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N ratio on nitrogen removal and microbial communities of CANON process in membrane bioreactors
Bioresource Technology 189 (2015) 302–308
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Effect of COD/N ratio on nitrogen removal and microbial communities of CANON process in membrane bioreactors Xiaojing Zhang ⇑, Hongzhong Zhang, Changming Ye, Mingbao Wei, Jingjing Du Henan Collaborative Innovation Center of Environmental Pollution Control and Ecological Restoration, School of Material and Chemical Engineering, Zhengzhou University of Light Industry, Zhengzhou 450001, China
h i g h l i g h t s COD/N in low ratio could improve nitrogen removal of CANON. The suppressing threshold of COD/N ratio on CANON was 1.7. Biodiversity of AOB and AAOB both decreased with COD increasing. Denitrifiers and AAOB showed different relationship under different COD/N ratio. Strategies for treating sewage with different COD/N ratio were proposed.
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Article history: Received 20 February 2015 Received in revised form 1 April 2015 Accepted 2 April 2015 Available online 8 April 2015 Keywords: CANON COD/N Membrane bioreactor (MBR) Microbial community Denitrification
a b s t r a c t In this study, the effect of COD/N ratio on completely autotrophic nitrogen removal over nitrite (CANON) process was investigated in five identical membrane bioreactors. The five reactors were simultaneously seeded for 1 L CANON sludge and be operated for more than two months under same conditions, with influent COD/N ratio of 0, 0.5, 1, 2 and 4, respectively. DGGE was used to analyze the microbial communities of aerobic ammonia-oxidizing bacteria (AOB) and anaerobic ammonia-oxidizing bacteria (AAOB) in five reactors. Results revealed the harmonious work of CANON and denitrification with low COD concentration, whereas too high COD concentration suppressed both AOB and AAOB. AOB and AAOB biodiversity both decreased with COD increasing, which then led to worse nitrogen removal. The suppressing threshold of COD/N ratio for CANON was 1.7. CANON was feasible for treating low COD/N sewage, while the high sewage should be converted by anaerobic biogas producing process in advance. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Completely autotrophic nitrogen removal over nitrite (CANON) process has been developed in recent years as the most efficient and economical method to remove ammonia from wastewater (Sliekers et al., 2002; Third et al., 2001). With the development of anaerobic treatment process, most organic compounds in wastewater are converted to biogas without consumption of ammonia (Kartal et al., 2010), and CANON process was suggested to be an alternative process to treat the effluent with high concentration of ammonia (Liang et al., 2014). Recent researches indicated that CANON process is very sensitive to environmental conditions such as temperature, pH, salinity and presence of inhibitors including nitrite, free ammonia and organic material (OM, expressed as COD in this study) (Gilbert et al., 2014; Kimura et al., 2011; Liu ⇑ Corresponding author. Tel.: +86 15237133016. E-mail address: [email protected] (X. Zhang). http://dx.doi.org/10.1016/j.biortech.2015.04.006 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
et al., 2008; Ni et al., 2012; Tang et al., 2010a). Specially, one factor could not be ignored was the presence of OM since CANON process was a completely autotrophic reaction without organic carbon consumption. The functional organisms including aerobic ammonia-oxidizing bacteria (AOB) and anaerobic ammonia-oxidizing bacteria (AAOB) both utilize CO2 as the source of carbon for ammonia oxidation. Generally, wastewaters containing ammonia are not free from OM, and the anaerobic process for biogas production could not totally remove the organic carbon, such as anaerobic digestion process. Previous studies have reported the adverse effect of the presence of OM on the growth of both AOB and AAOB. On one hand, the presence of COD would result in the survival of heterotrophic bacteria, which would compete DO with AOB (Ji et al., 2012; Zhi and Ji, 2014). Furthermore, previous research provided evidence that the organic carbon source affects the make-up of AOB community in mixed cultures (Racz et al., 2010). Researchers are unanimous when it comes to the inhibitory effect
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of high COD concentration on AAOB. As much, AAOB are not able to compete with denitrifying bacteria (Jia et al., 2012; Ni et al., 2012). Further study revealed that both COD concentrations and COD to N ratio (COD/N) affect the performance of AAOB (Chamchoi et al., 2008; Hao and Loosdrecht, 2004; Molinuevo et al., 2009). Considering the adverse effect on both AOB and AAOB, the COD and COD/N ratio would also significantly affect the nitrogen removal performance by CANON. Only one or two studies focused on the relative low COD/N ratio influencing CANON so far. Chen (Chen et al., 2009) observed a significant reduction of the nitrogen removal efficiency (NRE) from 79% to below 52% when influent COD/N ratio was increased from 0.5 to 0.75, while Lackner and Horn (Lackner and Horn, 2013) reached 80–85% NRE with an influent COD/N ratio of 1 in a sequencing batch reactor. The available studies have not mentioned the performance of CANON process under a higher COD/N ratio, and it could not be conclusive to determine the COD/N ratio effects from the limited studies. In addition, since the reactor performance relies on the functional bacteria (Zhi et al., 2014), it is important to know the responses of microbial communities to the presence of different COD/N ratio. However, to the best of our knowledge, limited work has been carried out to explore the performance and microbial communities of CANON with different COD/N level. The main goal of this study was to investigate the effect of influent COD/N ratio on the nitrogen removal performance and microbial communities of CANON process. Membrane bioreactor (MBR) was chosen to be the experimental equipment in this study due to its effectiveness for CANON process (Zhang et al., 2013b). Five identical MBRs with same seed sludge were simultaneously started-up, and fed with different COD/N levels. The nitrogen removal and denitrification ratio of the five MBRs were compared, and the microbial communities of each reactor were analyzed using DGGE and clone-sequencing. 2. Methods 2.1. Reactors and seed sludge Five identical lab-scale MBRs (effective volume: 1 L, diameter: 100 mm, height: 200 mm, Supplementary material) were adopted to this study, named R0, R1, R2, R3 and R4, respectively. Each reactor was installed with a hollow fiber membrane module (material: PVDF; pore size: 0.1 lm; effective area: 0.05 m2; water permeability: 9 L h1). For each reactor, it was entirely placed in a water bath to ensure a constant reaction temperature (25 °C). Effluent was continuously extracted from the reactor using a peristaltic pump, while the synthetic wastewater was fed into the reactor. Oxygen was supplied continuously from an aeration ring locating at the bottom of the reactor which was connected to an air blower, and the aeration was controlled by the gas flow meter. A mixer was set in each reactor to assure homogeneous condition. A running MBR with identical setup was adopted as the seeding reactor, it showed an excellent nitrogen removal ability with the NRR of 0.6–0.7 kg m3 d1 before seeding. The five experimental MBRs were simultaneously inoculated with 1 L CANON sludge from the running MBR. The mixed liquor suspended solid and mixed liquor volatile suspended solid of the seed sludge were 4.1 and 3.1 g L1, respectively.
(0.136), MgSO47H2O (0.3) and 1 mL L1 of trace element solution (van de Graaf et al., 1996). Influent ammonia for the five reactors were 200 mg L1 during the whole experiment, the only difference was the COD concentration in influent. As a logical choice of organic substrate, glucose was considered as the harmless one to AOB (Ni et al., 2012). So glucose was added to the influent of R1, R2, R3 and R4, to adjust the influent COD concentration to 100, 200, 400 and 800 mg L1, with COD/N ratios of 0.5, 1, 2 and 4, respectively. For comparison, no OM was added to R0. The five reactors were running in a continuous mode at a HRT of 6.4 h. The pH in each reactor was all around 7.8 without adjustment. It was demonstrated that AAOB had a long growth cycle and low growth rate (l = 0.0027 h1, doubling time = 10.6 d) (Strous et al., 1998; Zhang et al., 2013a), so this study were conducted for more than two months. Sludge samples were obtained from each reactor on day 61, for DGGE analysis. Since previous studies (Zhang et al., 2013a,b) showed the prominent performance when DO was 0.15 mg L1, and the seeding reactor was also operated under this value for a long time, DO was controlled as 0.15 mg L1 in the five experimental MBRs, to eliminate the oxygen impact on the reactor performance. Other operational conditions of the reactors are summarized as Table 1. 2.3. Analytical methods According to the methods mentioned by APHA, concentrations of NH+4-N and NO 2 -N were daily measured using visible spectrophotometer (Shanghai Yoke Instrument Co., 722S, China) with different colorimetric methods. NO 3 -N was analyzed using ultraviolet spectrophotometric (Shanghai Yoke Instrument Co., UV755B, China). The temperature, DO and pH were detected using online instruments with specific probes (WTW, Oxi1296TriOxmatic700-7 and pH296-Sensolyt 650-7, Germany). COD was detected by digestion instrument (Lianhua Technology Co., 5B-6CV8.0, China). Denitrification ratio was defined as the ratio of nitrogen removal by denitrification to the total nitrogen removal. The nitrogen removal by denitrification was equal to the decreased amount of NO 3 -N when compared to the amount generated by CANON reaction. Denitrification ratio was calculated as Eq. (1). þ NH4 -N inf NHþ4 -N eff 0:11 NO3 -N eff Denitrification ratioð%Þ ¼ ½TNinf ½TNeff ð1Þ 2.4. DNA extraction, PCR–DGGE, cloning and sequencing Since DGGE was suitable for determining the specie difference and the biodiversity variation of the same kind of bacteria, it was chosen to characterize the microbial communities of AOB and AAOB under different influent COD/N ratio in this study. Some mixed liquor was respectively collected from the five reactors on day 61. DNA was extracted using a bacterial genomic mini extraction kit (Sangon, China) according to the manufacturer’s manual and was detected by 0.8% (w/V) agarose gel electrophoresis.
