Bioresource Technology 159 (2014) 258–265
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Advanced nitrogen removal from landfill leachate using real-time controlled three-stage sequence batch reactor (SBR) system Lei Miao, Kai Wang, Shuying Wang ⇑, Rulong Zhu, Baikun Li, Yongzhen Peng, Dongchen Weng Engineering Research Center of Beijing, Beijing University of Technology, Beijing, China
h i g h l i g h t s A novel three-stage SBR process was firstly developed to treat landfill leachate. The real landfill leachate without diluted by tap-water was used in this study.
þ
The ratio of NO2 :NH4 in the Anammox influent was not needed to be adjusted. The modified continuous filling mode minimized the nitrite inhibition effects. Quantitative PCR analysis of Anammox showed Anammox gene ratio was less than 5%.
a r t i c l e
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Article history: Received 7 January 2014 Received in revised form 12 February 2014 Accepted 14 February 2014 Available online 24 February 2014 Keywords: Landfill leachate SBR Anammox Nitrogen removal Real-time control
a b s t r a c t A three-stage sequencing batch reactor (SBR), comprising pretreating SBR (SBRpre), nitritation SBR (SBRni), and anaerobic ammonium oxidation (Anammox) SBR (SBRana), was developed for the nitrogen removal from mature landfill leachate. The concentrations of ammonia and chemical oxygen demand (COD) in the leachate were 2000 ± 100 and 2200 ± 200 mg/L, respectively. About 100 mg/L of organic substance was removed from SBRpre to reduce the negative effect on the Anammox process under real-time control. After acclimation for 40 days, the nitrite to nitrogen oxide ratio (NO 2 /NOx) in SBRni was above 0.95. The nitrogen removal efficiency reached 90% in SBRana, and nitrogen load rate and nitrogen removal rate were 0.81 and 0.76 kg N/(m3 d), respectively. The continuous filling process was used to avoid the nitrite inhibition on the Anammox activity. The quantitative PCR analysis of Anammox indicated the average Anammox gene ratio increased from 0.23% to 4.77% after 220 days operation. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Landfill leachate contains high concentrations of organic/inorganic contaminants and may cause severe environmental pollution if not treated properly. There are three classes of landfill leachate in terms of landfill age: the young, middle-age, and mature landfill leachate (Bernard et al., 1997). Mature landfill leachate contains a relatively high concentration of ammonia and low concentration of biodegradable organic substances. In general, the ammonia concentration is above 1000 mg/L, chemical oxygen demand (COD) is below 3000 mg/L, and a ratio of biological oxygen demand (BOD5) to COD is below 0.1 (Chen, 1996; Kulikowska and Klimiuk, 2008). The low C:N ratio and low biodegradability of landfill leachate poses high challenges for treatment. The biological treatment (e.g. nitrification/denitrification) is used as the main treatment ⇑ Corresponding author. Tel./fax: +86 10 67392627. E-mail addresses: [email protected] (L. Miao), [email protected] (S. Wang). http://dx.doi.org/10.1016/j.biortech.2014.02.058 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
