A twilight for the complete nitrogen removal via synergistic partial-denitrification, anammox, and DNRA process

Start-up phase

To 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).

Furthermore, 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 grow^33,34,35. 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 NO_x⁻–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 growth^13,14. 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.

From 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 activity^5,36. 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 loading

After 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.

Nitrogen 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 bacteria

Effects of nZVI and MF on the functional genes

So 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 concentrations^37,38,39. 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 bacteria^22,23,45. 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.

Further 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 microorganisms^28,47, 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 communities

It 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.

The 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 system^50,51. 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 < 0.01. But, significantly higher (chi test, p < 0.01) relative abundance (10.9%) of Planctomycetacia in R2 clearly indicated the positive effects of nZVI on the anammox-related class Planctomycetacia R1 (3%), R3 (2.9%), and R4 (3.5%) after adaptation of sludge to nZVI (Fig. 5b). The Planctomycetacia is an important class that contains anammox bacteria such as Candidatus Brocadia⁵³ while class Betaproteobacteria (15.7% in R1, 14.6% in R2, 11.5% in R3, and 14.6% in R4) contain many nitrifying, denitrifying and other nitrogen-related bacteria. Other dominated classes in R1–R4 were Anaerolineae (22.5%, 24.3%, 21.1%, and 27.4%), Chlorobia (13.1%, 1.8%, 16.9%, and 10.7%), Ignavibacteria (1.3%, 5%, 2.2%, and 5%), Phycisphaerae (5.3%, 4.0%, 5.4%, and 3.2%). However, the role of Anaerolineae, Chlorobia, Phycisphaerae in anammox reactor is not confirmed yet. With regard to Ignavibacteria, it is reported that the player of class Ignavibacteria might have the ability to performed partial DNRA bacteria^15,17.

The key OTUs (top 50 genera) were selected and analyzed using a heat map to know the in-depth community structure of four reactors (Fig. 5c). The color reflects the abundance of OTUs in the corresponding sample (green indicated lower abundance and red showed higher abundance). The OTUs associated with Candidatus Brocadia were more abundant in R2 and R3, while less abundant in R1 and R4. But, in terms of nitrogen removal efficiency, R4 showed the highest removal efficiency, thanks to synergetic bacteria co-existence such as DNRA and denitrifiers. The difference of anammox abundance may be caused due to the difference in the ecological niche and physiological characteristics of anammox genera⁵⁴. The proliferation of other nitrogen-cycle related bacteria such as nitrifiers, denitrifiers, and DNRA played an essential role in increasing the nitrogen removal of anammox reactor^55,56,57. The Nitrosomonas is the dominant AOB and was more abundant in R1 and R2. The denitrifying genus Thauera had a higher relative abundance in R4 than R1–R3. The abundance of Geobacter was higher in R3 and R1, while Ignavibacterium was more abundant in R4. The genera included Thauera and Denitratisoma are denitrifying genera^58,59, and Geobacter has reported DNRA bacteria which can reduce nitrate into ammonium^60,61,62. The genus Ignavibacterium is also responsible for the P-DNRA process¹⁷. Overall, the diversity of microbial genera involving anammox bacteria such as Candidatus Brocadia has the ability to perform the DNRA process^16,54,63. As it can be seen from Fig. 5a, b, Proteobacteria and Planctomycetes were among the abundant phyla, respectively. Thus, the denitrifying bacteria (Thauera and Denitratisoma), DNRA (Geobacter and Ignavibacterium), and anammox were co-existed in R4 with more abundance.

According to the literature, Thauera is mostly autotrophic denitrifying bacteria, which can be used for short-cut denitrification (only conversion of NO₃⁻–N to NO₂⁻–N)^53,64,65. The role of partial denitrification cannot be ruled out in the anammox reactor^54,66,67,68. The Denitratisoma performed the reduction of NO₃⁻–N⁶³ and was also reported as heterotrophic denitrifying bacteria in an anammox reactor. The DNRA bacteria might fuel the anammox process with the conversion of NO₃⁻ to NH₄⁺⁶⁹. Although anammox enrichment was carried out with synthetic wastewater, the presence of denitrifying bacteria is the same as in earlier reports^70,71. In this study, NO₃⁻–N produced from anammox reaction is considered as an essential substrate for denitrifying and DNRA bacteria. Further, the decay of biomass under the pressure of unfavorable environmental conditions is inevitable. Also, the extracellular polymeric substances released by anammox and other bacteria provide the organic matter for reducing NO3–N and NO2–N by denitrifying bacteria (44) and converting NO₃⁻–N or NO₂⁻–N to NH₄⁺ by DNRA bacteria¹⁵. Thus, a synergistic partial-denitrification, anammox, and DNRA (SPDAD) process was created coincidentally.