Table 1 Operational conditions of the five experimental reactors.
2.2. Feed and experimental setup
Reactor
CODinf (mg L1)
CODeff (mg L1)
HRT (h)
Aeration rate (L min1)
DO (mg L1)
pH –
The effect of COD/N on CANON process was tested with synthetic wastewater. To avoid influent impact, the influent for the five experimental reactors were totally same as the seeding MBR. The main synthetic wastewater used in this study contained (in g L1): (NH4)2SO4 (0.942), NaHCO3 (3.357), KH2PO4 (0.136), CaCl2
R0 R1 R2 R3 R4
0 97.9 198.2 399.2 800.2
0 13.72 32.56 74.51 252.4
6.4 6.4 6.4 6.4 6.4
0.2 0.25 0.3 0.35 0.4
0.15 0.15 0.15 0.15 0.15
7.84 7.80 7.78 7.86 7.88
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Using the extracted DNA as template, the 16S rDNA fragments of AOB was amplified for DGGE. Primers CTO 189fA/B and CTO 189fC of a 2:1 ratio, together with the reverse primer CTO654r were used for the first round PCR of AOB. Then the PCR products were used as templates for a second round PCR using universal primer set F338 (with GC-clamp)/R518. Primers Pla46F/630R and Amx368f (with GC-clamp)/Amx820r were respectively used for the first and second round PCR of Planctomycetales-like AAOB. The primes and PCR conditions used in this study was same as previously reported (Liang et al., 2014). The amplified DNA fragments were separated on 8% polyacrylamide gels with a 30–60% linear gradient of denaturant, for DGGE. The gel was conducted at 60 °C, 120 V for 5 h (AOB) or 7 h (AAOB) on Dcode Universal Mutation Detection System (Bio-Rad). After electrophoresis, the gel was stained using silver-staining method (Bio-Rad) (Bassam et al., 1991) followed by taking photos on Gel Doc XR 192 system. Diversity statistics (Shannon–Wiener diversity index (H) and Simpson’s diversity index (D)) were calculated from the DGGE profile by using the number and intensity of bands in each DGGE profile, as previously reported (Zhang et al., 2013a). Prominent bands in gel were excised and dissolved in 50 lL 1 TE buffer at 4 °C overnight. 1 lL TE solution as template was reamplified with the primer pairs F338/R518 and Amx368f/Amx820r using the same methods as described before, then the PCR products were cloned by pMD19-T plasmid vector system (TaKaRa, Japan). Sequencing was performed on an ABI 3730 DNA sequencer by a commercial service (Sangon, China). All sequences were compared with the reference microorganisms available in Genbank by BLAST tool and submitted to GenBank database. 3. Results and discussions 3.1. Reactor performance without organic carbon addition The control experiment was carried out at a HRT of 6.4 h without addition of COD in R0. The operational condition was totally same as the seeding reactor. The performance of R0 after seeded was depicted in Fig. 1. In the initial days after seeded, both AOB and AAOB showed poor bioactivity, leading to a lower NRR. Then the organisms seemed to be adapted to the new reactor with the operation, NRR went up gradually and then achieved to the steady state after several days. R0 was run for two months with high substrate removal, the average effluent ammonia was 15 mg L1 during the steady state, with the ammonia removal efficiency of 92%. Due to the nitrate production by CANON reaction, the TN removal efficiency (NRE) was only 80.2%, and NRR reached 0.61 kg m3 d1. Moreover, no nitrite accumulated in the reactor, indicating the excellent bioactivity of AAOB, which could timely consume the nitrite produced by AOB. The ratio of nitrate production to ammonia consumption (Dnitrate/Dammonia) was constantly around 0.11, which is the theoretical reaction ratio of CANON process (Sliekers et al., 2002). This result revealed that no excess nitrate was produced by NOB, indicating the effective inhibition or totally washout of NOB in the seeding sludge. The stable NRR was similar to the seeding reactor, suggesting no adverse effect of the new reactor. 3.2. Nitrogen removal with different influent COD/N ratio The nitrogen removal performance of the four reactors with COD feeding were shown in Fig. 2. With an influent COD/N ratio of 0.5, R1 showed a similar performance as R0 without COD feeding, indicating the slight inhibition on AOB and AAOB by COD in this COD/N ratio. Some previous reports suggested that COD would
inhibit AOB bioactivity since the oxygen competition with heterotrophic bacteria, Zhu and Chen (Zhu and Chen, 2001) observed a 70% reduction of AOB activity in a nitrification reactor when influent COD/N ratio was between 1.8 and 3.5. However, AOB was not restricted in this reactor, which was consistent with the previous study that the AOB bioactivity went up with increasing influent COD/N ratio (Jenni et al., 2014). The possible reason was that the COD concentration was too low to perform inhibition on AOB. Another possible reason was the oxygen supply was sufficient, the resulted DO was the same as the seeding reactor and R0 without COD feeding. After operation for 60 days, no nitrite or excess nitrate accumulated in the reactor, indicating the prominent bioactivity of both AOB and AAOB. Moreover, nitrate production in effluent showed a slight reduction about 10 mg L1 than the theoretical value, this perhaps was due to the consumption by denitrification, which was successfully induced due to the COD feeding. Finally NRR achieved to 0.63 kg m3 d1, which was a little higher than that of R0. This result demonstrated that a small supply of organic substrate could induce denitrification, which could further improve the nitrogen removal. For R2, COD addition in influent was 200 mg L1, with a COD/N ratio of 1. As shown in Fig. 2, more nitrite accumulated in the reactor initially, indicating the more intensive inhibition on AAOB by COD. The TN removal ability of this reactor began to increase from day 15, which was slower than R1, further indicating the inhibition on microorganisms by COD feeding. After 30 days’ operation, nitrite accumulation disappeared, and the NRR reached a stable range. Moreover, denitrification showed an obvious enhancement when compared to that of R1, the denitrification ratio increased to 10.1% (Table 2). No nitrite or nitrate was detected in the effluent during the last several days, finally NRR achieved to 0.69 kg m3 d1, which was the highest one in the five experimental reactors. Moreover, NRE reached 92.3%, which was higher than the theoretical CANON removal efficiency. The nitrate produced by CANON was totally consumed by denitrification, as a result, no nitrite or nitrate accumulated in the effluent, with the effluent ammonia around 13 mg L1. This result demonstrated the cooperative work of CANON and denitrification in this reactor with feeding COD/N ratio of 1. In R3 with the feeding COD/N ratio of 2, it showed a worse nitrogen removal than R2. Nitrite accumulated in the reactor during the whole experiment, indicating the continuous inhibition on AAOB. On the other hand, the limitation of AAOB bioactivity led to the nitrite accumulation, and the nitrite accumulation would suppress AAOB bioactivity in return (Bettazzi et al., 2010). Effluent nitrate reduced to around 1.6 mg L1. Since the CANON reaction would led to nitrate production about 20 mg L1 in this reactor, the decreased nitrate was undoubtedly consumed by denitrifiers, indicating the existence of denitrification. However, NRR finally achieved to only 0.57 kg m3 d1, which was lower than R0 without COD feeding. The possible reason of this result was the inhibition on AAOB by COD and nitrite or the oxygen competition of heterotrophic bacteria with AOB. Although the denitrification ratio was the highest, the limitation of autotrophic nitrogen removal resulted in the poor NRR. For R4 with COD/N ratio of 4, it showed the worst nitrogen removal performance. About 37 mg L1 nitrite accumulated in the reactor during the whole experiment, and effluent ammonia was more than 45 mg L1. Finally R4 achieved a NRR of 0.43 kg m3 d1 with the NRE of 57.4%. The comparisons between the five reactors showed that when influent COD/N was low, the COD feeding could elevate the nitrogen removal, no nitrite or nitrate accumulated in the reactor, AOB and AAOB both showed profitable bioactivity, CANON process and denitrification performed a cooperative work. However, when influent COD/N was too high, CANON was inhibited by the COD feeding, which limited
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0.5
0.8
0.4
Eff. nitrate
0.3
-3
0.6 100 0.4
0.2
Δ nitrate/ Δ ammonia
Eff. nitrite
-1
150
NRR (kg m d )
Inf. ammonia Eff. ammonia NRR Δ nitrate/ Δ ammonia
-1
Nitrogen concentration (mg L )
200
50
0 0
10
20
30
40
50
0.2
0.1
0.0
0.0
60
Time (d) Fig. 1. Nitrogen removal performance of the reactor without COD feeding (R0).