for landfill leachate (Renou et al., 2008). However, for the mature landfill leachate with low C:N ratio, the conventional nitrification/denitrification process needs an external carbon source, which makes the treatment systems complicated and expensive. Anaerobic ammonium oxidation (Anammox) is a cost-effective process with a great potential system (Kalyuzhnyi et al., 2006; Mulder et al., 1995; Kartal et al., 2010). Compared to conventional nitrification/denitrification processes, Anammox does not need external carbon sources and the power consumption for nitritation-Anammox process can be reduced 50% substantially. Even though Anammox is suitable for the treatment of mature landfill leachate with a low carbon-to-nitrogen (C/N) ratio (Shen et al., 2012; Chamchoi et al., 2008; Ganigué et al., 2009), there are some obstacles for real-world application. First, biodegradable organic substances have an adverse effect on Anammox (Chamchoi et al., 2008; Dapena-Mora et al., 2007; Molinuevo et al., 2009), thus nitritation and COD removal should be achieved prior to Anammox. Second, the doubling time of Anammox bacteria is longer than 11 days (Schmidt et al., 2003), so that long sludge retention time
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(SRT) is needed to prevent the loss of Anammox sludge (Chamchoi and Nitisoravut, 2007). Third, a high concentration of nitrite inhibits Anammox (Strous et al., 1999; Lotti et al., 2012). The activity of Anammox decreases when the nitrite concentration in the landfill is higher than 100 mg/L (Strous et al., 1999), and operational modes should be modified to alleviate the adverse effects of nitrite. Anammox has been studied for nitrogen removal from mature landfill leachate (Liang and Liu, 2008), and was coupled with partial nitritation (PN) in an upflow anaerobic sludge blanket (UASB) (Liu et al., 2010; Sri Shalini and Joseph, 2012; Anfruns et al., 2013). Although the organic substances can be removed by PN, the ratio of nitrite to ammonia suitable for Anammox could not be maintained. Until now, the studies on the Anammox process for the treatment of mature landfill leachate either used diluted landfill leachate (Liu et al., 2010), or employed complicated operaþ tional modes to maintain the ratio of NO 2 :NH4 (Anfruns et al., 2013). Therefore, it is critical to investigate the advanced nitrogen removal process for the treatment of real mature landfill leachate without diluting or adding extra chemicals. Sequencing batch reactor (SBR) has distinct advantages of space-saving, flexible operational mode, and auto-control capability (Chamchoi and Nitisoravut, 2007). It has been extensively used for biological nutrient removal (BNR) in municipal and industrial wastewaters (Wang et al., 2013). However, only a few researches applied SBR for Anammox process. In fact, SBR is suitable for Anammox enrichment due to its simplicity, efficient biomass retention, high stability over a long period of operation, and good BNR efficiency under substrate-shortage condition (Chamchoi and Nitisoravut, 2007; Strous et al., 1998). Therefore, this study aimed at developing a novel three-stage SBR process, comprising pretreatment SBR (SBRpre), nitritation SBR (SBRni), and Anammox SBR (SBRana) for nitrogen removal from real mature landfill leachate. Specifically, the biodegradable organic substances were removed in the SBRpre to reduce the negative effect on the Anammox process. Ammonia was oxidized to nitrite in the SBRni, and the effluents of the SBRpre and SBRni were mixed together for the SBRana. In addition, the pH profiles and redox potential (ORP) profiles that could distinguish different operational stages (e.g. nitrification, dinitrification) in SBR cycles (Peng et al., 2008) were used for real-time control to achieve high Anammox efficiency and save operational energy. Finally, the Anammox microbial communities were determined using quantitative PCR and correlated with nitrogen removal efficiency and SBR operational conditions.
