing bacteria (NOB), carrying nxr gene, can contribute to nitrate production from nitrite in an anammox reactor31. Besides, concentrations of NO2−–N in each reactor’s effluent showed a different degree of an increasing trend. On the 11th day, the NO2−–N concentration in the effluent of R1–R4 reached 60.5, 52.6, 62.8, and 54.1 mg/L, respectively (Fig. 1b).Fig. 1: Nitrogen removal performance of four reactors during the start-up period.a NH4+–N concentration (mg/L), b NO2−–N concentration (mg/L), c NO3−–N concentration (mg/L).Full size imageFurthermore, the DO of each reactor was also monitored, and it was found that DO values of four reactors in the first 20 days were varied from 0.20 to 0.50 mg/L. The oxygen half-saturation constant of AOB has been reported in the range of 0.2–0.4 mg/L, and the growth rate of AOB can reach 0.5–1.0 per day32. Thus, AOB and archaea, and some NOB adapt to the lower DO concentration and could grow33,34,35. So, the higher NO3−–N values in the start-up phase can be justified by the growth of nitrifying bacteria and some other bacteria carrying the nxr gene. However, as the incubation time increased, some bacteria which may not be adapted to the conditions might be degraded. The denitrifying bacteria could propagate on the carbon source provided by degraded biomass12. The use of NOx−–N by denitrifying bacteria contributed to the nitrogen removal rate (Figs. 1 and 2c) and can be called denitrifying propagation phase. Due to unfavorable environmental conditions, the degraded bacteria could have provided a carbon source for denitrification bacteria’s growth13,14. It has been seen from Fig. 2c that in the initial, nitrogen removal efficiency showed a decreasing trend, which is caused by consumption of endogenous organic matter, which slowly consumed and excluded the dominancy of the denitrification process. The conditions in the reactors are beneficial to anammox growth and eventually led to the washout of denitrifiers. The high TN removal in the latter phase was attributed to the enrichment of anammox abundance.Fig. 2: Nitrogen removal performance of the four reactors in the stabilization period.a NH4+–N concentration (mg/L), b NO2−–N concentration (mg/L), c removal efficiency (%) of total nitrogen.Full size imageFrom the 29th day, the effluent concentrations of NH4+–N and NO2−–N in R3 declined sharply, and the NO2−–N and NH4+–N removal ratio increased from 0.52 to 1.24, close to the theoretical ratio of 1.32, which indicates a rapid increase in the anammox bacterial activity5,36. The stoichiometric ratios of R1, R2, and R4 reached close to theoretical value on days 50, 50, and 41, respectively. The activity enhancing phase started around 22 days earlier in R3 when compared with R1, which clearly showed the positive effects of MF to short the start-up period of anammox. Though R4 showed a more extended start-up period than R3, it was still shorter when compared with R1. It was observed that R2 with the only nZVI did not show a clear difference from the control reactor. On the 34th day, the NH4+–N and NO2−–N removal rates of R3 were 95.5% and 88.1%, respectively, which maintained at 85% from days 41 to 60. Compared with the control, the start-up time of R3 was shortened from 60 days to 34 days under the action of MF, and the efficiency was increased by 43.3% in the start-up period. The start-up time of the reactor under the combined effects of nZVI and MF was ~50 days, and the efficiency increased by 16.7%. On the other hand, the start-up time of anammox in R1 and R2 showed no noticeable difference.Reactors’ performance under increased nitrogen loadingAfter the successful start-up of all reactors, the influent NH4+–N and NO2−–N concentrations were increased progressively to observe the reactors’ stability under different loading rates. As shown in Fig. 2, on day 69, the influent concentrations of NH4+–N and NO2−–N were increased from 50 to 100 mg/L and 60 to 120 mg/L (nitrogen loading rate was 2.64 kg/L/d), respectively. The response of R2 against increased N loading was almost comparable to R1. The concentration of effluent NO2−–N in R1–R3 was slightly increased. The removal performance of R4 was somewhat better than R1. Similarly, the removal of NH4+–N and NO2−–N in R4 was better than R3 (Fig. 2a, b). This implies that the increase in nitrogen loading does not have a deterioration impact on the operation of the four reactors after the successful build-up of anammox activity.The influent concentration of NH4+–N and NO2−–N was again amplified to 200 and 240 mg/L (nitrogen loading rate was 5.28 kg/m3/d) on the 91st day. After the second increment in N loading, the effluent NH4+–N concentration in R1 and R4 was increased to 69.2 and 56.3 mg/L, respectively, and the effluent NO2−–N concentration was increased to 55.5 and 37.4 mg/L in R1 and R4, respectively. The increasing trend in effluent NO2−–N concentration in R1 was observed till the 106th day, and the highest value of effluent NO2−–N concentration was 134.8 mg/L which decreased the removal rate in R1 about 42.4%. Likewise, considerable fluctuations were