Introduction

The pollution of nitrate in surface water has increasingly become a serious environmental problem in modern world due to the increasing anthropogenic activities, such as discharge of industrial and domestic sewage, leaching of animal manure from the farming industry1 and excessive utilization of fertilizers in agriculture2. One significant concern of the nitrate pollution is eutrophication3, 4, which is represented by the thriving of aquatic plants and algae in water, resulting in the oxygen depletion and death of aquatic animals5. Besides, the accumulation of nitrate in water also increases the health risk to human. It was reported that high concentrations of nitrate (e.g. 10 mg/L) in the drinking water may increase the risk of the methaemoglobinaemia to human, a disease also named as Blue Baby Syndrome6. Furthermore, high levels of nitrate in drinking water was also showed positive associations to the occurrence of cancer, e.g. stomach cancer7 and gastric cancer8. Though much effort has been done to purify the polluted water, the level of nitrate in the surface water was still high according to the recent studies. In an investigation in Osona region of northeast Spain, approximately 70% of wells tested contained at least 50 mg/L of nitrate9. In North China Plain, the level of nitrate pollution ranges from 0.9 mg/L to 131 mg/L in the surface water10. Therefore, efficient methods are needed urgently to purify the nitrate contaminated surface water.

Traditionally, the nitrate removal is performed through a heterotrophically anaerobic denitrification (ANDN) process, where the nitrate is reduced to gaseous nitrogen forms in the absence of oxygen gas, such as the nitrous oxide (N2O), nitric oxide (NO) and dinitrogen gas (N2)11. However, there is a problem in the ANDN process, which is the exclusion of oxygen. Oxygen is a crucial factor affecting the transformation of nitrogen in water, e.g. the oxidation of ammonia and reduction of nitrate. Ammonia is another toxic nitrogen contaminant in surface water, which can be removed through being oxidized to nitrate (nitrification) in the presence of oxygen gas12. So the different requirement of oxygen determines the aerobic nitrification and anaerobic denitrification processes cannot be operated under the same condition. It is known that the surface water in nature, e.g. flowing river, is often an aerobic environment13. The oxygen scarcity often occurs when the eutrophication happens3, which is a situation needed to be avoided. Thus, the ANDN process is not suitable for the water purification in situ before the concentration of the contaminated nitrate exceeds the threshold and leads to the occurrence of eutrophication. Furthermore, many evidences indicated that the denitrification also occurred under the aerobic conditions14. Just as its name implies, the aerobic denitrification is a process which can simultaneously use the both oxygen and nitrate as the electron acceptors and therefore is capable of reducing the nitrate in the presence of oxygen gas15. Therefore, some researchers tried to isolate and apply the aerobic denitrifying bacteria in the removal of nitrate through the aerobic denitrification process16.

Some aerobic denitrifying bacteria have been isolated from different niches, such as the Bacillus methylotrophicus L7 isolated from wastewater17, Pseudomonas putida AD-21 isolated from soil18, Pseudomonas stutzeri TR2 isolated from domestic wastewater19 and Zoogloea sp. N299 isolated from the drinking water reservoir20. However, little is known on the community diversity of the culturable aerobic denitrifying bacteria in the surface water system. Therefore, in this study, the aerobic denitrifying bacterial strains were isolated from 3 types of niches in the Liangshui River, including the overlying water where the bacteria live in planktonic mode, biofilm phase where the microorganisms live in a sessile mode and sediment where high concentrations of nutrients, e.g. nitrogen and phosphorus, were usually deposited and absorbed from the overlying water phase21, 22. The classification and compositions of the aerobic denitrifying consortiums were also studied.

Results

Screening of the aerobic denitrifying bacteria from the river

Three types of the samples, including the overlying water, sediment and biofilms on supporting materials, were collected from the Liangshui River which is the receiving water of the effluents of Xiaohongmen wastewater treatment plant. The sampling points were locating at 500 m downstream of the outfall point of the wastewater treatment plant (Fig. S1). Samples of Liangshui River were collected in March 2015 at three niches: overlying water (at 50 cm depth), sediment (at 100 cm depth) and biofilm (at 50 cm depth). The average nitrogen concentrations in forms of ammonia and nitrate in sampling sites were tested to be 24.5 mg/L and 18.6 mg/L respectively, and the range of dissolved oxygen level was 1–4 mg/L. After the screening, a total of 116 strains of bacteria were tested to have the denitrifying ability under the aerobic conditions, all of which showed the positive reaction with the BTB (Fig. S2). An OTU based analysis of the 16S rDNA was conducted. At cutoff of 0.03, which is generally considered as the label for Species23, a total of 24 OTUs were clustered (Supplementary Table S1). The classification of the OTUs against to the RDP and NCBI 16S ribosome RNA reference databases was shown in Table 1. The rarefaction curve and OTU coverage analysis indicated that the isolated bacterial strains covered approximately 80–90% of the culturable bacterial community in each of the three niches, implying the data here were representative.

