Achieving fast start-up of anammox process by promoting the growth of anammox bacteria with FeS addition

Effects of FeS on nitrogen removal

The start-up period could be divided into two phases based on the operating strategy of the reactor, as illustrated in Table 1. The first phase was characterized by high HRT and low substrate concentration (days 0–18), in which the HRT was 48 h and the concentrations of influent NH₄⁺-N and NO₂⁻-N were 50 and 60 mg L⁻¹, respectively. The second phase was characterized by low HRT and high substrate concentration (days 24–68), in which the HRT was 36 h and the theoretical concentrations of influent NH₄⁺-N and NO₂⁻-N were 100 and 120 mg L⁻¹, respectively.

The effluent ammonium concentration was significantly higher than that of influent at the beginning of the reactor operation shown in Fig. 1a. On the first day, the effluent NH₄⁺-N concentration of R1 and R2 reached 106.0 and 80.6 mg L⁻¹, respectively, nearly twice as high as the influent NH₄⁺-N concentration. This is mainly due to the fact that some microorganisms were unable to adapt to the new environmental conditions, inducing cellular lysis²¹. At the same time, effluent NO₂⁻-N concentration of R1 and R2 on the fourth day were 18.4 and 17.3 mg L⁻¹, respectively, with the removal efficiency of more than 70% (Fig. 1b); and NO₃⁻-N accumulated in the effluent. The high-throughput results showed that Nitrospirae, which contained massive nitrite-oxidizing bacteria (NOB), accounted for a higher proportion in the inoculation sludge (Supplementary Fig. 1)²². qPCR results also indicated that NOB abundance was higher in the inoculation sludge as shown in the section “Effect of FeS on functional bacteria abundance”. Therefore, the removal of NO₂⁻-N in the beginning might be attributed to the role of nitrification. Denitrification also might promote the decrease of NO₂⁻-N through using the organic matter which was released by decay of biomass²³. From day 7 to day 10, effluent NH₄⁺-N of R1 and R2 decreased rapidly from 38.1 and 49.4 mg L⁻¹ to 6.8 and 6.8 mg L⁻¹, respectively, however the removal rate of NO₂⁻-N did not change much. From day 1 to day 18, the accumulation of NO₃⁻-N in R1 and R2 gradually decreased from 10 mg L⁻¹ to 0 mg L⁻¹. These phenomena indicated that NOB was gradually eliminated in the low-oxygen environment and the activity of anammox bacteria was increasing. In addition, microbial metabolism and decay of biomass will release organic carbon, which can be used as carbon sources by denitrifying bacteria²³. From day 4 to day 18, the total nitrogen removal efficiency (TNRE) of R1 and R2 increased from 30.4% and 22.2% to 96.0% and 98.3%, respectively. On day 18, the values of removed NO₂⁻-N/NH₄⁺-N and produced NO₃⁻-N/removed NH₄⁺-N were 1.14 and 0 in R1 while these were 1.17 and 0 in R2, which was the result of the combined action of nitrifying bacteria, denitrifying bacteria and anammox bacteria.