Inf. ammonia
Eff. ammonia
Eff. nitrite
Eff. nitrate
NRR
1.0
1.0 200 180
140 120 100 80 60
nitrogen concentration (mg L-1)
0.8
0.6
0.4
0.2
40 20 0
10
20
30
40
50
60
Time (d)
1.0
120 100 80 60
0.6
0.4
0.2
40 20
0.0
0 10
20
30
40
50
60
Time (d)
0.4
80 60
0.2
40 20
0.0 10
20
30
40
50
60 1.0
200
nitrogen concentration (mg L-1)
NRR (kg m-3 d-1)
nitrogen concentration (mg L-1)
0.8
140
0
100
180
R3:C/N=2
160
0.6
120
Time (d)
200 180
0.8
140
0 0
0.0
0
R2:C/N=1
160
NRR (kg m-3 d-1)
R1:C/N=0.5
160
NRR (kg m-3 d-1)
nitrogen concentration (mg L-1)
180
R4:C/N=4
0.8
160 140
0.6
120 100
0.4
80 60
NRR (kg m-3 d-1)
200
0.2
40 20 0 0
0.0 10
20
30
40
50
60
Time (d)
Fig. 2. Nitrogen removal performances of reactors with different influent COD/N ratio.
Table 2 Nitrogen removal and denitrification ratio in steady state (last 10 days) of the five CANON reactors. Reactor 3
1
NRR (kg m d ) Denitrification ratio (%)
R0
R1
R2
R3
R4
0.61 0
0.63 4.87
0.69 10.1
0.57 11.17
0.43 13.18
the nitrogen removal. Denitrifiers and AAOB showed different competitions under different COD/N ratio. Thus, CANON process was feasible for treating sewage with low COD/N ratio.
3.3. Denitrification ratio and threshold COD/N ratio for CANON process The effect of organic loading on denitrification ratio and TN removal in steady state of the five reactors was illustrated in Fig. 3. In order to quantify the effect of organic loading on nitrogen removal more precisely, a COD/N threshold ratio for CANON inhibitory was defined when TN removal dropped to lower than the R0 without organic feeding. As shown in Fig. 3, the denitrification ratio went up with the COD increasing, indicating the COD contribution to denitrification. The threshold ratio was 1.7,
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14
Denitrification ratio
NRR
0.70
12
0.60
8 6
0.55
4 0.50
NRR (kg m-3 d-1)
Denitrification ratio (%)
0.65 10
2 0.45
0
0.40
-2 0
1
2
3
4
C/N ratio Fig. 3. Effect of COD/N ratio on the denitrification ratio and NRR.
corresponding to 340 mg L1 COD concentration. Chamchoi (Chamchoi et al., 2008) found the complete suppression of AAOB activity at COD concentration over 300 mg L1 or COD/N ratio over 2.0, which was similar to the result about the CANON process in the present study. Thus, the suppression on CANON process perhaps was depended on the suppression on AAOB. As shown in Table 2, both CANON and denitrification processes contributed to TN removal in the four reactors with COD feeding. Though high COD concentrations resulted in lower TN removal, a low concentration of COD could lead to higher TN removal (Fig. 3). Under the low COD strength, the fluctuation of AOB and AAOB activity was limited while the produced nitrate was reduced to nitrogen gas, resulting in higher TN removal (R1 and R2). These findings indicate that although COD feeding reduced AAOB activity, denitrification enhanced nitrite and nitrate removal when low concentration of COD was presented in the wastewater.
3.4. Microbial community of AOB and AAOB Sludge sample was obtained from the five reactors on day 61 for DGGE analysis, the results were shown in Fig. 4. As shown in Fig. 4, there were seven bands in the AOB profile of R0. And in R1 with COD/N ratio of 0.5, band 8 disappeared. This perhaps was due to the COD inhibition on AOB. Even though the reactor showed a
higher NRR, the biodiversity of AOB decreased. And the denitrification enhanced the TN removal in R1. Moreover, band 8 was not bright in lane 0, indicating the no predominant role, which then disappeared with COD feeding. The AOB profile of R2 showed similar result as R1, which was consistent with the reactor performance that the two reactors both showed nitrogen removal increase when compared to R0. This result also proved that the disappearance of band 8 was due to the COD adoption. Then in R3, band 1 and 5 declined while a new band (band 3) emerged, indicating the significant shift while COD concentration or COD/N ratio was higher than the threshold. Correspondingly, the nitrogen removal showed a significant decrease when compared to the three reactors with lower COD feeding. And in R4, one more band (band 6) declined, indicating the biodiversity decreasing due to the high COD feeding. The Shannon–Wiener diversity index and Simpson’s diversity index of AOB in Table 3 also showed the consistent result, H and D both decreased with the COD increasing, and R4 presented the lowest biodiversity. DNA sequencing results of the eight bands were shown in Table 4. The four bands (band 1, 5, 6 and 8) which declined in the four reactors with COD feeding were all related to Nitrosomonas sp., this result suggested that the COD feeding performed significant suppression on this specie. In addition, the new band emerged in R3 and R4 with high COD feeding showed a 99% similarity to Nitrosomonas eutropha. N. eutropha was regarded as owning denitrification ability (Kumar and Lin, 2010), and R3 and R4 both showed remarkable denitrification ability, so its emergence in the two reactors could be explained. What’s more, band 2 and 4 that both affiliating with Nitrosomonas europaea existed in all the five reactors, indicating the slight impact of COD feeding on this specie. For AAOB, COD feeding performed slight effect on the community of the five reactors. There were four bands in R0, R1 and R2, and the biodiversity index H and D both showed similar value, which indicated the slight effect of COD at low concentration on the competition of AAOB. The four bands were related to three species: Candidatus Kuenenia stuttgartiensis, Candidatus Brocadia fulgida and Candidatus Brocadia sp., indicating the complex community compositions in these reactors. Ca. K. stuttgartiensis predominated in these reactors, which was consistent with the previous study in MBR (Zhang et al., 2013a). Then in R3 and R4, band 9 that related to Ca. K. stuttgartiensis declined, which was consistent with the previous study in a biofilter packed with volcanic rock with COD feeding (Liang et al., 2014). The absence of band 9 perhaps was due to the inhibitory effect of high COD concentration, since the high COD feeding would significantly limit the AAOB bioactivity (Chamchoi et al., 2008; Daverey et al., 2013; Guven et al., 2005; Molinuevo et al., 2009). The DGGE result of AAOB was consistent with the reactor performances. In R3 and R4, nitrite accumulation were observed during the whole experiment, although denitrification were active, the total nitrogen removal decreased. All of these results indicated the insufficient bioactivity of AAOB, which then deteriorate the CANON performance. Although AAOB was first identified in a denitrification reactor (Mulder et al., 1995), it would be significantly suppressed by
Table 3 Shannon–Wiener diversity index and Simpson’s diversity index of AOB and AAOB.