Table 1 The classification for the OTUs of culturable aerobic denitrifiers in surface water against to the NCBI 16S ribosome RNA reference database.
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The capability of total nitrogen removal was also tested through the incubation in the nitrate enriched medium (DM medium in this study) aerobically for these OTUs. The results showed that all the isolated OTUs had the aerobic denitrifying ability, where the removal efficiency of total nitrogen (TN) was in the range of 1.6–67% (Fig. 1). The top three aerobic denitrifying bacteria were OTU 7 (O. rhizosphaerae), OTU 2 (Pm. stutzeri) and OTU 4 (unclassified Ochrobactrum sp.), which reduced the TN by 66.63%, 66.17%, 50.30% respectively after the incubation for 96 hours. The TN removal efficiency of the rest OTUs were in the range of 6–50%, except for the OTU 16 (Pa. versutus) which had the TN reduction efficiency of 1.6%.

Figure 1
figure 1

The removal efficiency of total nitrogen (TN) for the representative strains of the isolated 24 OTUs. The removal efficiency reflected the aerobic denitrification capacity.

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Community of culturable aerobic denitrifiers in the overlying water

Among the 24 OTUs, 13 OTUs were isolated from the overlying water, 11 OTUs were isolated from the biofilms and 10 OTUs were isolated from the sediment phase samples (Fig. 2 and Supplementary Table S1). Therefore, the community structure was studied specifically in the three niches.

Figure 2
figure 2

The Venn diagram of OTUs at cutoff of 0.03 for the culturable aerobic denitrifying bacteria in the phases of biofilm, water and sediment. The numbers in the overlapping parts represent the shared OTUs in different phases.

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In the overlying water samples, all the 13 OTUs were composed of a group of 45 strains of bacteria (Fig. 3). After the sequence assignment against to the RDP and NCBI databases, they were clustered to 9 genera of bacteria, including Pseudomonas (Pm) sp., Comamonas sp., Stenotrophomonas sp., Pannonibacter (Pn) sp., Paracoccus sp., Pseudocrobactrum (Pc) sp., Ochrobactrum sp., Arthrobacter sp. and Gordonia sp. (Supplementary Fig. S3). In the genus of Pseudomonas, 17 strains of isolates, such as W1, W15, W36, W45, etc. were assigned to the Pm. stutzeri, 9 strains of isolates, including W6, W16, W35, etc. were classified to the Pm. mendocina (Fig. 3). In the genus of Gordonia, the strain W3 was classified to G. terrae while the W9 was assigned to G. malaquae (Fig. 3). In addition, in the overlying water, the culturable aerobic denitrifiers also included Pn. phragmitetus (W30 and W38), Pc. saccharolyticum (W14), O. rhizosphaerae (W24, W40 and W46), A. ureafaciens (W10), S. terrae (W8), S. acidaminiphila (W4), and unclassified Comamonas sp. (W2).

Figure 3
figure 3

The phylogenetic tree (circular mode) for the culturable aerobic denitrifying isolates in the water phase of Liangshui River. The vertical scale bar in the middle represents the distance (dissimilarity) of the isolates and reference strains.

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Community of culturable aerobic denitrifiers in the biofilm

In this study, 11 OTUs, composed of a group of 41 strains of bacteria, were isolated from the biofilm samples and screened to have the denitrifying ability under the aerobic condition (Fig. 4). Against to the RDP and NCBI databases, 8 genera of bacteria were assigned, including the Pseudomonas (Pm) sp., Rheinheimera (Rh) sp., Gemmobacter sp., Pannonibacter (Pn) sp., Rhizobium (Rz) sp., Ochrobactrum sp., Arthrobacter sp. and Rhodococcus (Rc) sp. (Supplementary Fig. S4). The Pseudomonas sp. were the most abundant culturable aerobic denitrifiers in the biofilm phase, which were composed of 11 strains of Pm. Mendocina (B17, B19, B44, etc.), 1 strain of Pm. monteilii (B45), 5 strains of Pm. putida (B4, B10, B30, etc.), 5 strains of Pm. stutzeri (B1, B15, B36, etc.) and 9 strains of unclassified Pseudomonas sp. (B34, B40, B42, etc.) that all had a very close relation with the Pm. toyoyomensis HT-3, Pm. Oleovorans RS1 and Pm. chengduensis MBR (Fig. 4). The Rheinheimera were the second abundant genus which all were hit to the Rh. pacifica (B24, B32, B33, B39). In addition, 4 species including G. caeni (B8), Pn. phragmitetus (B16), Rz. pusense (B38), A. soil (B5) and Rc. Pyridinivorans (B7) were also isolated. The strain B12 had the same similarity (99.85%) to each of the O. anthropi ATCC 49188, O. lupine LUP21 and O. cytisi ESC1, which was therefore cannot be identified at the Species level just based on the 16S rDNA.