On the 21st day, when influent NH₄⁺-N and NO₂⁻-N concentrations increased to 100.3 and 138.1 mg L⁻¹, effluent NH₄⁺-N and NO₂⁻-N concentrations of R1 increased to 6.5 and 24.2 mg L⁻¹, respectively; while those of R2 increased to 2.6 and 19.9 mg L⁻¹. On the 24th day, when HRT decreased from 48 h to 36 h, effluent NH₄⁺-N and NO₂⁻-N continued to increase. At this time, the abundance of anammox bacteria in the reactors was relatively low and had not played a dominant role. Meanwhile, the cell lysis phase was over and denitrifying bacteria activity began to decrease with the continuous consumption of organic substance²³. Therefore, the NH₄⁺-N and NO₂⁻-N removal efficiencies fluctuated widely when the nitrogen loading rate (NLR) increased. Moreover, the higher removal rate of NH₄⁺-N and NO₂⁻-N in R2 can be attributed to the promotion effect of FeS on anammox growth. On the 27th day, effluent NO₂⁻-N concentration of R1 and R2 reached the highest values (81.8 mg L⁻¹, 71.1 mg L⁻¹); the TNRE was the lowest, which were 52.8% and 61.0%, respectively. After this point, the NH₄⁺-N and NO₂⁻-N removal efficiencies of both R1 and R2 gradually increased and there were significant differences in total nitrogen removal capability between the two reactors. As shown in Fig. 1c, the TNRE of R2 on the 30th day increased to 73.3%; R1 achieved a TNRE of over 70% 12 days later, while the TNRE of R2 reached over 80% at this time. On the 45th day, the accumulation of nitrate appeared again in the effluent of the two reactors, meaning anammox was predominant. On the 51st day, the NH₄⁺-N and NO₂⁻-N removal in R2 reached more than 85% simultaneously, and the values of removed NO₂⁻-N/NH₄⁺-N and produced NO₃⁻-N/removed NH₄⁺-N were 1.12 and 0.17, respectively, closing to the theoretical stoichiometric ratio of anammox reaction, which marks that anammox reactor was started up successfully²¹. Based on Eq. (1) and the experimental data on day 51, an assumed transformation model was constructed to reflect the pathways of the nitrogen conversions in the system as shown in Supplementary Fig. 2. Due to the lack of oxygen and organic matter and the inhibition of denitrification by FeS, anammox played a dominant role. The same phenomenon occurred in R1 on day 56. Bi et al. studied the effect of Fe(II) concentration on the start-up of anammox process with a HRT of 12 h and found that the start-up time of anammox process could be shortened from 70 to 58 days when the concentration of Fe(II) ranged from 1.68 to 3.36 mg L⁻¹²¹. Because the concentration of Fe(II) was relatively lower than previous study, the influence was relatively less but this method is more convenient. The heme c content at day 50 in R2 was higher than that in R1 as shown in the section “Fe²⁺ release and Heme c content”, demonstrating that the activity of anammox bacteria in R2 was higher than that in R1. In summary, FeS effectively shortened the start-up time and improved the nitrogen removal performance.

On the 71st day, when influent NH₄⁺-N and NO₂⁻-N concentrations increased to 150 mg L⁻¹ and 180 mg L⁻¹, respectively, the NH₄⁺-N and NO₂⁻-N removal rates in the two reactors decreased. On the 75th day, effluent NH₄⁺-N concentrations of R1 and R2 increased to 37.1 and 35.3 mg L⁻¹, meantime effluent NO₂⁻-N concentration increased to 93.3 and 84.8 mg L⁻¹. Although the nitrogen removal rate of the two reactors decreased obviously after the NLR was increased, it quickly recovered to the original level. As shown in Fig. 1a, b, on day 81, effluent NH₄⁺-N in R1 and R2 decreased to 11.1 and 7.1 mg L⁻¹ and effluent NO₂⁻-N concentrations decreased to 16.5 and 6.2 mg L⁻¹. The TNRE increased to about 90%. This indicated that the reactors have a certain capacity in resistance to weak shock loading due to the enrichment of anammox bacteria. And, when influent NH₄⁺-N and NO₂⁻-N were further increased, effluent NH₄⁺-N and NO₂⁻-N concentrations of R2 were significantly lower than these of R1. Meantime, the responses caused by the unit intensity of shock (R) of R2 was substantially lower than these of R1 as shown in Supplementary Table 1, indicating that R2 had more resistance to shock loading rate. The same trend was observed when HRT were further shortened to 36 h and 12 h, suggested that the stability of anammox reactors can be improved with the addition of FeS.

During the start-up period, the NO₃⁻-N concentration in R2 was substantially higher than that in R1 as shown in Supplementary Fig. 3, which might be attributed to the inhibition of denitrification process in R2 by FeS^24,25. However, in the stabilization period, the NO₃⁻-N concentration in R2 was substantially lower than that in R1. This was due to the lack of organic matter in R1 which inactivated denitrifying bacteria. Meantime, the presence of FeS in R2 might promote sulfur autotrophic denitrification and DNRA to reduce nitrate. The KEGG function prediction result as shown in the section “Effect of FeS on microbial community” verified this inference.