Fig. 4. DGGE results of AOB and AAOB in the five experimental reactors (0: COD/ N = 0; I: COD/N = 0.5; II: COD/N = 1; III: COD/N = 2; IV: COD/N = 4).
COD/N ratio
AOB
AAOB
Number of bands
H
D
Number of bands
H
D
COD/N = 0 COD/N = 0.5 COD/N = 1 COD/N = 2 COD/N = 4
7 6 6 5 4
1.942 1.786 1.604 1.605 1.381
0.856 0.831 0.798 0.798 0.747
4 4 4 3 3
1.384 1.385 1.381 1.092 1.092
0.749 0.749 0.747 0.662 0.662
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X. Zhang et al. / Bioresource Technology 189 (2015) 302–308 Table 4 DGGE results AOB and AAOB in the five reactors. Band
Closest relative
Identity (%)
Access. No.
Phylum (classifier)
Access. No.
1 2 3 4 5 6 7 8 9 10 11 12
Nitrosomonas sp. Nitrosomonas europaea Nitrosomonas eutropha Nitrosomonas europaea Nitrosomonas sp. Nitrosomonas sp. Nitrosomonas sp. Nitrosomonas sp. Candidatus Kuenenia stuttgartiensis Candidatus Brocadia fulgida Candidatus Brocadia sp. Candidatus Kuenenia stuttgartiensis
99 99 99 99 99 99 99 98 98 98 99 99
HF678378 NR_074774 JX545090 HE862405 AJ621029 HF678378 AF272415 HF678378 KF429801 JX243380 AM285341 CT573071
b-Proteobacteria b-Proteobacteria b-Proteobacteria b-Proteobacteria b-Proteobacteria b-Proteobacteria b-Proteobacteria b-Proteobacteria Planctomycetia Planctomycetia Planctomycetia Planctomycetia
KM649686 KM649687 KM649689 KM649690 KM649691 KM649692 KM649693 KM649695 KP663624 KP663625 KP663626 KP663627
denitrifying communities under high OM content due to the weaker competition for nitrite (electron acceptor) and living space (Tang et al., 2010b).The high COD feeding led to the bioactivity and biodiversity decreasing of both AOB and AAOB, which then further resulted in the lower NRR in R3 and R4. In addition, band 10 that existed in all the five reactors showed a 98% similarity to Ca. B. fulgida, which could consume organic substrate (Winkler et al., 2012). This result was consistent with the previous report (Jenni et al., 2014) that the addition of glucose performed no influence on the dominant role of Ca. B. fulgida in a sequencing batch reactor. Thus, Ca. B. fulgida could be cultivated to treat the sewage with COD. 3.5. Strategies for treating wastewater with different COD/N ratio Combining the nitrogen removal performances and microbial communities of the five reactors, CANON process was strongly suggested to treat sewage with low COD/N ratio. The presence of OM with low concentration could induce denitrification, which could reduce the produced nitrate and then improve the total nitrogen removal. And since the COD/N ratio was relative low, it could not suppress the CANON process, the autotrophic nitrogen removal by AOB and AAOB and heterotrophic nitrogen removal by denitrifiers could cooperatively work with each other. Consequently, the sewage in the side-stream with low COD/N ratio could be directly treated by CANON process, such as the nitrogen fertilizer wastewater. For sewage with high COD/N ratio such as domestic sewage in main-stream, the COD/N ratio was always about 3–4. The denitrifiers would compete with AAOB and the high OM would inhibit both the bioactivity of AOB and AAOB. As a result, the autotrophic nitrogen removal would be significantly suppressed during a longterm operation. So the anaerobic treatment coupled with the subsequent CANON process would be a better alternative technology for treating sewage with high COD. In this two stages process, COD was converted to biogas without ammonia consumption in the anaerobic treatment, and then the nitrogen was removed in the CANON stage. The biogas production would be used as the power energy in the process. It could on one hand convert the redundant COD to biogas for energy reuse, which was feasible for the demand of energy autarkic wastewater treatment plants (WWTP), and on the other hand control the effluent to be with a COD/N ratio lower than 1.7. As a result, it could achieve the simultaneous removal of COD and nitrogen efficiently and economically. Moreover, from the results by DGGE, organic feeding performed slight impact on N. eutropha of AOB and Ca. B. fulgida of AAOB. So if these two species could be cultivated specifically in one single system, it could be a possible alternative to treat the sewage with high COD/N ratio.
Whilst the application of autotrophic nitrogen removal technologies in the side-stream is at present state of the art, the feasibility of this energy-efficient process at mainstream conditions is still under development. The results of this study were necessary and useful for the conclusive information about the COD/N effect on CANON process, which pointed a feasible way for the application of CANON process in mainstream. 4. Conclusions Denitrification could be successfully induced by organic feeding, and COD/N ratio in low range was favorable for nitrogen removal. The high COD/N ratio performed suppression on the bioactivity and biodiversity of both AOB and AAOB, which then led to the decreasing of nitrogen removal. The suppressing threshold of COD/N ratio on CANON was 1.7. For sewage with low COD/N ratio, it could be directly treated by CANON process. For sewage with high COD, it should be pretreated with anaerobic biogas production process. A cultivated system with enrichment of N. eutropha and Ca. B. fulgida could be a possible alternative. Acknowledgement This work was supported by Ph.D. Programme of Zhengzhou University of Light Industry – China (2014BSJJ055). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.04. 006. References Bassam, B.J., Caetano-Anollés, G., M. Gresshoff, P., 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem. 19 (6), 80–83. Bettazzi, E., Caffaz, S., Vannini, C., Lubello, C., 2010. Nitrite inhibition and intermediates effects on Anammox bacteria: a batch–scale experimental study. Process Biochem. 45, 573–580. Chamchoi, N., Nitisoravut, S., Schmidt, J.E., 2008. Inactivation of ANAMMOX communities under concurrent operation of anaerobic ammonium oxidation (ANAMMOX) and denitrification. Bioresour. Technol. 99 (9), 3331–3336. Chen, H., Liu, S., Yang, F., Xue, Y., Wang, T., 2009. The development of simultaneous partial nitrification, ANAMMOX and denitrification (SNAD) process in a single reactor for nitrogen removal. Bioresour. Technol. 100 (4), 1548–1554. Daverey, A., Hung, N.T., Dutta, K., Lin, J.G., 2013. Ambient temperature SNAD process treating anaerobic digester liquor of swine wastewater. Bioresour. Technol. 141, 191–198. Gilbert, E.M., Agrawal, S., Karst, S.M., Horn, H., Nielsen, P.H., Lackner, S., 2014. Low temperature partial nitritation/anammox in a moving bed biofilm reactor treating low strength wastewater. Environ. Sci. Technol. 48 (15), 8784–8792. Guven, D., Dapena, A., Kartal, B., Schmid, M.C., Maas, B., van de Pas-Schoonen, K., Sozen, S., Mendez, R., Op den Camp, H.J.M., Jetten, M.S.M., Strous, M., Schmidt, I.,
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Racz, L., Datta, T., Goel, R., 2010. Effect of organic carbon on ammonia oxidizing bacteria in a mixed culture. Bioresour. Technol. 101 (16), 6454–6460. Sliekers, A.O., Derwort, N., Campos-Gomez, J.L., Strous, M., Kuenen, J.G., Jetten, M.S.M., 2002. Completely autotrophic nitrogen removal over nitrite in one single reactor. Water Res. 36 (10), 2475–2482. Strous, M., Heijnen, J.J., Kuenen, J.G., Jetten, M.S.M., 1998. The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammoniumoxidizing microorganisms. Appl. Environ. Microbiol. 50 (5), 589–596. Tang, C.J., Zheng, P., Hu, B.L., Chen, J.W., Wang, C.H., 2010a. Influence of substrates on nitrogen removal performance and microbiology of anaerobic ammonium oxidation by operating two UASB reactors fed with different substrate levels. J. Hazard. Mater. 181 (1–3), 19–26. Tang, C.J., Zheng, P., Wang, C.H., Mahmood, Q., 2010b. Suppression of anaerobic ammonium oxidizers under high organic content in high-rate Anammox UASB reactor. Bioresour. Technol. 101 (6), 1762–1768. Third, K.A., Sliekers, A.O., Kuenen, J.G., Jetten, M.S.M., 2001. The CANON system (completely autotrophic nitrogen-removal over nitrite) under ammonium limitation: interaction and competition between three groups of bacteria. Syst. Appl. Microbiol. 24 (4), 588–596. van de Graaf, A.A., de Bruijn, P., Robertson, L.A., Jetten, M.S.M., Kuenen, J.G., 1996. Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor. Microbiology U.K. 142, 2187–2196. Winkler, M.K., Kleerebezem, R., van Loosdrecht, M.C., 2012. Integration of anammox into the aerobic granular sludge process for main stream wastewater treatment at ambient temperatures. Water Res. 46 (1), 136–144. Zhang, X., Li, D., Liang, Y., He, Y., Zhang, Y., Zhang, J., 2013a. Autotrophic nitrogen removal from domestic sewage in MBR–CANON system and the biodiversity of functional microbes. Bioresour. Technol. 150, 113–120. Zhang, X., Li, D., Liang, Y., Zhang, Y., Fan, D., Zhang, J., 2013b. Application of membrane bioreactor for completely autotrophic nitrogen removal over nitrite (CANON) process. Chemosphere 93 (11), 2832–2838. Zhi, W., Ji, G., 2014. Quantitative response relationships between nitrogen transformation rates and nitrogen functional genes in a tidal flow constructed wetland under C/N ratio constraints. Water Res. 64, 32–41. Zhi, W., Ge, Z., He, Z., Zhang, H., 2014. Methods for understanding microbial community structures and functions in microbial fuel cells: a review. Bioresour. Technol. 171, 461–468. Zhu, S., Chen, S., 2001. Effects of organic carbon on nitrification rate in fixed film biofilters. Aquacult. Eng. 25 (1), 1–11.