Figure 4
figure 4

The phylogenetic tree (circular mode) for the culturable aerobic denitrifying isolates from the biofilm phase of Liangshui River. The vertical scale bar in the middle represents the distance (dissimilarity) of the isolates and reference strains.

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Community of culturable aerobic denitrifiers in the sediment

The sediment in the river is often composed of the soil, sands, deteriorated materials and the benthic microbial communities24. The nutrient levels are usually very high in the sediment, due to the deposition and absorption of the organics and inorganics from the overlying water phase21, 22. On the other hand, the sediment is also a reservoir which released the nutrient and microorganisms to the overlying water phase. Therefore, the aerobic denitrifying bacteria were also isolated from the sediment phase in Liangshui River. In total, 10 OTUs which were composed of 30 strains of aerobic denitrifying bacteria were acquired from the sediment. These OTUs can be grouped into 6 genera, including the Pseudomonas (Pm) sp. Brevundimonas sp., Rhizobium (Rz) sp., Ochrobactrum sp., Gordonia sp. and Rhodococcus (Rc) sp. (Fig. 5 and Supplementary Fig. S5). Similarly with the phases of water and biofilm, the Genus Pseudomonas dominated the whole culturable aerobic denitrifying community, which contained 5 strains of Pm. putida (S10, S15, S22, etc.), 4 strains of Pm. mendocina (S18, S25, S27 and S32), 4 strains of Pm. resinovorans (S7, S9, S11 and S12), 2 strains of Pm. stutzeri (S1 and S2), 3 strains of Pm. monteilii (S29, S30 and S33) and 3 strains of unclassified Pseudomonas sp. (S24, S26 and S31). The Genus Ochrobactrum were the second abundant culturable aerobic denitrifiers in sediment, which included 4 strains of O. tritici (S5, S13, S14 and S16) and 1 strain of O. anthropi (S4). In addition, some rare species were also isolated, including B. diminuta (S20), Rz. pusense (S19), G. alkanivorans (S3) and Rc. canchipurensis (S6).

Figure 5
figure 5

The phylogenetic tree (circular mode) for the culturable aerobic denitrifying isolates from the sediment phase of Liangshui River. The vertical scale bar in the middle represents the distance (dissimilarity) of the isolates and reference strains.

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Distribution of aerobic denitrifiers in overlying water, biofilm and sediment

In order to compare the habitat-preference of the culturable aerobic denitrifiers, the beta-diversity of the communities was studied between the overlying water, biofilm and sediment. Among them, the biofilm and sediment samples shared the highest number of OTUs (6 OTUs), in comparison with the group of biofilm and water (4 OTUs) and group of sediment and water (3 OTUs) (Fig. 2). Only 3 OTUs were found in all the 3 phases of samples, which included OTU 1 (Pm. mendocina), OTU 2 (Pm. stutzeri) and OTU 3 (Pm. putida). Examined by the cluster analysis, the culturable aerobic denitrifying communities had only 38.1% of Bray-Curtis similarity in the phases of water, biofilm and sediment. Therein, the communities of biofilm and sediment had a closer relationship (55.2% in Bray-Curtis similarity) relative to the aerobic denitrifying bacteria in water (Fig. 6). Examined through the “SIMPER” analysis by the PRIMER 6.0, the Pm. mendocina contributed most to the Bray-Curtis similarity (29.8% in contribution), followed by the Pm. putida (28.2% in contribution) and Pm. stutzeri (21.9% in contribution), which together contributed 79.9% to the Bray-Curtis similarity (Fig. 7). This implied these three species may be the universal aerobic denitrifiers in the phases of overlying water, biofilm and sediment of the Liangshui River system.

