An integrated fixed-film activated sludge (IFAS) process (G1) and an activated sludge anoxic–oxic process (G2) were operated at nitrate liquor recirculation ratio (R) of 100, 200 and 300% to investigate the feasibility of enhancing nitrogen removal efficiency (RTN) and reducing R by improving simultaneous nitrification and denitrification (SND) in the IFAS process. The results showed that the effluent NH4+-N and total nitrogen (TN) of G1 at R of 200% were less than 1.5 and 14.5 mg/L, satisfying the Chinese discharge standard (NH4+-N < 5 mg/L; TN < 15 mg/L). However, the effluent NH4+-N and TN of G2 at R of 300% were higher than 8.5 and 15.3 mg/L. It indicated that better RTN could be achieved at a lower R in the IFAS process. The polymerase chain reaction–denaturing gradient gel electrophoresis results implied that nitrifiers and denitrifiers co-existed in one microbial community, facilitating the occurrence of SND in the aerobic reactor of G1, and the contribution of SND to TN removal efficiency ranged 15–19%, which was the main reason that the RTN was improved in the IFAS process. Therefore, the IFAS process was an effective method for improving RTN and reducing R. In practical application, this advantage of the IFAS process can decrease the electricity consumption for nitrate liquor recirculation flow, thereby saving operational costs.

INTRODUCTION

Biological nitrogen removal (BNR) processes are generally accepted in existing wastewater treatment plants (WWTPs) for nitrogen removal due to their economic advantages compared with chemical treatment methods (Fan et al. 2009). Among these BNR processes, the anoxic–oxic (A/O) process utilizes activated sludge (AS) technology that has been widely applied in most WWTPs on account of rich practical experiences obtained in its long-term application (Chan et al. 2009).

During the A/O process, the nitrogen removal is dependent on aerobic nitrification (ammonium is converted into nitrate) and anoxic denitrification (the nitrate is reduced to N2). Higher nitrification capability can produce more nitrates as electron acceptor for denitrification, thereby improving nitrogen removal efficiency. Thus, nitrification is a prerequisite for nitrogen removal. However, it is difficult to maintain the steady and high-efficiency nitrification in an AS system, because the slow-growing nitrifiers may be washed out of the system if the sludge retention time (SRT) is shorter than that needed for their proliferation (Nogueira et al. 2002). In addition, the nitrate recirculation flow from the aerobic to the anoxic zones is one of the important factors affecting nitrogen removal efficiency in the A/O process (Baeza et al. 2004). Higher nitrate recirculation flow would bring more nitrates back to the anoxic zone for denitrification. Nevertheless, this can increase the electricity consumption for recirculation and the operational cost. Hence, an effective method is required to modify the A/O process with AS technology for improving nitrification and reducing the nitrate recirculation flow, in order to ensure effluent nitrogen concentration satisfying the increasingly stringent discharge standard, and responding to requirement of energy conservation.

The integrated fixed-film activated sludge (IFAS) process is a hybridized involving microorganisms both in suspended sludge and on the biofilm carriers, and is particularly attractive for nitrogen removal because the slow-growing nitrifiers could be retained on the biofilm, which favors achieving longer SRT to strengthen nitrification (Regmi et al. 2011; Bassin et al. 2012). In addition, Fu et al. (2010) has verified that nitrifiers and denitrifiers can co-exist in one microbial community in a single aerobic reactor with free-floating carriers, facilitating the occurrence of simultaneous nitrification and denitrification (SND). As a novel BNR technology, SND can remove a part of nitrogen in the oxic reactor, which may reduce nitrate recirculation flow from the aerobic reactor to the anoxic reactor.

Therefore, to verify the feasibility of enhancing N removal and reducing the nitrate recirculation flow by improving SND in the IFAS process, an IFAS process and an A/O process were operated in parallel at different nitrate liquor recirculation ratios to investigate the nitrogen removal efficiency. In addition, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and fluorescence in situ hybridization (FISH) were used to analyze the mechanism for SND from the molecular biology perspective, and to evaluate the relative quantity variations of functional bacteria at different nitrate liquor recirculation ratios, respectively.