FeS structure change

The appearance of FeS with dark brown color, particle size between 1 and 5 mm and compact texture before being added to the reactor was observed (Supplementary Fig. 4). After 180 days of reactor operation, the FeS materials remaining in R2 were found to be covered with a layer of sludge. And the appearance displayed clear differences: most of the color changed from dark brown to khaki and the texture was sparse, which may be caused by the oxidation of FeS. Moreover, the red anammox granule sludge as shown in Supplementary Fig. 4 was observed in R2. Touching these red anammox granule sludge felt that the interior was relatively hard, which was made of FeS particles. FeS may promote the formation of anammox granular sludge.

To further understand the structure change, the morphology of FeS before and after reaction were observed by SEM at different magnifications. As shown in Fig. 2c, d, there were many honeycomb style holes on the surface and inside of the FeS particles after the reaction. The voids formed on the surface may facilitate the attachment of microorganisms, which acted like microbial carriers. Therefore, anammox granular sludge containing FeS as inert cores formed in R2. In addition, Fe²⁺/Fe³⁺ produced by oxidation and ionization of FeS could weaken the electrostatic repulsion among negatively charged anammox cells and promote the aggregation of anammox bacteria into zoogloea by the effect of salt bridge²⁶. Thus, the addition of FeS could promote the formation of anammox granular sludge, then improve the stability of the reactor. Figure 2e, f showed that many plate-shaped secondary minerals were produced after the reaction of FeS. In the presence of dissolved oxygen (DO), O₂ can diffuse into the FeS surface and oxidize Fe²⁺ to Fe³⁺ (Eq. (5))⁶. The formation of these secondary minerals may hinder the release of iron ions from FeS²⁷.

Effect of FeS on functional bacteria abundance

The abundance of anammox bacteria in the two reactors were monitored during the period of their operation. As shown in Fig. 3a, the copy numbers of anammox 16S rRNA gene in the inoculation sludge was 3.31 × 10⁶ copies per ng DNA. After 150 days of cultivation, the copy numbers of anammox 16S rRNA gene in R1 and R2 (1.21 × 10⁷, 1.42 × 10⁷copies per ng DNA) were significantly higher than that in the inoculation sludge. The data demonstrate that although the content of anammox in the inoculation sludge was low, anammox bacteria can be rapidly enriched and the reactor could be properly started-up as long as the cultural conditions for anammox bacteria growth were suitable. The anammox 16S rRNA gene copy numbers of R1 and R2 were 5.68 × 10⁶ and 7.04 × 10⁶ copies per ng DNA on day 70, respectively. Compared with R1, the abundance of anammox bacteria in R2 was increased by 29%. The contrast in cell quantities between R1 and R2 indicated that the addition of FeS with this concentration promoted the growth of anammox bacteria. Combined with the water quality results, the faster growth rate of anammox bacteria in R2 was responsible for the higher removal efficiencies of NH₄⁺-N and NO₂⁻-N and shorter start-up time of reactor.