Figure 6
figure 6

The OTU compositions and relationship of aerobic denitrifying bacteria in biofilm, water and sediment. The clustering tree on the left half was constructed based on the Bray-Curtis similarity of OTUs compositions which is on the right half of the figure, in which cluster 1 was the combination of biofilm and sediment and cluster 2 included cluster 1 and water.

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Figure 7
figure 7

The contributions of different OTUs to the Bray-Curtis similarity of culturable aerobic denitrifiers in the phases of water, biofilm and sediment. The values of contributions were calculated in the PRIMER 6.0.

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On the other hand, the culturable aerobic denitrifiers presented 61.9% of dissimilarity among the phases of overlying water, biofilm and sediment. The PCA plot indicated that the OTU 5 (Rh. pacifica) and OTU 9 (unclassified Pseudomonas sp.) preferred to inhabit in biofilms rather than the water and sediment (Fig. 8). For example, the OTU 9 (unclassified Pseudomonas sp.) accounted for 22.0% and 10% of the culturable aerobic denitrifiers in biofilms and sediment respectively, but no strains of OTU 9 were isolated from the phase of water (Fig. 6). Especially, the OTU 5 (Rh. pacifica) was only isolated from the biofilm phase (Fig. 6). In the sediment, OTU 4 (unclassified Ochrobactrum sp.) and OTU 6 (Pm. resinovorans) contributed most to the dissimilarity between the sediment phase and the other two phases (Fig. 8). In addition, OTU 12 (G. alkanivorans), OTU 14 (B. diminuta) and OTU 18 (Rc. canchipurensis) were only isolated from the sediment, which all had the relative abundance of 3.3% in the sediment community. Examined by the PCA plot, among the OTUs in water phase, the OTU 2 (Pm. stutzeri) and OTU 7 (O. rhizosphaerae) contributed most to the dissimilarity of culturable denitrifying community between the overlying water and other two phases (biofilm and sediment) (Fig. 8). Specifically, the OTU 2 (Pm. stutzeri) accounted for 37.8% of the culturable aerobic denitrifiers in overlying water but only had the percentage of 12.2% and 6.7% in the phases of biofilm and sediment respectively (Fig. 6). The OTU 7 (O. rhizosphaerae) was the fourth abundant culturable aerobic denitrifier in water which occupied 6.7% of the relative abundance. Nonetheless, the strains of OTU 7 (O. rhizosphaerae) were not isolated from the biofilm and sediment samples.

Figure 8
figure 8

The PCA plot and dominant OTUs corresponding to the dissimilarity of aerobic denitrifying bacterial communities in the phases of biofilm, water and sediment. The red circle, blue square and green triangle represent the culturable communities in biofilm, sediment and water respectively. The black branches represent the dominant OTUs which contributed most to the dissimilarity among the communities in the 3 environmental sites. The blank circle indicating a maximal vector which contains all the base variables, and it is a default setting in PRIMER 6.0.

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Discussion

In order to purify the nitrate contaminated river, it is necessary to isolate the efficient denitrifying bacteria which can adapt to different niches. In this study, three species of the genus Pseudomonas, including the Pm. stutzeri, Pm. mendocina and Pm. putida, were found to be the common abundant aerobic denitrifying bacteria in the phases of overlying water, biofilm and sediment, indicating they may be the universal dominant culturable aerobic denitrifiers. The conclusion here was supported by the previous studies, where the Pm. stutzeri, Pm. mendocina and Pm. putida were all found to have the denitrifying ability under different aerobic conditions15, 18, 25. For example, the Pm. stutzeri had been found to distribute widely in the environment, such as the wastewater and sediment of fishponds16, 26, soil of silver mine27, groundwater, marine water and wastewater treatment plants28. Furthermore, a strain of Pseudomonas stutzeri isolated from the wastewater treatment plants, Pm. stutzeri TR2, was also reported to promote the transformation of N2O to N2 and therefore decrease the emission of N2O15, which is a significant contributor to the global warming29. Thus, the dominant existence of the Pm. stutzeri in the culturable aerobic denitrifying community in this study indicated that the Pm. stutzeri may be used in the nitrate removal from the surface water and play an important role in the prevention of global warming. The Pm. putida was another denitrifying species that had been proven to tolerate 5.0–6.0 mg/L of the oxygen18. The detection of nitrate reductase from the periplasm of Pm. putida was a further demonstration of the denitrifying ability for this bacterium30. The Pm. monteilii was reported to be highly similar to the Pm. putida based on the 16S rDNA and can only be differentiated by the different substrate utilization of inositol, α-aminobutyrate, and o-/m-hydroxybenzoate31. This conclusion was also supported by this study, where the dissimilarity of the 16S rDNA was below 1% between the Pm. putida and Pm. monteilii (Figs 3–5). Further, the data here showed another common character of the Pm. monteilii and Pm. putida, which is the nitrate reducing property.