MATERIALS AND METHODS

Experimental set-up and operating conditions

The IFAS process (G1) consisted of an anoxic reactor (6 L) and an aerobic reactor (9 L), and the same apparatus was used as the control process (G2, A/O process with AS technology) (Figure 1). The free-floating carriers with the size of diameter 7 × 9 mm and the density of 0.97 g/cm3 were added into the aerobic reactor of the IFAS process with 30% of the packing ratio. In both processes, the influent flow was fixed at 36 L/d, resulting in the hydraulic retention time being 4 h for the anoxic reactor and 6 h for the aerobic reactor, respectively. The anoxic reactor was equipped with a mechanical stirrer to efficiently mix the suspended liquor. A blower aerated the aerobic reactor through the porous stone diffusers. Dissolved oxygen (DO) concentration in aerobic reactors was kept in the range of 2.0–2.5 mg/L. The processes were operated at a constant sludge recirculation ratio of 70%. To evaluate the effect of nitrate liquor recirculation ratio (R: the ratio of nitrate liquor recirculation flow to influent flow) on nitrogen removal, R was controlled at 100%, 200% and 300%, respectively. The duration at each R was at least 40 days. The SRT and the temperature were kept at 7–8 days and 20–23 °C during the experimental period, respectively.
Figure 1

Schematic diagram of the IFAS (G1) and the control (G2) processes.

Figure 1

Schematic diagram of the IFAS (G1) and the control (G2) processes.

Wastewater and seed sludge

The artificial wastewater contained glucose and ammonium chloride as the chemical oxygen demand (COD) and nitrogen, respectively. The characteristics of the synthetic wastewater fed into the system are shown in Table 1. The AS was obtained from a secondary sedimentation tank of the WWTP located in Dalian, China.

Table 1

Characteristics of the influent

Parameter Range Average 
pH 7.2–7.6 7.4 
COD (mg/L) 171.6–213.9 199.9 
NH4+-N (mg/L) 60.3–68.8 65.2 
NO2-N (mg/L) 0–0.46 0.35 
NO3-N (mg/L) 0–0.94 0.61 
TN (mg/L) 62.5–70.3 67.8 
MLSS (mg/L) 3,264–4,064 3,761 
Parameter Range Average 
pH 7.2–7.6 7.4 
COD (mg/L) 171.6–213.9 199.9 
NH4+-N (mg/L) 60.3–68.8 65.2 
NO2-N (mg/L) 0–0.46 0.35 
NO3-N (mg/L) 0–0.94 0.61 
TN (mg/L) 62.5–70.3 67.8 
MLSS (mg/L) 3,264–4,064 3,761 

Analytical methods

Temperature, DO and pH were measured by a WTW Multi 340i meter with DO and pH probes (WTW Company, Germany). COD (SM 5220), total nitrogen (TN) (SM 4500-N), NH4+-N (SM 4500-NH4+), NO2-N (SM 4500-NO2), NO3-N (SM 4500-NO3), mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS) (SM 2540) were analyzed according to Standard Methods (APHA 2005).

DNA extraction and PCR-DGGE analysis

The mature carriers obtained from the reactor were kept in 1.0 mM phosphate buffer solution (PBS). The biofilm was peeled from the carriers and homogenized using ultrasonic vibration for 10 min. The carriers were subsequently washed three times in PBS and then centrifuged at 12,000 g for 10 min at 4 °C to collect cells.