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Effect of COD/N ratio on nitrogen removal and microbial communities of CANON process in membrane bioreactors Xiaojing Zhang ⇑, Hongzhong Zhang, Changming Ye, Mingbao Wei, Jingjing Du Henan Collaborative Innovation Center of Environmental Pollution Control and Ecological Restoration, School of Material and Chemical Engineering, Zhengzhou University of Light Industry, Zhengzhou 450001, China
h i g h l i g h t s COD/N in low ratio could improve nitrogen removal of CANON. The suppressing threshold of COD/N ratio on CANON was 1.7. Biodiversity of AOB and AAOB both decreased with COD increasing. Denitrifiers and AAOB showed different relationship under different COD/N ratio. Strategies for treating sewage with different COD/N ratio were proposed.
a r t i c l e
i n f o
Article history: Received 20 February 2015 Received in revised form 1 April 2015 Accepted 2 April 2015 Available online 8 April 2015 Keywords: CANON COD/N Membrane bioreactor (MBR) Microbial community Denitrification
a b s t r a c t In this study, the effect of COD/N ratio on completely autotrophic nitrogen removal over nitrite (CANON) process was investigated in five identical membrane bioreactors. The five reactors were simultaneously seeded for 1 L CANON sludge and be operated for more than two months under same conditions, with influent COD/N ratio of 0, 0.5, 1, 2 and 4, respectively. DGGE was used to analyze the microbial communities of aerobic ammonia-oxidizing bacteria (AOB) and anaerobic ammonia-oxidizing bacteria (AAOB) in five reactors. Results revealed the harmonious work of CANON and denitrification with low COD concentration, whereas too high COD concentration suppressed both AOB and AAOB. AOB and AAOB biodiversity both decreased with COD increasing, which then led to worse nitrogen removal. The suppressing threshold of COD/N ratio for CANON was 1.7. CANON was feasible for treating low COD/N sewage, while the high sewage should be converted by anaerobic biogas producing process in advance. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Completely autotrophic nitrogen removal over nitrite (CANON) process has been developed in recent years as the most efficient and economical method to remove ammonia from wastewater (Sliekers et al., 2002; Third et al., 2001). With the development of anaerobic treatment process, most organic compounds in wastewater are converted to biogas without consumption of ammonia (Kartal et al., 2010), and CANON process was suggested to be an alternative process to treat the effluent with high concentration of ammonia (Liang et al., 2014). Recent researches indicated that CANON process is very sensitive to environmental conditions such as temperature, pH, salinity and presence of inhibitors including nitrite, free ammonia and organic material (OM, expressed as COD in this study) (Gilbert et al., 2014; Kimura et al., 2011; Liu ⇑ Corresponding author. Tel.: +86 15237133016. E-mail address: [email protected] (X. Zhang). http://dx.doi.org/10.1016/j.biortech.2015.04.006 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
et al., 2008; Ni et al., 2012; Tang et al., 2010a). Specially, one factor could not be ignored was the presence of OM since CANON process was a completely autotrophic reaction without organic carbon consumption. The functional organisms including aerobic ammonia-oxidizing bacteria (AOB) and anaerobic ammonia-oxidizing bacteria (AAOB) both utilize CO2 as the source of carbon for ammonia oxidation. Generally, wastewaters containing ammonia are not free from OM, and the anaerobic process for biogas production could not totally remove the organic carbon, such as anaerobic digestion process. Previous studies have reported the adverse effect of the presence of OM on the growth of both AOB and AAOB. On one hand, the presence of COD would result in the survival of heterotrophic bacteria, which would compete DO with AOB (Ji et al., 2012; Zhi and Ji, 2014). Furthermore, previous research provided evidence that the organic carbon source affects the make-up of AOB community in mixed cultures (Racz et al., 2010). Researchers are unanimous when it comes to the inhibitory effect
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of high COD concentration on AAOB. As much, AAOB are not able to compete with denitrifying bacteria (Jia et al., 2012; Ni et al., 2012). Further study revealed that both COD concentrations and COD to N ratio (COD/N) affect the performance of AAOB (Chamchoi et al., 2008; Hao and Loosdrecht, 2004; Molinuevo et al., 2009). Considering the adverse effect on both AOB and AAOB, the COD and COD/N ratio would also significantly affect the nitrogen removal performance by CANON. Only one or two studies focused on the relative low COD/N ratio influencing CANON so far. Chen (Chen et al., 2009) observed a significant reduction of the nitrogen removal efficiency (NRE) from 79% to below 52% when influent COD/N ratio was increased from 0.5 to 0.75, while Lackner and Horn (Lackner and Horn, 2013) reached 80–85% NRE with an influent COD/N ratio of 1 in a sequencing batch reactor. The available studies have not mentioned the performance of CANON process under a higher COD/N ratio, and it could not be conclusive to determine the COD/N ratio effects from the limited studies. In addition, since the reactor performance relies on the functional bacteria (Zhi et al., 2014), it is important to know the responses of microbial communities to the presence of different COD/N ratio. However, to the best of our knowledge, limited work has been carried out to explore the performance and microbial communities of CANON with different COD/N level. The main goal of this study was to investigate the effect of influent COD/N ratio on the nitrogen removal performance and microbial communities of CANON process. Membrane bioreactor (MBR) was chosen to be the experimental equipment in this study due to its effectiveness for CANON process (Zhang et al., 2013b). Five identical MBRs with same seed sludge were simultaneously started-up, and fed with different COD/N levels. The nitrogen removal and denitrification ratio of the five MBRs were compared, and the microbial communities of each reactor were analyzed using DGGE and clone-sequencing. 2. Methods 2.1. Reactors and seed sludge Five identical lab-scale MBRs (effective volume: 1 L, diameter: 100 mm, height: 200 mm, Supplementary material) were adopted to this study, named R0, R1, R2, R3 and R4, respectively. Each reactor was installed with a hollow fiber membrane module (material: PVDF; pore size: 0.1 lm; effective area: 0.05 m2; water permeability: 9 L h1). For each reactor, it was entirely placed in a water bath to ensure a constant reaction temperature (25 °C). Effluent was continuously extracted from the reactor using a peristaltic pump, while the synthetic wastewater was fed into the reactor. Oxygen was supplied continuously from an aeration ring locating at the bottom of the reactor which was connected to an air blower, and the aeration was controlled by the gas flow meter. A mixer was set in each reactor to assure homogeneous condition. A running MBR with identical setup was adopted as the seeding reactor, it showed an excellent nitrogen removal ability with the NRR of 0.6–0.7 kg m3 d1 before seeding. The five experimental MBRs were simultaneously inoculated with 1 L CANON sludge from the running MBR. The mixed liquor suspended solid and mixed liquor volatile suspended solid of the seed sludge were 4.1 and 3.1 g L1, respectively.