In addition to the dominant Pseudomonas species, bacteria of other genera also presented the denitrifying ability in the presence of oxygen gas, such as the Ochrobactrum, Pannonibacter (Pn), Gordonia and Stenotrophomonas. The nitrate reduction ability of O. tritici and O. anthropi was also reported in a previous study32. Interestingly, the Pn. Phragmitetus had been proven to perform denitrification under anaerobic condition33, but this study indicated that this species was also capable of reducing the nitrate in the presence of oxygen gas. For the genus of Gordonia sp., it was known that one species of G. sihwensis was aerobic nitrate reducing bacterium34. However, few reports showed the G. terrae and G. malaquae were both capable of aerobic denitrification. So this study is the first report to show the aerobic denitrifying ability of G. terrae and G. malaquae. Two species of Stenotrophomonas, including S. terrae and S. acidaminiphila, were isolated from the overlying water in this study. The nitrate reducing character of S. terrae had already been reported35, while the S. acidaminiphila was first reported to be an aerobic denitrifier in this study. Some other bacteria were also first reported to have denitrifying ability in this study, including the Pc. Saccharolyticum, O. rhizosphaerae, A. ureafaciens and Rh. Pacifica. An important phenomenon needs to be noticed that some aerobic denitrifying bacteria were reported to often have the ammonia oxidizing ability, such as Pm. stutzeri 26, Pm. putida 36, Paracoccus denitrificans 37 and Klebsiella pneumoniae 38. This facilitates the simultaneously aerobic nitrification and denitrification processes, which are different from the traditional nitrogen removal processes through the aerobic nitrification and anaerobic denitrification. The strains of Pm. stutzeri and Pm. putida were also isolated in this study. However, the simultaneously nitrifying and denitrifying ability of these isolates have not been tested. Therefore, a task in the future work may be to determine the ammonia oxidizing capability of these isolated aerobic denitrifying bacteria.

The isolation of the aerobic denitrifying bacteria is a premise for the bioaugmentation-based water purification. Bioaugmentation is a strategy to promote the degradation of contaminants through introducing the highly efficient pollutant-degrading bacteria to the environment or wastewater treatment plant39. For the in situ remediation of polluted river through bioaugmentation, two environmental sites including the sediment and overlying water are usually selected. The site selection determines the way of bioaugmentation, e.g. depositing the pollutant-degrading bacteria immobilized gel beads into the sediment or pouring the solution of pollutant-degrading bacteria into the contaminated water directly40. In addition, it was known that some bacteria in aquatic phase can attached onto certain surface to form the biofilms41. Biofilm is a microbial sessile living mode where the bacteria aggregate together onto the surface to develop a three-dimensional structure and were regulated by the quorum sensing mechanisms41. In the biofilm structure, the bacteria usually owned some different characters which are different from the planktonic living bacteria in water, e.g. secretion of extracellular polysaccharides41. The formation of biofilms may increase the biomass and duration of bacteria in water. Thus it was also regarded as a bioaugmentation method by introducing the biofilm bed to the water phase42. In combination, the selection of environmental sites and bioaugmentation modes determine the types of bacteria which would be used to conduct the purification function in surface water. The unevenly distribution of richness shown by the Venn diagram (Fig. 2) indicated that the structure of aerobic denitrifiers may be different between the phases of biofilm, sediment and water. Examined by the cluster analysis of the 16S rDNA, the culturable aerobic denitrifying communities had only 38.1% in Bray-Curtis similarity among the three phases of Liangshui River (Fig. 6), which was a confirmation that the culturable aerobic denitrifiers had different community structures in the three phases of river. Moreover, the aerobic denitrifying community in biofilm and sediment had a closer relationship (55.2% in Bray-Curtis similarity) relative to the aerobic denitrifying bacteria in water. This is consistent with the data of richness distribution, implying the community of culturable aerobic denitrifiers had more similar community compositions in the biofilm and sediment phases, in comparison with the planktonic living phase in overlying water. It is known that the bacteria own a sessile living mode in the biofilm, where a surface is needed for the attachment of bacteria. This is the reason why we provided a polypropylene supporting material for the growth of biofilm (Supplementary Fig. S6). The sediment is usually composed of microorganisms, humic substances, soil, sands, etc. So the soil and sands in sediment may offer the surface for the bacteria to form biofilm structures. This may explain why the culturable denitrifiers in the biofilms on polypropylene supporting material had more similar community structure to the sediment rather than the overlying water.