Genomic DNA extraction was performed according to the methods described by Lakay et al. (2007). The universal primers for bacteria were 341F (forward primer, containing a 40-bp GC clamp) and 907R (reverse primer). Appropriately sized PCR products were confirmed by using 1% agarose gel electrophoresis in 0.5 × TAE buffer, followed by staining with Genfinder (Dalian TaKaRa, China). The PCR products were applied onto a 6% polyacrylamide DGGE gel with a linear denaturing gradient ranging from 30 to 60% (e.g., 100% denaturing gradient contains 7 M urea and 40% formamide). Electrophoresis was performed at a constant voltage of 180 V for 6 h in the 1 × TAE buffer. Subsequently, the gels were stained with SYBR Gold (Dalian TaKaRa, China) in 1 × TAE buffer for 40 min, and gel digital images were obtained using the Gel Doc 2000 System (Bio-Rad Laboratories, USA). The 16S rRNA fragments were sequenced with sequencing system ABI PRISM 3730 (Applied Biosystems, USA). Community similarities in band patterns were calculated using the Dice coefficients and displayed graphically in the form of a UPGMA (unweighted pair group method with arithmetic mean) dendrogram. The gene sequences obtained were compared with the reference microorganisms available in the GenBank by BLAST search.

FISH analysis

The sludge samples of the suspended sludge and the biofilm biomass were hybridized for 3 h with a 5 ng/μL oligonucleotide probe at 46 °C (Coskuner et al. 2005). Afterwards, the samples were stained with 4′,6′-diamidino-2-phenylindole (DAPI) in the dark for 10 min. The oligonucleotide probes used for FISH and the corresponding hybridization conditions are given in Table 2. All of the probes were synthesized by Dalian TaKaRa (China). The samples were immediately visualized with a fluorescence microscope (OLYMPUS IX71, Japan). A semi-quantitative analysis was conducted using the software Image-Pro Plus 6.0 (Media Cybernetics, USA) (Fu et al. 2010).

Table 2

Oligonucleotide probes for FISH analysis

Probes Sequence (5′–3′) Target References 
EUB338 GCTGCCTCCCGTAGGAGT Total bacteria Amann et al. (1990)  
Nso190 CGATCCCCTGCTTTTCTCC Ammonia-oxidizers Betaproteobacteria Egli et al. (2003)  
Nitspa662 GGAATTCCGCGCTCCTCT Nitrospira sp. Daims et al. (2001)  
Probes Sequence (5′–3′) Target References 
EUB338 GCTGCCTCCCGTAGGAGT Total bacteria Amann et al. (1990)  
Nso190 CGATCCCCTGCTTTTCTCC Ammonia-oxidizers Betaproteobacteria Egli et al. (2003)  
Nitspa662 GGAATTCCGCGCTCCTCT Nitrospira sp. Daims et al. (2001)  

Calculation of nitrogen removal through SND

The amount of nitrogen removal through SND and the SND efficiency (RSND) could be calculated according to Equations (1)–(4), respectively. 
formula
1
 
formula
2
 
formula
3
 
formula
4
where Rden, Rassi and RSND were the efficiencies of nitrogen removal through denitrification, biomass assimilation and SND, respectively; RTN was TN removal efficiency; Ninf and Neff were the amounts of TN in influent and effluent, respectively (g N/d); Aninf and Aneff were the TN in the influent and effluent of anoxic reactor, respectively. R was nitrate liquor recirculation ratio (%); Q was the influent flow (L/d); MLSSsurplus was the surplus sludge concentration (g/L); Vsurplus was the daily discharge amount of surplus sludge (L); fVSS/SS was the ratio of MLVSS to MLSS in surplus sludge, and the value of fVSS/SS was around 0.45; fN/biomass represented the nitrogen ratio of the total biomass (12.39%) (Henze et al. 2000).

RESULTS AND DISCUSSION

COD removal efficiencies of G1 and G2 at different nitrate liquor recirculation ratios

The COD removal efficiencies of the IFAS process (G1) and the control process (G2) under different R are illustrated in Figure 2. Although the influent COD fluctuated between 171.6 and 213.9 mg/L during the whole study, the effluent COD of G1 and G2 at R of 100%, 200% and 300% was found to be in the range of 25–38 mg/L and 23–50 mg/L, respectively, which were below the 50 mg/L discharge standard. It indicated that there was no significant effect on COD removal performance by the alteration of the nitrate liquor recirculation ratio.
Figure 2

COD removal profiles of G1 and G2 at different nitrate liquor recirculation ratios.