also observed in the removal rate of NH4+–N, where more variation was noted in R1. A little higher nitrogen removal efficiency of R3 on day 91 (80%) and on day 106 (71.18%) were witnessed compared to R4 (67% and 66%, respectively). Similarly, a little high nitrogen removal efficiency was registered in R2 on days 88–91 and on days 97–100 as compared to R4. These variations might be caused due to the calculation error. The presence of MF and nZVI decreased the influence of substrate shock on the anammox performance. Although the removal rates of NH4+–N and NO2−–N in R4 were also decreased, but the fluctuation was slighter than R1 and R3. On the 115th day, the removal rates of NH4+–N and NO2−–N in R4 were recovered to over 80% and gradually reached over 90% afterward. Comparable results have also been observed by Wang et al.14 in ABBR. However, Chen et al.6 reported different results, which showed anammox was failed to adapt to the higher nitrogen loading rate. In addition, the increase in nitrogen loading also showed a negative impact on the nitrogen removal performance of R3. As shown in Fig. 2c, total nitrogen removal efficiency (about 80%) of R4 was always better than other reactors from the 90th to 180th day.Fig. 3: Nitrogen mass balance of R4 on day 166.This mass balance is drawn on the base of anammox reaction stoichiometry. The black arrows showed the amount of nitrogen to effluent, the red lines indicated anammox process, the green line indicated partial nitrification process, and the pink arrows indicate partial denitrification or partial-DNRA process.Full size imageNitrogen mass balance (Fig. 3) on day 166 proved the coupling of nitrogen removal bacteria on the basis of reported anammox stoichiometric values of NO2−–N/NH4+–N (1.32) and NO3−–N/NH4+–N (0.26). So, if there is only an anammox process, ammonium and nitrite consumption should be around 196 and 258 mg/L, respectively, to meet the reported stoichiometry of the anammox process and effluent nitrate concentration should be approximately 51 mg/L. However, the stoichiometric values of NO2−–N/NH4+–N and NO3−–N/NH4+–N obtained in this study were around 1.15 and 0.19, respectively, in R4 (coupled effects of nZVI and MF) on day 166. This means, 227 mg/L of NO2−–N and 196 mg/L of NH4+–N consumed by anammox, and the remaining amount is removed by other nitrogen cycle bacteria. The NH4+, NO2−, and NO3− attributed to various groups of microorganisms by keeping R1 (1.15) and R2 (0.19) values by using Eqs. 4 and 5. Overall, the proposed nitrogen mass balance shows a clear coupling of anammox with other nitrogen cycle bacteriaEffects of nZVI and MF on the functional genesSo as to study the combined effect of nZVI and MF on functional genes of anammox bacteria, the 16S rRNA gene copy numbers of anammox and other nitrogen cycle bacteria at different stages of the experiment were determined by the qPCR technique. The copy numbers of hzo (hydrazine oxidoreductase) functional gene were also recorded.The anammox 16S rRNA copy number was increased gradually with time (Fig. 4a). Initially, the copy number of anammox 16S rRNA was 1.01 × 106 copies/ng DNA. R1 had the lowest anammox copy number among the four reactors on the 60th day, 120th, and 180th day. The highest copy number of anammox 16S rRNA was recorded in R4 (2.13 × 106), which was followed by R3 (2.10 × 106) and R2 (1.70 × 106) on the 60th day. Nevertheless, R4 and R3 have almost similar gene copy number on the 60th day, which made us propose that MF positively influence the anammox activity, which reduced the start-up period of anammox. It has been reported that bacterial activity is suppressed in the presence of incompatible nZVI concentrations37,38,39. The adaptive capacity of microbes depends on the nZVI concentration40. The inhibition in anammox activity at higher nZVI concentration (3 g/L in this experiment) is also supported by a comparable study41. Interestingly, at day 180, the copy numbers of anammox 16S rRNA in R2 (7.17 × 106 copies/ng of DNA), R3 (6.98 × 106 copies/ng of DNA), and R4 (7.8 × 106 copies/ng of DNA) were significantly higher than R1 (5.56 × 106 copies/ng of DNA). It has also been claimed that the optimum concentration of nZVI can improve the proliferation of anammox cells after adaptation41. It is reported that nZVI lost the reactivity after 3 and 60 days in the presence and absence of oxygen in the wastewater treatment system, respectively42. It is an accepted fact the nZVI released Fe2+ and H2 in the solution43, which can be stored by anammox. Anammox can store iron ions for future haem synthesis and haem-containing enzymes involved in the electron transport chain44. Further, according to the previous study, the addition of nZVI enhanced the abundance of anammox bacteria22,23,45. The above explanation supports the conclusion about the higher anammox gene copy number in R2 in the later stage. Compared with R1, the percent increase of anammox gene copy numbers under a higher nitrogen loading rate were 29.0%, 25.5%, and 40.3% in R2–R4, respectively, on day 180.Fig. 4: The qPCR results of anammox enrichment