In biofilm phase, the OTU 1 (Pm. mendocina) had the highest relative abundance, implying the Pm. mendocina may have the property to inhabit in biofilm. This is consistent to the previous studies, in which the Pm. mendocina was demonstrated to be the initial colonizers of biofilm and promoted the growth of biofilm aggregations through the production of cell bounded extracellular polysaccharides43. For the OTU 5 (Rh. pacifica) it was only isolated and enriched in the biofilm samples. This may indicate the Rh. pacifica was the rare organisms in water and sediment but had strong biofilm forming ability, leading to the thriving and enrichment of the OTU in biofilms. These findings also implied that the Pm. mendocina and Rh. pacifica may be suitable to be used in the biofilm-based bioaugmentation. Similarly, the OTU 6 (Pseudomonas resinovorans) and OTU 14 (Brevundimonas diminuta) may be better to be introduced into the sediment, while the OTU 2 (Pm. stutzeri) and OTU 7 (O. rhizosphaerae) may give a better growth in the overlying water. It was also noted that some OTUs can only be detected in one phase, such as the OTUs 6, 12, 14 and 18 isolated from sediment, the OTUs 7, 11, 17, 19 and 21 isolated from overlying water and the OTUs 5, 22, 23 and 24 isolated from biofilm. However, it may be hard to judge that there were no such bacteria in the other two phases of Liangshui River, as the abundance of these microorganisms may be quite low which increased the difficulty to isolate them. Therefore, the knowledge here is helpful to select the appropriate niche in the isolation of aerobic denitrifying bacteria and may increase the possibility to get the targeted bacteria.

In conclusion, a total of 116 strains of aerobic denitrifying bacteria were isolated from the sediment, overlying water and biofilms on supporting materials in Liangshui River of Beijing, which were classified into 14 genera based on the 16S rDNA, such as Pseudomonas, Rheinheimera, and Pannonibacter. Three species of Pseudomonas, including the Pm. stutzeri, Pm. mendocina and Pm. putida, were the dominant and universal culturable aerobic denitrifying bacteria in all the phases of river. These culturable aerobic denitrifiers had different distribution characteristics among the 3 phases of Liangshui River. The isolation of aerobic denitrifiers would facilitate the purification of the nitrate polluted surface water, e.g. contaminated river and lake. However, the optimal working conditions of these denitrifiers, such as pH, ratio of carbon to nitrogen (C/N) and removal efficiency, had not been revealed. Therefore, the future works may focus on the determination and optimization of removal efficiencies and working conditions. Furthermore, the screening of the ammonia oxidizing ability also needs to be done for these isolated aerobic denitrifiers, in order to find the simultaneous nitrifying and aerobic denitrifying bacteria.

Methods

Sample collection and measurement of water quality

The sampling points were selected at 500 meters (m) downstream of the outfall point of Xiaohongmen wastewater treatment plant, which located near the Liangshui River of Beijing and drain off the treated water to Liangshui River (Fig. S1). Three types of samples, which included the sediment, overlying water and biofilms, were collected in Liangshui River as the microbial reservoir to isolate the aerobic denitrifying bacteria. Samples of Liangshui River were collected in March 2015 at three niches: overlying water (at 50 cm depth), sediment (at 100 cm depth) and biofilm (at 50 cm depth). In order to isolate aerobic denitrifying bacteria from the biofilm samples, pieces of polypropylene supporting material had been immersed and suspended in the Liangshui River for 6 months (Supplementary Fig. S6). The water quality in the Liangshui River of Beijing in China was determined based on the contamination of ammonia and nitrate. The concentration of ammonia in the water was measured based on the Nessler’s reaction44. The concentration of nitrate was quantified by the thymol solution45.