Figure 2

COD removal profiles of G1 and G2 at different nitrate liquor recirculation ratios.

Nitrogen removal efficiency of G1 and G2 at different nitrate liquor recirculation ratios

Figure 3 illustrates the NH4+-N removal profile of G1 and G2 operating at different R. As is evident from Figure 3, the effluent NH4+-N concentration of G1 was low compared to that of G2. The possible explanation of this phenomenon could be that the SRT of nitrifiers in the AS system was about 10–15 days (Whang & Park 2006), whereas the SRT in this study was controlled at 7–8 days, which might be not enough for the proliferation of nitrifiers, leading to the G2 being difficult to obtain higher nitrification efficiency. Thus, the nitrification efficiency of G2 was less than 85%, with the effluent NH4+-N concentration in the range of 5.4–11.5 mg/L. However, due to the advantage of the IFAS process (G1), most of the slow-growing nitrifiers could be retained on the biofilm, increasing the number of nitrifiers in the IFAS process (Regmi et al. 2011). Consequently, the higher nitrification efficiency was achieved in G1, which was kept above 97%, and the NH4+-N concentration in the effluent of G1 was below 1.5 mg/L (Figure 3). In addition, it could be found that the effluent NH4+-N concentration of G2 was increased significantly at R of 300% (Figure 3), and the value of NH4+-N concentration was higher than 8.5 mg/L. Jimenez et al. (2011) has reported that the increase of nitrate liquor recirculation ratio would shorten the retention time of the slow-growing nitrifiers in an AS system. Hence, it is difficult to maintain a sufficient number of nitrifiers for nitrification at a higher R, which results in the nitrification efficiency of G2 being decreased at R of 300%.
Figure 3

NH4+-N removal profile of G1 and G2 at different nitrate liquor recirculation ratios.

Figure 3

NH4+-N removal profile of G1 and G2 at different nitrate liquor recirculation ratios.

As shown in Figure 4, TN removal efficiency (RTN) of G1 and G2 was increased from 67.9% and 60.4% at R of 100% to 86.9% and 78.1% at R of 300%, respectively. It is well known that R plays an important role that affects TN removal when the system is operated in A/O operational mode. Increasing the nitrate liquor recirculation flow from the aerobic reactor to the anoxic reactor will bring more nitrates as electron acceptors for denitrification, thereby improving TN removal efficiency. In this study, TN removal efficiencies via denitrification (Rden) of G1 and G2 were increased from 46.4% and 49.0% at R of 100% to 60.7% and 66.9% at R of 300%, respectively (Table 3). Consequently, RTN of G1 and G2 exhibited an incremental trend with the increase of R. It is noteworthy that there was a distinct difference of TN between anoxic effluent (Aneff) and aerobic effluent (Oeff) of G1 (Table 3), indicating that SND took place in the aerobic reactor. Puznava et al. (2000) reported that in the aerobic reactor with the bio-carriers, the anoxic micro-environment could appear in the inner of the biofilm owing to the variation of DO concentration gradients. In this way, nitrifiers could grow in the outer of the biofilm for nitrification, and denitrifiers could grow in the inner of the biofilm for denitrification, respectively, thus facilitating the occurrence of SND. The TN removal efficiency via SND (RSND) of G1 was 15.0–19.0% (Table 3). Table 3 also shows that there is no significant difference in the value of Rden and biomass assimilation (Rassi) of G1 and G2 at the same R. However, RTN of G1 was found to be high compared to that of G2 at the same R. This could be explained by the enhancement of SND that ultimately improved RTN of G1 (IFAS process). As a result, the effluent TN concentration (14.1 mg/L) of G1 at R of 200% could satisfy the discharge standard of China (TN <15 mg/L), whereas the effluent TN concentration of G2 (control process) at R of 300% was only 15.3 mg/L. These results demonstrated that, although the IFAS process is operated at relatively lower R, it could still obtain the higher nitrogen removal efficiency. In the actual project, this advantage of the IFAS process is conducive to reduce the nitrate liquor recirculation flow, which enables decrease of the energy consumption for recirculation, and ultimately saves operational cost.
Table 3