process.a Anammox 16S rRNA copy numbers at different days (0, 60, 120, and 180) and b functional gene copy numbers of AOB, denitrifying bacterial, and anammox on day 180. Data indicate average, and error bars represent standard deviation of the results from three independent samplings, each tested in triplicate.Full size imageFurther investigation was done to analyze the functional gene hzo of anammox and other nitrogen-cycle related bacteria in the reactors (Fig. 4b) on day 180. The co-existence of AOB and denitrifying bacteria with anammox is reported to improve nitrogen removal efficiency46. The copy number of hzo gene in R1–R4 were 2.39 × 105 and 3.87 × 105, 3.29 × 105, and 4.01 × 105 copies/ng DNA, respectively. The coupled effects of nZVI and MF on anammox functional gene copy number were obvious, which might be responsible for the higher nitrogen removal under increasing nitrogen loading. The contents of amoA, nirK, and nirS in R3 were less, different from other reactors. The difference in amoA, nirK, and nirS gene copy numbers implies that microbes behave differently under the influence of MF. The optimum range of MF intensity is dissimilar for different microorganisms28,47, which can be considered a possible reason for the difference in gene contents among all four reactors. The lower nirK and nirS gene contents in R3 indicated that MF of this intensity (65 ± 10 mT) might have inhibitory effects on denitrifying bacteria. Furthermore, higher nirK gene contents in R4 (nZVI + MF) originate a premise that nZVI might have neutralized the adverse effects of MF on denitrifying bacteria. As it can be seen from Fig. 4b, the denitrifying functional genes nirK and nirS were higher in nZVI reactor. Further, the lower amoA gene content signifies anammox and denitrifying bacteria as major contributors in nitrogen removal.Effects of nZVI and MF on microbial communitiesIt is an established fact that community structure and abundance considerably affect the stability and performance of the anammox process. Therefore, the community composition of the four reactors was analyzed after 180 days by high-throughput sequencing.The sludge samples from all four reactors were collected at the end of the experiment, and operational taxonomic units (OTUs) and four indices (ACE, Chao1, Simpson, and Shannon) of each sample were calculated (Table 1). The OTUs varied from 1599 to 1889 for different treatments. The ACE and Chao1 indices reflect the community’s richness, while Simpson and Shannon’s indices reflect the community’s diversity48. Generally speaking, the greater the Chao1 or ACE index, the higher the abundance of the community. The Shannon diversity index comprehensively considers the richness and uniformity of the community. The higher the Shannon index value, the higher the diversity of the community. The Simpson index is also one of the commonly used indexes for evaluating community diversity. The higher the Simpson index value, the higher the community diversity. The value of ACE (1600) and Chao1 (1600) indices of R1 was the minimum in four reactors reflecting the lowest community richness in R1. On the other hand, the maximum community richness was observed in R3 (MF reactor) as reflected by the highest value of ACE (2180) and Chao1 (2145) indices followed by R2 (nZVI reactor) and R4 (nZVI + MF reactor). In short, the effect of MF on community richness was more pronounced than the effect of nZVI and the combination of nZVI and MF. The Simpson indices of all samples were not different. However, the Shannon indices of R4 were slightly higher than other samples, which showed a little higher diversity of community than other reactors.Table 1 The OTU numbers and bacterial diversity indices of sludge samples.Full size tableThe effects of nZVI and MF treatments on community structure distribution of sludge samples at different classification levels are presented in Fig. 5. The Chloroflexi, Proteobacteria, Cholorobi, and Planctomycetes were the abundant phyla in all samples with little variation among different treatments (Fig. 5a). The presence of phyla such as Chloroflexi, Proteobacteria, and Cholorobi in an anammox reactor was also reported previously49. Relative abundance of Chloroflexi in R1–R4 was 24.3%, 29.5%, 23.4% and 30.0%, respectively. The phylum Chloroflexi has also been detected in the anammox reactor and single-step autotrophic nitrogen removal system50,51. Some genera’s proposed role belongs to Chloroflexi in the anammox reactor is to consume the dead organic material and avoid their accumulation52. The relative abundance of Proteobacteria in R1–R4 was 25.2%, 28.5%, 22.5%, and 27.5%, respectively. According to the previous literature, mostly nitrifying and denitrifying bacteria (Thauera, Denitratisoma, and Geobacter) belong to the phylum Proteobacteria and could use NO2+–N and NH4+–N for their metabolism and proliferation activities4. As far as the relative abundance of phylum Planctomycetes in different reactors is concerned, no significant difference in R1 (8.4%), R3 (8.4%), and R4 (8.5%) was noted in this regard (chi test, p More