Media, isolation and screening of the aerobic denitrifying bacteria

Three types of media were used in the isolation and screening of the aerobic denitrifying bacteria, including the enrichment medium (EM), denitrifying medium (DM), and screen medium (GN). The EM was composed of 0.5 g/L (NH4)2SO4, 0.36 g/L KNO3, 4.0 g/L sodium citrate, 0.05% (ratio of volume) of the trace element solution which was composed of 6.5 g/L K2HPO4·3H2O, 2.5 g/L MgSO4·7H2O, 2.5 g/L NaCl, 0.05 g/L FeSO4·7H2O, 0.04 g/L MnSO4·H2O. The final pH of the EM was adjusted to 7.0. The DM was composed of 0.36 g/L KNO3, 10.55 g/L Na2HPO4·12H2O, 1.5 g/L KH2PO4, 0.1 g/L MgSO4·7H2O, 4.0 g/L sodium citrate, 0.2% (volume ratio) of trace element solution which included 50.0 g/L EDTA-Na2, 2.2 g/L ZnSO4, 5.5 g/L CaCl2, 5.06 g/L MnCl2·4H2O, 5.0 g/L FeSO4·7H2O, 1.57 g/L CuSO4·5H2O, 1.61 g/L CoCl2·6H2O. The final pH of the DM was adjusted to 7.0. The GN for the denitrifying bacteria was composed of 1.0 g/L KNO3, 8.5 g/L sodium citrate, 1.0 g/L L-asparagine, 1.0 g/L KH2PO4, 1.0 g/L MgSO4·7H2O, 0.2 g/L CaCl2·6H2O, 0.05 g/L FeCl3·6H2O, 0.1% (volume ratio) of the 1% (ratio of weight/volume) alcoholic dissolved bromothymol blue (BTB)46. The final pH of the DM was adjusted to 7.0. The solid GN was made by adding 2% of agar powder to the liquid GN.

The isolation and screening of denitrifying bacteria were based on the production of hydroxide ion (OH−) due to the reduction of nitrate in denitrification process. A complete denitrifying process is composed of 4 steps which were shown as the Eqs (1–4):

$$8{{\rm{NO}}}_{3}^{-}+2{{\rm{CH}}}_{3}{\rm{COOH}}\to 8{{\rm{NO}}}_{2}^{-}+4{{\rm{CO}}}_{2}+4{{\rm{H}}}_{2}{\rm{O}}$$
(1)
$$8{{\rm{NO}}}_{2}^{-}+{{\rm{CH}}}_{3}{\rm{COOH}}+2{{\rm{H}}}_{2}{\rm{O}}\to 8{\rm{NO}}+2{{\rm{CO}}}_{2}+8{{\rm{OH}}}^{-}$$
(2)
$$8{\rm{NO}}+{{\rm{CH}}}_{3}{\rm{COOH}}\to 2{{\rm{CO}}}_{2}+4{{\rm{N}}}_{2}{\rm{O}}+2{{\rm{H}}}_{2}{\rm{O}}$$
(3)
$$4{{\rm{N}}}_{2}{\rm{O}}+{{\rm{CH}}}_{3}{\rm{COOH}}\to 2{{\rm{CO}}}_{2}+4{{\rm{N}}}_{2}+2{{\rm{H}}}_{2}{\rm{O}}$$
(4)

Overall, the denitrifying process can be displayed as the Eq. (5)

$$8{{\rm{NO}}}_{3}^{-}+5{{\rm{CH}}}_{3}{\rm{COOH}}\to 4{{\rm{N}}}_{2}+10{{\rm{CO}}}_{2}+6{{\rm{H}}}_{2}{\rm{O}}+8{{\rm{OH}}}^{-}$$
(5)

In the equations, the acetate (CH3COOH) is used as the carbon source and electron donor for the denitrification and the OH− is produced as a byproduct of the reduction of nitrite to nitric oxide, which will increase the pH of the reaction system47. The GN in this study was composed of nutrient elements and the pH indicating dye BTB, which would change the color from green to blue when the pH increases from 7.0 to high levels. If the bacteria growing on the solid GN (pH 7.0) have the denitrifying ability, the pH in the medium surrounding the bacterial colonies would increase, resulting in the color of the colonies turns to the blue19.

Samples from the phases of water, biofilm and sediment were transferred and inoculated into 250 mL EM and incubated at 30 °C with shaking at 150 r/min for 30 days (d). During the enrichment stage, 25 mL of mixed solution of EM and microorganisms were sucked out and replaced with the fresh EM every 48 hours (hr). From the d 31, 25 mL of the mixed solution of medium and microorganisms were sucked out and replaced with the fresh DM every 24 hr. After the incubation with DM for 14 d, the isolation and screening of the aerobic denitrifying bacteria was conducted. For the samples of water phase, 1 mL of the mixed cultures were diluted and spread on solid GN. For the isolation from the biofilm phase, 30 mL of the mixed solutions of DM and biofilm samples were transferred to sterile 50 mL tubes which contained sterile glass beads. The tubes containing biofilm samples were shaken at 5000 r/min on a vortex mixer (VORTEX-GENIE 2) to detach the bacteria from the biofilms. Next, 1 mL of mixed solution of DM and detached bacteria were diluted to spread on the solid GN. The procedure of bacteria isolation from sediment was similar to the biofilm samples, where the biofilm samples were replaced by the sediment. The solid GN plates were incubated at 30 °C. During the incubation, the blue colonies were separately picked out and incubated in liquid GN at 30 °C with shaking at 150 r/min to confirm the change of color (Supplementary Fig. S6).