(0.136), MgSO47H2O (0.3) and 1 mL L1 of trace element solution (van de Graaf et al., 1996). Influent ammonia for the five reactors were 200 mg L1 during the whole experiment, the only difference was the COD concentration in influent. As a logical choice of organic substrate, glucose was considered as the harmless one to AOB (Ni et al., 2012). So glucose was added to the influent of R1, R2, R3 and R4, to adjust the influent COD concentration to 100, 200, 400 and 800 mg L1, with COD/N ratios of 0.5, 1, 2 and 4, respectively. For comparison, no OM was added to R0. The five reactors were running in a continuous mode at a HRT of 6.4 h. The pH in each reactor was all around 7.8 without adjustment. It was demonstrated that AAOB had a long growth cycle and low growth rate (l = 0.0027 h1, doubling time = 10.6 d) (Strous et al., 1998; Zhang et al., 2013a), so this study were conducted for more than two months. Sludge samples were obtained from each reactor on day 61, for DGGE analysis. Since previous studies (Zhang et al., 2013a,b) showed the prominent performance when DO was 0.15 mg L1, and the seeding reactor was also operated under this value for a long time, DO was controlled as 0.15 mg L1 in the five experimental MBRs, to eliminate the oxygen impact on the reactor performance. Other operational conditions of the reactors are summarized as Table 1. 2.3. Analytical methods According to the methods mentioned by APHA, concentrations of NH+4-N and NO 2 -N were daily measured using visible spectrophotometer (Shanghai Yoke Instrument Co., 722S, China) with different colorimetric methods. NO 3 -N was analyzed using ultraviolet spectrophotometric (Shanghai Yoke Instrument Co., UV755B, China). The temperature, DO and pH were detected using online instruments with specific probes (WTW, Oxi1296TriOxmatic700-7 and pH296-Sensolyt 650-7, Germany). COD was detected by digestion instrument (Lianhua Technology Co., 5B-6CV8.0, China). Denitrification ratio was defined as the ratio of nitrogen removal by denitrification to the total nitrogen removal. The nitrogen removal by denitrification was equal to the decreased amount of NO 3 -N when compared to the amount generated by CANON reaction. Denitrification ratio was calculated as Eq. (1). þ NH4 -N inf NHþ4 -N eff 0:11 NO3 -N eff Denitrification ratioð%Þ ¼ ½TNinf ½TNeff ð1Þ 2.4. DNA extraction, PCR–DGGE, cloning and sequencing Since DGGE was suitable for determining the specie difference and the biodiversity variation of the same kind of bacteria, it was chosen to characterize the microbial communities of AOB and AAOB under different influent COD/N ratio in this study. Some mixed liquor was respectively collected from the five reactors on day 61. DNA was extracted using a bacterial genomic mini extraction kit (Sangon, China) according to the manufacturer’s manual and was detected by 0.8% (w/V) agarose gel electrophoresis.
Table 1 Operational conditions of the five experimental reactors.
2.2. Feed and experimental setup
Reactor
CODinf (mg L1)
CODeff (mg L1)
HRT (h)
Aeration rate (L min1)
DO (mg L1)
pH –
The effect of COD/N on CANON process was tested with synthetic wastewater. To avoid influent impact, the influent for the five experimental reactors were totally same as the seeding MBR. The main synthetic wastewater used in this study contained (in g L1): (NH4)2SO4 (0.942), NaHCO3 (3.357), KH2PO4 (0.136), CaCl2
R0 R1 R2 R3 R4
0 97.9 198.2 399.2 800.2
0 13.72 32.56 74.51 252.4
6.4 6.4 6.4 6.4 6.4
0.2 0.25 0.3 0.35 0.4
0.15 0.15 0.15 0.15 0.15
7.84 7.80 7.78 7.86 7.88
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Using the extracted DNA as template, the 16S rDNA fragments of AOB was amplified for DGGE. Primers CTO 189fA/B and CTO 189fC of a 2:1 ratio, together with the reverse primer CTO654r were used for the first round PCR of AOB. Then the PCR products were used as templates for a second round PCR using universal primer set F338 (with GC-clamp)/R518. Primers Pla46F/630R and Amx368f (with GC-clamp)/Amx820r were respectively used for the first and second round PCR of Planctomycetales-like AAOB. The primes and PCR conditions used in this study was same as previously reported (Liang et al., 2014). The amplified DNA fragments were separated on 8% polyacrylamide gels with a 30–60% linear gradient of denaturant, for DGGE. The gel was conducted at 60 °C, 120 V for 5 h (AOB) or 7 h (AAOB) on Dcode Universal Mutation Detection System (Bio-Rad). After electrophoresis, the gel was stained using silver-staining method (Bio-Rad) (Bassam et al., 1991) followed by taking photos on Gel Doc XR 192 system. Diversity statistics (Shannon–Wiener diversity index (H) and Simpson’s diversity index (D)) were calculated from the DGGE profile by using the number and intensity of bands in each DGGE profile, as previously reported (Zhang et al., 2013a). Prominent bands in gel were excised and dissolved in 50 lL 1 TE buffer at 4 °C overnight. 1 lL TE solution as template was reamplified with the primer pairs F338/R518 and Amx368f/Amx820r using the same methods as described before, then the PCR products were cloned by pMD19-T plasmid vector system (TaKaRa, Japan). Sequencing was performed on an ABI 3730 DNA sequencer by a commercial service (Sangon, China). All sequences were compared with the reference microorganisms available in Genbank by BLAST tool and submitted to GenBank database. 3. Results and discussions 3.1. Reactor performance without organic carbon addition The control experiment was carried out at a HRT of 6.4 h without addition of COD in R0. The operational condition was totally same as the seeding reactor. The performance of R0 after seeded was depicted in Fig. 1. In the initial days after seeded, both AOB and AAOB showed poor bioactivity, leading to a lower NRR. Then the organisms seemed to be adapted to the new reactor with the operation, NRR went up gradually and then achieved to the steady state after several days. R0 was run for two months with high substrate removal, the average effluent ammonia was 15 mg L1 during the steady state, with the ammonia removal efficiency of 92%. Due to the nitrate production by CANON reaction, the TN removal efficiency (NRE) was only 80.2%, and NRR reached 0.61 kg m3 d1. Moreover, no nitrite accumulated in the reactor, indicating the excellent bioactivity of AAOB, which could timely consume the nitrite produced by AOB. The ratio of nitrate production to ammonia consumption (Dnitrate/Dammonia) was constantly around 0.11, which is the theoretical reaction ratio of CANON process (Sliekers et al., 2002). This result revealed that no excess nitrate was produced by NOB, indicating the effective inhibition or totally washout of NOB in the seeding sludge. The stable NRR was similar to the seeding reactor, suggesting no adverse effect of the new reactor. 3.2. Nitrogen removal with different influent COD/N ratio The nitrogen removal performance of the four reactors with COD feeding were shown in Fig. 2. With an influent COD/N ratio of 0.5, R1 showed a similar performance as R0 without COD feeding, indicating the slight inhibition on AOB and AAOB by COD in this COD/N ratio. Some previous reports suggested that COD would
inhibit AOB bioactivity since the oxygen competition with heterotrophic bacteria, Zhu and Chen (Zhu and Chen, 2001) observed a 70% reduction of AOB activity in a nitrification reactor when influent COD/N ratio was between 1.8 and 3.5. However, AOB was not restricted in this reactor, which was consistent with the previous study that the AOB bioactivity went up with increasing influent COD/N ratio (Jenni et al., 2014). The possible reason was that the COD concentration was too low to perform inhibition on AOB. Another possible reason was the oxygen supply was sufficient, the resulted DO was the same as the seeding reactor and R0 without COD feeding. After operation for 60 days, no nitrite or excess nitrate accumulated in the reactor, indicating the prominent bioactivity of both AOB and AAOB. Moreover, nitrate production in effluent showed a slight reduction about 10 mg L1 than the theoretical value, this perhaps was due to the consumption by denitrification, which was successfully induced due to the COD feeding. Finally NRR achieved to 0.63 kg m3 d1, which was a little higher than that of R0. This result demonstrated that a small supply of organic substrate could induce denitrification, which could further improve the nitrogen removal. For R2, COD addition in influent was 200 mg L1, with a COD/N ratio of 1. As shown in Fig. 2, more nitrite accumulated in the reactor initially, indicating the more intensive inhibition on AAOB by COD. The TN removal ability of this reactor began to increase from day 15, which was slower than R1, further indicating the inhibition on microorganisms by COD feeding. After 30 days’ operation, nitrite accumulation disappeared, and the NRR reached a stable range. Moreover, denitrification showed an obvious enhancement when compared to that of R1, the denitrification ratio increased to 10.1% (Table 2). No nitrite or nitrate was detected in the effluent during the last several days, finally NRR achieved to 0.69 kg m3 d1, which was the highest one in the five experimental reactors. Moreover, NRE reached 92.3%, which was higher than the