TN removal performance of each reactor in G1 and G2 at different nitrate liquor recirculation ratios

  R (%) Ninf (mg/L) Aninf (mg/L) Aneff (mg/L) Oeff (mg/L) fVSS/SS RTN (%) RDN (%) Rassi (%) RSND (%) 
G1 100 67.0 44.3 28.8 21.5 0.41 67.9 46.3 6.2 15.4 
200 66.7 31.6 20.1 14.1 0.45 78.9 51.7 7.6 17.8 
300 69.8 24.3 16.3 9.1 0.50 86.9 60.7 7.5 18.7 
G2 100 67.0 44.0 27.6 26.5 0.40 60.4 49.0 7.1 4.3 
200 66.7 34.2 20.9 19.1 0.48 71.4 59.6 7.7 4.1 
300 69.8 28.7 17.1 15.3 0.47 78.1 66.9 7.3 3.9 
  R (%) Ninf (mg/L) Aninf (mg/L) Aneff (mg/L) Oeff (mg/L) fVSS/SS RTN (%) RDN (%) Rassi (%) RSND (%) 
G1 100 67.0 44.3 28.8 21.5 0.41 67.9 46.3 6.2 15.4 
200 66.7 31.6 20.1 14.1 0.45 78.9 51.7 7.6 17.8 
300 69.8 24.3 16.3 9.1 0.50 86.9 60.7 7.5 18.7 
G2 100 67.0 44.0 27.6 26.5 0.40 60.4 49.0 7.1 4.3 
200 66.7 34.2 20.9 19.1 0.48 71.4 59.6 7.7 4.1 
300 69.8 28.7 17.1 15.3 0.47 78.1 66.9 7.3 3.9 

R is the nitrate liquor recirculation ratio, Ninf is the influent TN, Aninf is the influent TN of anoxic reactor, Aneff is the effluent TN of anoxic reactor, Oeff is the effluent TN of aerobic reactor, fVSS/SS is the ratio of MLVSS to MLSS, RTN is TN removal efficiency; Rden, Rassi and RSND are the efficiencies of nitrogen removal through denitrification, biomass assimilation and SND, respectively.

Figure 4

TN removal profile of G1 and G2 at different nitrate liquor recirculation ratios.

Figure 4

TN removal profile of G1 and G2 at different nitrate liquor recirculation ratios.

PCR-DGGE analysis

In order to investigate the mechanism of SND from the perspective of molecular biology, PCR-DGGE technology was applied to evaluate the microbial community in the IFAS process (G1) and the control process (G2), and the fingerprints are presented in Figure 5. Hierarchical cluster analysis was used to demonstrate the similarities in the banding profiles of the samples, and the results are presented in the form of UPGMA dendrograms (Figure 5(b)). As shown in Figure 5, the samples (S1 and S2) from the AS of G1 and G2 show a similarity in the community structure; however, the microorganism composition in the sample of the biofilm was different from that in the AS. The specific bands 8–11 were only detected in the sample of the biofilm from G1 (Figure 5(a)). To investigate the composition of the microbial community, these bands were sequenced by sequencing system ABI PRISM 3730. The gene sequences obtained were compared with the reference microorganisms available in the GenBank by BLAST search, and the results are summarized in Table 4.
Table 4