In addition to anammox, the contents of ammonia-oxidizing bacteria (AOB), NOB and denitrifying bacteria also affect the start-up time and nitrogen removal capacity of anammox reactor. Compared with the inoculation sludge, the expression levels of amoA (NH₄⁺ → NO₂⁻) and nirS (NO₃⁻ → NO₂⁻) genes in both R1 and R2 were increased, while the expression levels of Nitrospira spp. 16S rRNA genes (NO₂⁻ → NO₃⁻) and nirK (NO₃⁻ → NO₂⁻) genes were decreased (Fig. 3b). The expression levels of Nitrospira spp. 16S rRNA genes could reflect the content of NOB in anammox reactor²⁸. As anammox was cultured in an anaerobic environment, which was not conducive to the growth of NOB, the content of NOB was gradually decreased with the increase of culture time. And the expression level of Nitrospira spp. 16S rRNA genes in the inoculated sludge was 2.14 × 10⁶ copies per ng DNA, which was consistent with the higher nitrite removal efficiency initially. On day 70, the expression levels of amoA gene in R1 and R2 were 1.34 × 10⁴ and 2.07 × 10³ copies per ng DNA, while anammox 16S rRNA gene expression level was 5.68 × 10⁶ and 7.04 × 10⁶ copies per ng DNA. It was clear that the content of anammox was two or three orders of magnitude higher than AOB. The qPCR results also demonstrated that the anammox bacteria were dominant after 70 days of operation, at which time the removal of ammonium nitrogen was mainly from anammox. In addition, the expression level of amoA gene in R2 was much lower than that of R1, and the NOB content of both reactors was higher than AOB content on day 70 (Fig. 3b). FeS could react with dissolved oxygen (DO) in the reactor, leading to an inhibitory effect on the growth of AOB⁶. But Nitrospira-like NOB has higher hypoxia tolerance ability than AOB. Liu et al. reported that when the reactor was operated under low oxygen conditions (0.16 mg DO L⁻¹) for a long time, some of Nitrospira-like NOB can adapt to anaerobic environment and maintain activity²⁹. Both nirS and nirK are functional genes of denitrifying bacteria. The expression level of nirS gene in R2 (2.05 × 10⁶ copies per ng DNA) was higher than that of R1 (1.11 × 10⁶ copies per ng DNA), while the expression of nirK gene in R2 (3.27 × 10⁶ copies per ng DNA) was slightly lower than that of R1 (3.65 × 10⁶ copies per ng DNA). According to previous reports, the nirK gene encodes copper-containing nitrite reductase and the nirS gene encodes heme-containing cd₁ nitrite reductase which contains heme d as its catalytic center³⁰. And iron ions are involved in the synthesis of various types of heme. It is reasonable to speculate that the synthesis of cd₁ nitrite reductase in microorganisms was promoted after adding FeS into the reactor.

Fe²⁺ release and Heme c content

The effluent Fe²⁺ and intracellular heme c concentrations were determined and illustrated in Fig. 4. Initially, the Fe²⁺ content in the effluent of R1 and R2 was similar because FeS particles with compact texture had a smaller specific surface area (Fig. 2a, b) and released less iron ions (Fig. 4a). After the reactor was operated for a period, the effluent Fe²⁺ concentration of R2 was significantly higher than that of R1. On the 30th day, the effluent Fe²⁺ concentration of R1 and R2 were 0.132 and 1.762 mg L⁻¹, respectively. The results on days 45 and 60 also showed that there was a significant difference in effluent Fe²⁺ concentration between R1 and R2. During this period, massive holes were corroded on the surface and inside of FeS particles as shown in Fig. 2, the specific surface area of FeS increased and the activity of FeS was higher, contributing to more release of iron ions. On day 70, the content of heme c in R1 and R2 was 7.2 and 11.8 μmol per g-protein, respectively (Fig. 4b). It has been reported that Fe²⁺ was involved in the formation of heme c, which was the active center of many enzyme proteins³¹. In many biochemical reactions, the transformation of substrate and intermediate is accomplished by the catalysis and electron transfer of c-type heme protein^32,33. Anammox cells contain a large amount of multi-heme cytochromes, for example one subunit of hydroxylamine oxidoreductase enzyme (HAO) binds 8 heme c³⁴. In this experiment, the positive correlation between Fe²⁺ and heme c confirmed that the concentration of Fe²⁺ in the reactor could be increased with the addition of FeS, then promoting the synthesis of heme c. On the 75th and 90th days, the Fe²⁺ content in the effluent of both reactors became lower, probably because the abundance of anammox bacteria increased gradually, corresponding to an increased consumption of iron ions. At the same time, the results showed that the content of Fe²⁺ in R2 effluent did not differ much from that in R1 effluent. On one hand, as the reaction progress, secondary minerals and biofilm were formed on the surface of FeS (Fig. 2), which led to a decrease in FeS activity. On the other hand, the abundance of anammox bacteria in R2 was higher than that in R1 (Fig. 3), thus more iron ions would be consumed.