theoretical CANON removal efficiency. The nitrate produced by CANON was totally consumed by denitrification, as a result, no nitrite or nitrate accumulated in the effluent, with the effluent ammonia around 13 mg L1. This result demonstrated the cooperative work of CANON and denitrification in this reactor with feeding COD/N ratio of 1. In R3 with the feeding COD/N ratio of 2, it showed a worse nitrogen removal than R2. Nitrite accumulated in the reactor during the whole experiment, indicating the continuous inhibition on AAOB. On the other hand, the limitation of AAOB bioactivity led to the nitrite accumulation, and the nitrite accumulation would suppress AAOB bioactivity in return (Bettazzi et al., 2010). Effluent nitrate reduced to around 1.6 mg L1. Since the CANON reaction would led to nitrate production about 20 mg L1 in this reactor, the decreased nitrate was undoubtedly consumed by denitrifiers, indicating the existence of denitrification. However, NRR finally achieved to only 0.57 kg m3 d1, which was lower than R0 without COD feeding. The possible reason of this result was the inhibition on AAOB by COD and nitrite or the oxygen competition of heterotrophic bacteria with AOB. Although the denitrification ratio was the highest, the limitation of autotrophic nitrogen removal resulted in the poor NRR. For R4 with COD/N ratio of 4, it showed the worst nitrogen removal performance. About 37 mg L1 nitrite accumulated in the reactor during the whole experiment, and effluent ammonia was more than 45 mg L1. Finally R4 achieved a NRR of 0.43 kg m3 d1 with the NRE of 57.4%. The comparisons between the five reactors showed that when influent COD/N was low, the COD feeding could elevate the nitrogen removal, no nitrite or nitrate accumulated in the reactor, AOB and AAOB both showed profitable bioactivity, CANON process and denitrification performed a cooperative work. However, when influent COD/N was too high, CANON was inhibited by the COD feeding, which limited
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0.5
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Eff. nitrate
0.3
-3
0.6 100 0.4
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Δ nitrate/ Δ ammonia
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-1
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NRR (kg m d )
Inf. ammonia Eff. ammonia NRR Δ nitrate/ Δ ammonia
-1
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Time (d) Fig. 1. Nitrogen removal performance of the reactor without COD feeding (R0).
Inf. ammonia
Eff. ammonia
Eff. nitrite
Eff. nitrate
NRR
1.0
1.0 200 180
140 120 100 80 60
nitrogen concentration (mg L-1)
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nitrogen concentration (mg L-1)
NRR (kg m-3 d-1)
nitrogen concentration (mg L-1)
0.8
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180
R3:C/N=2
160
0.6
120
Time (d)
200 180
0.8
140
0 0
0.0
0
R2:C/N=1
160
NRR (kg m-3 d-1)
R1:C/N=0.5
160
NRR (kg m-3 d-1)
nitrogen concentration (mg L-1)
180
R4:C/N=4
0.8
160 140
0.6
120 100
0.4
80 60
NRR (kg m-3 d-1)
200
0.2
40 20 0 0
0.0 10
20
30
40
50
60
Time (d)
Fig. 2. Nitrogen removal performances of reactors with different influent COD/N ratio.
Table 2 Nitrogen removal and denitrification ratio in steady state (last 10 days) of the five CANON reactors. Reactor 3
1
NRR (kg m d ) Denitrification ratio (%)
R0
R1
R2
R3
R4
0.61 0
0.63 4.87
0.69 10.1
0.57 11.17
0.43 13.18
the nitrogen removal. Denitrifiers and AAOB showed different competitions under different COD/N ratio. Thus, CANON process was feasible for treating sewage with low COD/N ratio.
3.3. Denitrification ratio and threshold COD/N ratio for CANON process The effect of organic loading on denitrification ratio and TN removal in steady state of the five reactors was illustrated in Fig. 3. In order to quantify the effect of organic loading on nitrogen removal more precisely, a COD/N threshold ratio for CANON inhibitory was defined when TN removal dropped to lower than the R0 without organic feeding. As shown in Fig. 3, the denitrification ratio went up with the COD increasing, indicating the COD contribution to denitrification. The threshold ratio was 1.7,
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14
Denitrification ratio
NRR
0.70
12
0.60
8 6
0.55
4 0.50
NRR (kg m-3 d-1)
Denitrification ratio (%)
0.65 10
2 0.45
0
0.40
-2 0
1
2
3
4
C/N ratio Fig. 3. Effect of COD/N ratio on the denitrification ratio and NRR.
corresponding to 340 mg L1 COD concentration. Chamchoi (Chamchoi et al., 2008) found the complete suppression of AAOB activity at COD concentration over 300 mg L1 or COD/N ratio over 2.0, which was similar to the result about the CANON process in the present study. Thus, the suppression on CANON process perhaps was depended on the suppression on AAOB. As shown in Table 2, both CANON and denitrification processes contributed to TN removal in the four reactors with COD feeding. Though high COD concentrations resulted in lower TN removal, a low concentration of COD could lead to higher TN removal (Fig. 3). Under the low COD strength, the fluctuation of AOB and AAOB activity was limited while the produced nitrate was reduced to nitrogen gas, resulting in higher TN removal (R1 and R2). These findings indicate that although COD feeding reduced AAOB activity, denitrification enhanced nitrite and nitrate removal when low concentration of COD was presented in the wastewater.
3.4. Microbial community of AOB and AAOB Sludge sample was obtained from the five reactors on day 61 for DGGE analysis, the results were shown in Fig. 4. As shown in Fig. 4, there were seven bands in the AOB profile of R0. And in R1 with COD/N ratio of 0.5, band 8 disappeared. This perhaps was due to the COD inhibition on AOB. Even though the reactor showed a
higher NRR, the biodiversity of AOB decreased. And the denitrification enhanced the TN removal in R1. Moreover, band 8 was not bright in lane 0, indicating the no predominant role, which then disappeared with COD feeding. The AOB profile of R2 showed similar result as R1, which was consistent with the reactor performance that the two reactors both showed nitrogen removal increase when compared to R0. This result also proved that the disappearance of band 8 was due to the COD adoption. Then in R3, band 1 and 5 declined while a new band (band 3) emerged, indicating the significant shift while COD concentration or COD/N ratio was higher than the threshold. Correspondingly, the nitrogen removal showed a significant decrease when compared to the three reactors with lower COD feeding. And in R4, one more band (band 6) declined, indicating the biodiversity decreasing due to the high COD feeding. The Shannon–Wiener diversity index and Simpson’s diversity index of AOB in Table 3 also showed the consistent result, H and D both decreased with the COD increasing, and R4 presented the lowest biodiversity. DNA sequencing results of the eight bands were shown in Table 4. The four bands (band 1, 5, 6 and 8) which declined in the four reactors with COD feeding were all related to Nitrosomonas sp., this result suggested that the COD feeding performed significant suppression on this specie. In addition, the new band emerged in R3 and R4 with high COD feeding showed a 99% similarity to Nitrosomonas eutropha. N. eutropha was regarded as owning denitrification ability (Kumar and Lin, 2010), and R3 and R4 both showed remarkable denitrification ability, so its emergence in the two reactors could be explained. What’s more, band 2 and 4 that both affiliating with Nitrosomonas europaea existed in all the five reactors, indicating the slight impact of COD feeding on this specie. For AAOB, COD feeding performed slight effect on the community of the five reactors. There were four bands in R0, R1 and R2, and the biodiversity index H and D both showed similar value, which indicated the slight effect of COD at low concentration on the competition of AAOB. The four bands were related to three species: Candidatus Kuenenia stuttgartiensis, Candidatus Brocadia fulgida and Candidatus Brocadia sp., indicating the complex community compositions in these reactors. Ca. K. stuttgartiensis predominated in these reactors, which was consistent with the previous study in MBR (Zhang et al., 2013a). Then in R3 and R4, band 9 that related to Ca. K. stuttgartiensis declined, which was consistent with the previous study in a biofilter packed with volcanic rock with COD feeding (Liang et al., 2014). The absence of band 9 perhaps was due to the inhibitory effect of high COD concentration, since the high COD feeding would significantly limit the AAOB bioactivity (Chamchoi et al., 2008; Daverey et al., 2013; Guven et al., 2005; Molinuevo et al., 2009). The DGGE result of AAOB was consistent with the reactor performances. In R3 and R4, nitrite accumulation were observed during the whole experiment, although denitrification were active, the total nitrogen removal decreased. All of these results indicated the insufficient bioactivity of AAOB, which then deteriorate the CANON performance. Although AAOB was first identified in a denitrification reactor (Mulder et al., 1995), it would be significantly suppressed by
Table 3 Shannon–Wiener diversity index and Simpson’s diversity index of AOB and AAOB.