Identity of dominant DGGE bands

Band Closest sequences GenBank number Similarity (%) Phylogenetic division 
Rhizobium sp. BGM3 KF008229 96 α-Proteobacteria 
Nitrosomonas eutropha C91 NEU72670 98 β-Proteobacteria 
Uncultured bacterium clone FB_106. HQ911033 94   
Candidatus Nitrospira defluvii’ DQ059545 99 Nitrospira 
Uncultured Sphingobacteriaceae bacterium clone bf2-101 GU257891 97 Bacteroidetes 
Pseudoxanthomonas sp. P2P1 GU902288 97 γ-Proteobacteria 
Agrobacterium tumefaciens strain LRC6 GQ861464 97 α-Proteobacteria 
Nitrospira sp. clone b37 AJ224046 96 Nitrospira 
Thauera sp. DNT-1 AB066262 94 β-Proteobacteria 
10 Pseudomonas alcaligenes strain 07-1 EU596481 95 γ-Proteobacteria 
11 Uncultured Nitrospira sp. clone H4-R50P HQ198851 97 Nitrospira 
Band Closest sequences GenBank number Similarity (%) Phylogenetic division 
Rhizobium sp. BGM3 KF008229 96 α-Proteobacteria 
Nitrosomonas eutropha C91 NEU72670 98 β-Proteobacteria 
Uncultured bacterium clone FB_106. HQ911033 94   
Candidatus Nitrospira defluvii’ DQ059545 99 Nitrospira 
Uncultured Sphingobacteriaceae bacterium clone bf2-101 GU257891 97 Bacteroidetes 
Pseudoxanthomonas sp. P2P1 GU902288 97 γ-Proteobacteria 
Agrobacterium tumefaciens strain LRC6 GQ861464 97 α-Proteobacteria 
Nitrospira sp. clone b37 AJ224046 96 Nitrospira 
Thauera sp. DNT-1 AB066262 94 β-Proteobacteria 
10 Pseudomonas alcaligenes strain 07-1 EU596481 95 γ-Proteobacteria 
11 Uncultured Nitrospira sp. clone H4-R50P HQ198851 97 Nitrospira 
Figure 5

(a) DGGE fingerprints of microbial communities; (b) cluster analysis based on UPGMA method (S1: AS sample of G2; S2 and S3: samples from the AS and biofilm of G1, respectively).

Figure 5

(a) DGGE fingerprints of microbial communities; (b) cluster analysis based on UPGMA method (S1: AS sample of G2; S2 and S3: samples from the AS and biofilm of G1, respectively).

As shown in Table 4, bands 8 and 11 showed 96 and 97% sequence similarity to Nitrospira sp. clone b37 and uncultured Nitrospira sp. clone H4-R50P. Nitrospira sp., as the typical nitrifiers, might oxidize nitrite to nitrate (Wagner and Loy 2002; Bernet et al. 2004), which were only detected in the biofilm sample. This fact indicated that the nitrifying microorganisms were enriched on the biofilm of G1. Additionally, one clone showed 94% sequence similarity to Thauera sp. DNT-1 (band 9). Yoshifumi et al. (2004) has verified that Thauera sp. was able to reduce nitrate in anoxic condition. Another clone affiliated with γ-proteobacteria showed 95% sequence similarities to Pseudomonas alcaligenes (band 10). Bothe et al. (2000) has reported that the cluster group of Pseudomonas sp. could play an important role for denitrification in natural environments. These denitrifying microorganisms co-existed with the nitrifiers in one microbial community, which might be the main reason that SND occurred in the aerobic reactor of the IFAS process.

FISH analysis

To investigate the relative quantity variations of functional bacteria at different nitrate liquor recirculation ratios, the relative quantities of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in G1 and G2 were evaluated by FISH technology, and the results are summarized in Table 5.