Effect of FeS on microbial community

Through clustering analysis of OTU, the number of OTUs shared among samples and the number of OTUs unique to each sample can be intuitively observed. The number of OTUs shared by the R1 and R2 samples was 816, which accounted for 71.8% and 69.9% of the total OTUs, respectively; the number of OUT unique to R1 was 321 and that for R2 was 352 (Supplementary Fig. 5). The addition of FeS led to different species composition of the two communities. The shared OTUs number of R1 and R2 samples with inoculated sludge was 168, accounting for 14.8% and 14.4% of the total OTUs of R1 and R2 samples, respectively. Obviously, after domestication, the R1 and R2 samples were less similar to the inoculated sludge.

The ACE, Chao1, Simpson and Shannon listed in Table 2 are the alpha diversity indexes that reflect the richness and diversity of the community. The ACE and Chao 1 indexes are mainly used to indicate the richness of the community. In general, the larger the two index values are, the higher the richness of the community is. Comparing the ACE and Chao1 index values of R1 and R2 samples, the richness of R2 community was higher than that of R1. The Simpson and Shannon indexes account for the richness and evenness of the community. The larger the two index values are, the higher the diversity of the community is. As shown in Table 2, the two index values of R2 samples were higher than these of R1, so the diversity of R2 community was higher. In summary, the community of R2 sample had higher richness and diversity. During the cultivation and acclimation process, some species in the seed sludge couldn’t adapt to the new environmental conditions and were gradually washed out from the system. The addition of FeS reduced the tendency of some species to disappear under its role in facilitating the formation of granular sludge.

It can be seen from the results of microbial diversity analysis that the addition of FeS had a certain influence on the number of species of R1 and R2. The differences in microbial community composition at different classification levels with or without the presence of FeS were shown in Fig. 5.

The microbial community composition of R1 and R2 was similar at phylum classification level (Fig. 5a). The dominant phylum in two reactors was Protobacteria, accounting for 40.1% and 29.6%, respectively, followed by Chloroflexi (12.5% and 14.1%). Other reports also showed there were higher contents of Protobacteria and Chloroflexi in anammox reactor^35,36. The relative abundance of Planctomycetes which anammox belonged to in R1 and R2 was 9.1% and 9.9%, respectively. The values were not very high, mainly due to the small proportion of Planctomycetes in the inoculated sludge (Supplementary Fig. 1) and the slower growth rate of the anammox bacteria. The proportion of Acidobacteria in R1 and R2 showed obvious difference, with relative abundances of 7.0% and 11.9%, respectively. Several publications demonstrated that some microorganisms belonged to Acidobacteria have the ability to dissimilate iron reduction with various simple organic acids such as acetate as alternative electron donors under anaerobic conditions^37,38,39. In addition, the relative abundance of Nitrospirae which Nitrospira belonged to in R1 and R2 was extremely low compared with the inoculated sludge, which was reduced from 16.58% to 0.45% and 0.15%, respectively (Supplementary Fig. 1). This result was consistent with the water quality.

Figure 5b showed the genus of the top 9 abundance in the microbial community of R1 and R2. The most abundant genus in R1 was Halomonas, accounting for 9.7%. Most parts of the microbes belonged to Halomonas were denitrifying bacteria, which could reduce NO₃⁻-N to NO₂⁻-N⁴⁰. Denitratisoma with a high relative abundance (7.3%) in R1 is also one type of denitrifying bacteria⁴¹. The proportions of Halomonas and Denitratisoma in R2 was 6.5% and 4.3%, respectively, significantly lower than these in R1. The relative abundance of Thiobacillus, which was the major autotrophic denitrifier detected in most sulfur-based autotrophic denitrification reactors, increased from 0.012% in R1 to 0.041% in R2 with the addition of FeS^42,43. The most abundant genus in R2 was Clone_Anammox_20, accounting for 9.0%. Clone_Anammox_20 and Clone_Anammox_2 are a class of microorganisms with anammox function. The most abundant anammox genus obtained in both reactors was “Ca. Kuenenia” and the proportion was relatively close. In order to further explore the effect of FeS on the distribution of anammox bacteria, the composition of R1 and R2 samples on Brocadiaceae classification level was analyzed. The Brocadiaceae family in R1 consisted of three anammox genus, “Ca. Kuenenia”, “Ca. Brocadia” and “Ca. Jettenia”, accounting for 99%, 0.9%, and 0.1%, while the Brocadiaceae family in R2 consisted of two anammox genus, “Ca. Kuenenia” and “Ca. Brocadia”, accounting for 98% and 2%, respectively (Fig. 5c). The dominant anammox bacteria in R1 and R2 was “Ca. Kuenenia”, and the proportion of “Ca. Brocadia” in R2 was higher than in R1. Other works have found that some of the anammox bacteria under the genus “Ca. Kuenenia” and “Ca. Brocadia” could oxidize Fe²⁺ with NO₃⁻-N while anammox process occurred⁴⁴. Thus, FeS might affect the distribution of species and relative abundance of anammox genus but did not change the dominant status of the anammox bacteria in the community.