The correlation between the relative abundance of nitrogen-related functional genera and external forces was performed by redundancy analysis (RDA) (Fig. 6). The angle of the arrow represents the correlation between functional genera and external treatment. The DNRA bacteria were excluded in the RDA analysis plot because of their very low abundance. The RDA analysis showed that the genus Denitratisoma and Thauera positively correlated while genera Nitrosomonas and Truepera negatively correlated with nZVI and MF. The Candidatus Brocadia negatively correlated with both factors. The PCA analysis revealed the microbial communities’ difference between R1–R4 (Fig. S2). PCA analysis showed the difference and similarity of microbial community composition. Although nitrogen removal performance of R1 and R3 was different but R1 and R3 cluster together while R2 and R4 make their own distinct cluster. The results showed that nZVI caused the shift in the microbial community as compared to MF since two reactors containing nZVI have distinct microbial communities.

To gain deep insight into the molecular functions of microbiota in all four reactors, PICRUSt was used to envisage the changes in microbial function. The accessible annotated genes were imputed within the Kyoto Encyclopedia of Genes and Genomes (KEGG). Based on the KEGG analysis (Fig. 7), the lowest abundance was observed for the gene related to the biosynthesis of secondary metabolites, while higher abundance was observed for the gene related to energy, carbohydrate, and amino acid metabolism. Overall, there was not a clear difference in the abundance of the functional gene under different treatments except functional gene related to lipid metabolism, which was suppressed by MF. However, PICRUSt is associated with limitations such as this analysis depends on the accessibility of the reference genome in the database, which must be considered in future studies. Further, the gene might not be expressed, limiting the impact of their annotated functions. So, the microbial function derived from PICRUSt should be considered as the hypothesis that must be validated through in-depth functional assays.

Significance and practical implementation of synergistic partial-denitrification, anammox, and DNRA (SPDAD) process

The noticeable resistance by anammox to higher nitrogen loading shock in R4 can be justified by the growth of denitrifying bacteria (Thauera), responsible for the reduction of NO₃⁻ to NO₂⁻ and DNRA bacteria (Geobacter and Ignavibacterium), responsible for the reduction of NO₃⁻ or NO₂⁻ to NH₄⁺ in anammox reactor. Overall, possible inter-relationship among anammox, DN, and DNRA bacteria were depicted in Fig. 8. Anammox can produce NO or NH₂OH from NO₂⁻ with the little amount of NO₃⁻. NO or NH₂OH was further reduced to N₂H₄ and finally converted into N₂. As seen from the qPCR analysis, NO₂⁻ respiratory genes nirS and nirK, which convert NO²− into NO (an intermediate product of anammox metabolism), existed in all reactors. NO₂⁻ is the essential substrate of anammox and an inhibitor if it presents in a higher concentration. So, denitrifying bacteria and DNRA bacteria may provide the shield to anammox bacteria from the substrate’s inhibitory effects. Nevertheless, PD and DNRA can also use NO₂⁻ and can affect the removal efficiency negatively. So, it was assumed that the careful selection of environmental factors could improve the performance of anammox nitrogen removal with the association of other nitrogen cycle genera. Keren et al.¹⁵ also enriched anammox without any external carbon and observed the growth of denitrifying and DNRA bacteria where an overgrowth of DNRA bacteria decreased the performance of anammox bacteria. However, in this study, the nitrogen removal efficiency was higher in the presence of other nitrogen players, which means other nitrogenous bacteria positively influence the nitrogen removal performance of anammox. Some bacteria in the complex microbial community depend on the metabolites of other bacteria⁵². The possible reason for PD and DNRA bacteria’s growth is that the decaying biomass of some bacteria and EPS and some vitamins produced by anammox bacteria can be used by PD and DNRA as carbon sources. It has also been reported that the bacteria performing the DNRA process were also capable of performing the PD process¹⁵. The performance of SPDAD was more apparent in R4 (nZVI-MF as an external factor) than in other reactors. This phenomenon was observed coincidentally in this study; therefore, further exploration is needed to optimize the growth conditions and external environmental factors for a synergistic relationship among anammox, PDN, and DNRA bacteria. Overall, the diverse community of anammox, PDN, and DNRA will be the right choice for the complete removal of nitrogen from wastewater. SPDAD process may be suitable for the treatment of wastewaters loaded with a high concentration of NO₃⁻. DNRA bacteria can provide NH₄⁺ and NO₂⁻ to anammox from NO₃⁻ and DN bacteria can generate NO₂⁻ if a careful selection of environmental conditions can be achieved. Also, anammox itself has the ability to perform the DNRA process¹⁶.

This study unfolded the effects of external factors on the performance and microbial community of anammox. The MF was an effective external force to stimulate the anammox growth and shorten the start-up period by 43.3%. nZVI addition did not show considerable effects on the reduction of the start-up period. However, in a later stage, nZVI showed a positive influence on anammox growth. The combination of nZVI and MF shortened the start-up time by 16.7% and improved the anammox stability under a high nitrogen loading rate. The results of qPCR showed that nZVI coupled with MF enhanced the proliferation and community diversity. The presence of DNRA (Ignavibacterium, Geobacter) and denitrifying bacterial genera (Thauera and Denitratisoma) may also support the removal of nitrogen. This study shows a twilight for the complete nitrogen removal via synergistic partial-denitrification, anammox, and DNRA process (SPDAD).

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