Fig. 4. DGGE results of AOB and AAOB in the five experimental reactors (0: COD/ N = 0; I: COD/N = 0.5; II: COD/N = 1; III: COD/N = 2; IV: COD/N = 4).
COD/N ratio
AOB
AAOB
Number of bands
H
D
Number of bands
H
D
COD/N = 0 COD/N = 0.5 COD/N = 1 COD/N = 2 COD/N = 4
7 6 6 5 4
1.942 1.786 1.604 1.605 1.381
0.856 0.831 0.798 0.798 0.747
4 4 4 3 3
1.384 1.385 1.381 1.092 1.092
0.749 0.749 0.747 0.662 0.662
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X. Zhang et al. / Bioresource Technology 189 (2015) 302–308 Table 4 DGGE results AOB and AAOB in the five reactors. Band
Closest relative
Identity (%)
Access. No.
Phylum (classifier)
Access. No.
1 2 3 4 5 6 7 8 9 10 11 12
Nitrosomonas sp. Nitrosomonas europaea Nitrosomonas eutropha Nitrosomonas europaea Nitrosomonas sp. Nitrosomonas sp. Nitrosomonas sp. Nitrosomonas sp. Candidatus Kuenenia stuttgartiensis Candidatus Brocadia fulgida Candidatus Brocadia sp. Candidatus Kuenenia stuttgartiensis
99 99 99 99 99 99 99 98 98 98 99 99
HF678378 NR_074774 JX545090 HE862405 AJ621029 HF678378 AF272415 HF678378 KF429801 JX243380 AM285341 CT573071
b-Proteobacteria b-Proteobacteria b-Proteobacteria b-Proteobacteria b-Proteobacteria b-Proteobacteria b-Proteobacteria b-Proteobacteria Planctomycetia Planctomycetia Planctomycetia Planctomycetia
KM649686 KM649687 KM649689 KM649690 KM649691 KM649692 KM649693 KM649695 KP663624 KP663625 KP663626 KP663627
denitrifying communities under high OM content due to the weaker competition for nitrite (electron acceptor) and living space (Tang et al., 2010b).The high COD feeding led to the bioactivity and biodiversity decreasing of both AOB and AAOB, which then further resulted in the lower NRR in R3 and R4. In addition, band 10 that existed in all the five reactors showed a 98% similarity to Ca. B. fulgida, which could consume organic substrate (Winkler et al., 2012). This result was consistent with the previous report (Jenni et al., 2014) that the addition of glucose performed no influence on the dominant role of Ca. B. fulgida in a sequencing batch reactor. Thus, Ca. B. fulgida could be cultivated to treat the sewage with COD. 3.5. Strategies for treating wastewater with different COD/N ratio Combining the nitrogen removal performances and microbial communities of the five reactors, CANON process was strongly suggested to treat sewage with low COD/N ratio. The presence of OM with low concentration could induce denitrification, which could reduce the produced nitrate and then improve the total nitrogen removal. And since the COD/N ratio was relative low, it could not suppress the CANON process, the autotrophic nitrogen removal by AOB and AAOB and heterotrophic nitrogen removal by denitrifiers could cooperatively work with each other. Consequently, the sewage in the side-stream with low COD/N ratio could be directly treated by CANON process, such as the nitrogen fertilizer wastewater. For sewage with high COD/N ratio such as domestic sewage in main-stream, the COD/N ratio was always about 3–4. The denitrifiers would compete with AAOB and the high OM would inhibit both the bioactivity of AOB and AAOB. As a result, the autotrophic nitrogen removal would be significantly suppressed during a longterm operation. So the anaerobic treatment coupled with the subsequent CANON process would be a better alternative technology for treating sewage with high COD. In this two stages process, COD was converted to biogas without ammonia consumption in the anaerobic treatment, and then the nitrogen was removed in the CANON stage. The biogas production would be used as the power energy in the process. It could on one hand convert the redundant COD to biogas for energy reuse, which was feasible for the demand of energy autarkic wastewater treatment plants (WWTP), and on the other hand control the effluent to be with a COD/N ratio lower than 1.7. As a result, it could achieve the simultaneous removal of COD and nitrogen efficiently and economically. Moreover, from the results by DGGE, organic feeding performed slight impact on N. eutropha of AOB and Ca. B. fulgida of AAOB. So if these two species could be cultivated specifically in one single system, it could be a possible alternative to treat the sewage with high COD/N ratio.
Whilst the application of autotrophic nitrogen removal technologies in the side-stream is at present state of the art, the feasibility of this energy-efficient process at mainstream conditions is still under development. The results of this study were necessary and useful for the conclusive information about the COD/N effect on CANON process, which pointed a feasible way for the application of CANON process in mainstream. 4. Conclusions Denitrification could be successfully induced by organic feeding, and COD/N ratio in low range was favorable for nitrogen removal. The high COD/N ratio performed suppression on the bioactivity and biodiversity of both AOB and AAOB, which then led to the decreasing of nitrogen removal. The suppressing threshold of COD/N ratio on CANON was 1.7. For sewage with low COD/N ratio, it could be directly treated by CANON process. For sewage with high COD, it should be pretreated with anaerobic biogas production process. A cultivated system with enrichment of N. eutropha and Ca. B. fulgida could be a possible alternative. Acknowledgement This work was supported by Ph.D. Programme of Zhengzhou University of Light Industry – China (2014BSJJ055). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.04. 006. References Bassam, B.J., Caetano-Anollés, G., M. Gresshoff, P., 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem. 19 (6), 80–83. Bettazzi, E., Caffaz, S., Vannini, C., Lubello, C., 2010. Nitrite inhibition and intermediates effects on Anammox bacteria: a batch–scale experimental study. Process Biochem. 45, 573–580. Chamchoi, N., Nitisoravut, S., Schmidt, J.E., 2008. Inactivation of ANAMMOX communities under concurrent operation of anaerobic ammonium oxidation (ANAMMOX) and denitrification. Bioresour. Technol. 99 (9), 3331–3336. Chen, H., Liu, S., Yang, F., Xue, Y., Wang, T., 2009. The development of simultaneous partial nitrification, ANAMMOX and denitrification (SNAD) process in a single reactor for nitrogen removal. Bioresour. Technol. 100 (4), 1548–1554. Daverey, A., Hung, N.T., Dutta, K., Lin, J.G., 2013. Ambient temperature SNAD process treating anaerobic digester liquor of swine wastewater. Bioresour. Technol. 141, 191–198. Gilbert, E.M., Agrawal, S., Karst, S.M., Horn, H., Nielsen, P.H., Lackner, S., 2014. Low temperature partial nitritation/anammox in a moving bed biofilm reactor treating low strength wastewater. Environ. Sci. Technol. 48 (15), 8784–8792. Guven, D., Dapena, A., Kartal, B., Schmid, M.C., Maas, B., van de Pas-Schoonen, K., Sozen, S., Mendez, R., Op den Camp, H.J.M., Jetten, M.S.M., Strous, M., Schmidt, I.,
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