To further explore the influence mechanism of FeS on nitrogen transportation, PICRUSTs was used in this experiment to predict the contents of enzymes related to NO₂⁻-N conversion based on KEGG database. As shown in Fig. 6a, nitrite can be reduced to nitrogen (NO₂⁻-N→N₂) through denitrification and ammonia nitrogen (NO₂⁻-N→NH₄⁺-N) through dissimilatory nitrate reduction to ammonium (DNRA), in addition to being removed by anammox. The nitrite reductase (ammonia-forming) content of R2 was significantly higher than that of R1, while nitrite reductase (NO-forming) and nitric oxide reductase content of R2 was lower than that of R1. It had been reported that some DNRA bacteria can conduct DNRA process with sulfide (S²⁻) or elemental sulfur (S⁰) as electron donors⁴⁵. And sulfide had an inhibitory effect on nitrous oxide reductase and nitric oxide reductase, which can inhibit the denitrification reaction, have an inhibitory effect on nitrite reductase (NO-forming) due to the accumulation of NO and promote the nitrite reduction reaction by the DNRA process^24,25,46. In addition, heme was involved in the formation of nitrite reductase (ammonia-forming)⁴⁷. Robertson et al. found that the addition of Fe²⁺ improved DNRA and inhibited denitrification^48,49. It is postulated that the iron ions and sulfur ions released by FeS encouraged the occurrence of DNRA process and somehow decreased the denitrification reaction. Therefore, the removal rates of NO₂⁻-N in the two reactors were significantly different, and the removal rates of NH₄⁺-N were similar. This may also account for the relatively low abundance of denitrifying bacteria in R2. Moreover, Fig. 6b showed the predicted metabolism function of the two reactors’ communities, and the results indicated that the metabolic function of R2 was slightly higher than that of R1. It can be seen that the addition of FeS to the anammox reactor can increase microbial metabolism.

Engineering significance

As a new type of environmentally-friendly biological nitrogen removal process, the anammox process has been a research hotspot, but it still encounters some issues to limit its wider application. Anammox bacteria are slow-growing microorganisms, and are sensitive to environmental conditions, such as salinity and organic carbon⁵⁰. Another challenge of the anammox process system is the maintenance of effluent quality since about 10% nitrate would be produced in the anammox reaction, failing to meet discharge standards⁵¹.

In this study, the start-up time of the anammox reactor was shortened and the nitrogen removal rate was significantly increased with the addition of FeS. There were mainly two reasons: On one hand, FeS promoted the formation of anammox granular sludge and increased the abundance of anammox bacteria; on the other hand, FeS stimulated the synthesis of the heme c, which participated in the synthesis of a variety of enzymes. In addition, FeS can promote the DNRA process by inhibiting denitrification. Microbial oxidation of FeS, which links to the efficiency of denitrification, DNRA and anammox, plays an important role in the N cycle and S cycle¹⁵. According to previous report, FeS could function as an alternative electron donor for sulfur-dependent autotrophic denitrification⁵². Nitrate reduction was achieved by using pyrrhotite as the biofilter medium and autotrophic denitrifiers as seed in lab¹⁷. And DNRA process could occur due to HS⁻ release¹⁸. This study found that FeS could promote the start-up of anammox process and promote the nitrite reduction reaction by the DNRA process through inhibiting denitrification. Therefore, it is possible to couple anammox with sulfur-autotrophic DNRA or sulfur-autotrophic denitrification in full-scale application by adding FeS to improve the total nitrogen removal efficiency.

Source: Ecology - nature.com