Table 5

Relative quantities of AOB and NOB in G1 and G2 at the different R

S1
 
S2
 
S3
 
AOB (%) NOB (%) AOB (%) NOB (%) AOB (%) NOB (%) 
100% 11.6 ± 0.5 27.6 ± 1.0 10.5 ± 0.6 28.7 ± 0.9 9.4 ± 0.5 24.9 ± 0.9 
200% 11.0 ± 0.7 26.3 ± 1.3 10.8 ± 0.5 27.8 ± 1.1 9.8 ± 0.4 25.4 ± 1.1 
300% 6.1 ± 0.6 18.7 ± 0.8 6.3 ± 0.5 19.2 ± 1.3 9.5 ± 0.5 24.1 ± 1.3 
S1
 
S2
 
S3
 
AOB (%) NOB (%) AOB (%) NOB (%) AOB (%) NOB (%) 
100% 11.6 ± 0.5 27.6 ± 1.0 10.5 ± 0.6 28.7 ± 0.9 9.4 ± 0.5 24.9 ± 0.9 
200% 11.0 ± 0.7 26.3 ± 1.3 10.8 ± 0.5 27.8 ± 1.1 9.8 ± 0.4 25.4 ± 1.1 
300% 6.1 ± 0.6 18.7 ± 0.8 6.3 ± 0.5 19.2 ± 1.3 9.5 ± 0.5 24.1 ± 1.3 

S1 and S2 are the samples from AS of G2 and G1; S3 is the sample from the biofilm of G1.

The relative quantities of AOB and NOB in the samples (S1 and S2) from AS of G2 and G1 showed no obvious difference. For instance, the relative quantities of AOB in S1 and S2 at R of 100% were approximately 11.6 and 10.5%, and were about 27.6% and 28.7% for NOB, respectively. However, the relative quantities of AOB and NOB in the biofilm sample of G1 (S3) were 9.4 and 24.9%. Hence, the number of nitrifiers on the biofilm increased the total amount of nitrifiers in the aerobic reactor of G1. This might be the reason for the stable and higher nitrification efficiency of G1 compared to G2.

In addition, the relative quantities of AOB and NOB in the AS sample (S1) of G2 were decreased from 11.6% and 27.6% at R of 100% to 6.1% and 18.7% at R of 300%, respectively. It is possible that the flow velocity of the system was increased with the increase of R, which shortened the biomass retention time in the system. As a result, a large number of nitrifiers in the AS might be washed out of the system, leading to the decrease of NH4+-N removal efficiency of G2 at R of 300%. Although the relative quantities of AOB and NOB in the AS of G1 also presented a declining trend with the increase of R, the relative quantities of AOB and NOB in the biofilm sample (S3) of G1 were stabilized in the range of 9.4–9.8% and 24.1–25.4%, respectively, at each R, which guaranteed that G1 (IFAS process) was operated steadily, and maintained its nitrification efficiency above 97% during the whole experiment.

CONCLUSIONS

The main conclusions of the study are summarized as follows.

  1. The effluent NH4+-N and TN of the IFAS process at R of 200% were less than 1.5 and 14.5 mg/L, satisfying the Chinese discharge standard (NH4+-N < 5 mg/L; TN < 15 mg/L), respectively. However, the effluent NH4+-N and TN of G2 at R of 300% were higher than 8.5 mg/L and 15.3 mg/L, respectively. These results suggested that the IFAS process was able to achieve better nitrogen removal efficiency at lower R.

  2. PCR-DGGE results showed that nitrifiers and denitrifiers co-existed in one microbial community, facilitating the occurrence of SND in the aerobic reactor of the IFAS process, and the contribution of SND to TN removal efficiency was 15–19%.

  3. FISH results demonstrated that the relative quantity of nitrifiers on the biofilm increased the total amount of nitrifiers in the IFAS process, which resulted in the number of nitrifiers in the IFAS process being higher than in the control process. In conclusion, a steady and higher nitrification efficiency was achieved in the IFAS process.

  4. The results from this study indicated that the IFAS process was an effective BNR process for improving nitrogen removal efficiency and reducing nitrate liquor recirculation flow. Applying the IFAS process in a practice project will be conducive to decreasing the electricity consumption and saving operational costs.

ACKNOWLEDGEMENT

The research was supported by the National Science and Technology Major Project Water Pollution Control and Treatment (No. 2012ZX07202006).

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