Measurement and re-confirmation of aerobic denitrifying ability

24 isolates were selected as the representative strains of 24 OTUs (Table 1). For nitrate removal, a 10 mL bacteria suspension under logarithmic phase was centrifuged at 5000 rpm for 5 mins and then rinsed with 1 × PBS (phosphate buffered saline) for 3 times to remove the excess medium. Resuspended the bacteria with 1 × PBS, inoculated into 1 mL denitrifying medium (DM), and incubated at 150 rpm at 30 °C for 96 h. Each treatment was carried out in triplicate. The aerobic denitrification efficiency (i.e., the TN removal efficiency) were calculated by the Eq. (6):

$$\mathrm{TN\; removal\; efficiency}=\frac{\mathrm{CK}(\mathrm{TN})-\mathrm{EG}(\mathrm{TN})}{\mathrm{CK}(\mathrm{TN})}\times 100 \% $$
(6)

In the equation, the CK (TN) represents the initial total nitrogen of the treatments, the EG (TN) is the terminal total nitrogen after 96 h. Concentration of TN was determined by the method of Alkaline potassium persulfate digestion-uv spectrophotometer48.

Colony PCR and sequencing

The 16S rDNA sequences for the isolated aerobic denitrification bacteria were amplified through a colony PCR procedure49. The primers for the 16S rDNA were composed of 27 F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′)18. The PCR procedure was composed of a pre-denaturing step at 95 °C for 5 min followed by 35 cycles of denaturing at 95 °C for 30 s, annealing at 55 °C for 45 s and extending at 72 °C for 1.5 min and 1 cycle at 72 °C for 10 min. The PCR products were sequenced by the Sanger sequencing method. All the sequences had been deposit in the GenBank. The accession numbers are KT380544 – KT380588 for the isolates from the overlying water (Supplementary Table S2), KT380503 – KT380543 for the bacteria isolated from the biofilm samples (Supplementary Table S3) and KT380589 – KT380619 for the bacteria isolated from the sediment of the Liangshui River (Supplementary Table S4).

Sequence classification and phylogenetic analysis

All sequences acquired from the Sanger sequencing method were classified to the taxonomic ranks against to the RDP 16S rRNA training set 14 and NCBI 16S ribosome RNA databases respectively50. The information of the taxonomic ranks from Kingdom to genus was generated by the RDP classification at the confidence threshold of 95%. The closest species and strains were picked from the NCBI blasting results based on the highest score, sequence coverage and identity. The bootstrap tested neighbor joining phylogenetic trees were constructed in MEGA 4.0 for the sequences of isolated strains and reference strains. The bootstrap value for the sequence classification was set to be 60.

OTU assignment and community analysis

An OTU table based on the 16S rDNA was formed for the sequences of all isolated aerobic denitrifiers using the MOTHUR software. First, three different barcode sequences were added to the sequences of the water, sediment and biofilm communities separately to distinguish amplicon sequences from different communities. Next, the sequences of the three groups were merged together by the command “merge.files” and re-assigned to different groups by the process of “trim.seqs”51, 52, in which the barcode sequences were removed at the same time. The sequences were then aligned to the Silva bacterial reference data by the “align.seqs” and the nonsense columns which only contained the character of “.” were removed by the process of “filter.seqs”. Finally, after the calculation of the distances using the command “dist.seqs”, the sequences were clustered and assigned to OTUs at the cutoff of 0.03 through the processes of “cluster” and “make.shared”51, 52. The OTU-based similarity or dissimilarity between the culturable communities in 3 phases was calculated by “SIMPER” analysis in PRIMER 6.0 (PRIMER-E). The contributions of the OTUs to the similarity or dissimilarity were calculated based on the relative abundances of OTUs between the groups. Clustering trees and principal component analysis (PCA) plot were created between the samples based on the average Bray-Curtis similarity of OTUs compositions in the different groups of the overlying water, sediment and biofilm52.