2. Methods 2.1. Experimental setup and operational procedure The three-stage SBR system, comprising SBRpre, SBRni, and SBRana, was made of polymethyl methacrylate with a total volume capacity of 39 L, distributed as 12, 12, and 15 L for the SBRpre, SBRni, and SBRana, respectively (Fig. 1). The working volume capacities of the SBRpre, SBRni, and SBRana were 10, 10, and 13 L, respectively (Fig. 1). The SBRpre was equipped with pH meters, mechanical stirrers, and air diffusers. The SBRni was equipped with pH meters and air diffusers. The SBRana was equipped with a pH meter and a mechanical stirrer. An oxidation–reduction potential (ORP) meter was installed in the SBRpre to monitor the aeration/anoxic status. During the aeration period, the aeration intensity was maintained at 100 L/h. The operational temperatures for SBRpre, SBRni and SBRana were maintained at 25 °C, 25 °C and 35 °C using a temperature controller. In the three-stage SBR system, the SBRpre and SBRni were operated under the traditional mode: filling-aeration-settle-decant (5 h), whereas the SBRana was operated under the modified mode
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with continuous filling (5 h). The influent of the SBRana was the mixed effluents from the SBRpre and SBRni based on a ratio of þ NO 2 —N=NH4 —N of 1.3 (Fig. 1). The SBRana was operated under the modified mode: continuous filling (5 h) and stirring until the completion of the reaction. The terminal point of the Anammox was determined using the pH profile. The exchange volumetric rate of the SBRana was found to be 38%. The main operational conditions of the three SBRs in each period are shown in Table 1. 2.2. Influent and seed sludge During the acclimation period, the SBR system was fed with a synthetic wastewater consisting of KH2PO4 (10 mg/L), CaCl22H2O (5.6 mg/L), MgSO47H2O (300 mg/L), KHCO3 (1250 mg/L), trace element solution I (EDTA 5000 mg/L and FeSO4 5000 mg/L), and trace element solution II (EDTA 1000 mg/L, H3BO4 14 mg/L, MnCl24H2O 990 mg/L, CuSO45H2O 250 mg/L, ZnSO47H2O 430 mg/L, NiCl26H2O 190 mg/L, NaSeO410H2O 210 mg/L, NaMoO42H2O 220 mg/L) (Sliekers et al., 2002). NaNO2 and NH4Cl solutions were added to supply nitrite and ammonium for Anammox activities. The pH of the influent was controlled at 7.5 ± 0.2. After the acclimation period, the landfill leachate collected from the Liulitun Municipal Solid Waste (MSW) Sanitation Landfill Site (Beijing, China) was used as the feeding solution (Table 2). The raw landfill leachate was stored at 4 °C to preserve the characteristics of the landfill leachate. The excess sludge taken from the existing lab-scale nitrification SBR and Anammox UASB systems treating domestic wastewater was used as the inocula for the aerobic SBR (SBRpre and SBRni) and Anammox SBR (SBRana), respectively. The sludge from the lab-scale SBR was flocculent, while the sludge from the Anammox UASB system was the mixture of granular and flocculent. After being put in the three-stage SBR system, the mixed liquor suspended solid (MLSS) of the aerobic SBR and the Anammox sludge SBR were 3500 and 4200 mg/L, respectively. 2.3. Operational strategy of three-stage SBR process The operational strategies for each unit in the three-stage SBR system were different. The SBRpre had two periods: pre-A period (acclimation period) and pre-B period (combination with SBRana). In pre-A, the influent of the SBRpre was a mixture of the raw mature landfill leachate and tap water. In pre-B, the influent of the SBRpre was a mixture of the mature landfill leachate and effluent of the SBRana. The SBRni had four periods: (i) ni-A period (acclimation period), (ii) ni-B period (stable operation period), (iii) ni-C period (increasing load period), and (iv) ni-D period (the combination with SBRana). In ni-A, ni-B, and ni-C periods, the influent of the SBRni was a mixture of the raw mature landfill leachate and tap water. In ni-D period, the influent of the SBRni was the effluent of the SBRpre. The SBRana had four periods: (i) ana-A period (acclimation for synthetic wastewater), (ii) ana-B period (increasing nitrogen load), (iii) ana-C period (adding the effluent of SBRni), and (iv) ana-D period (combination of the SBRpre and SBRni). In ana-A and ana-B periods, the SBRana was fed with synthetic wastewater (Sliekers et al., 2002). In ana-C period, the nitrite in the influent of the SBRana was supplied by the effluent of the SBRni, and the ammonia was added in the form of NH4Cl. In ana-D period, the influent of the SBRana was the mixed effluents from the SBRpre and SBRni. 2.4. Analytical methods The dissolved oxygen (DO), pH, ORP, and temperature were monitored using a pH/Oxi 340i analyzer (WTW Company,
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Temperature controller
Stirrer ORP -300
Stirrer ORP -300
pH 7.00
pH 7.00
pH 7.00
ORP pH meter meter
ORP pH meter meter
pH meter
Heater
Heater
Sample outlets
Heater
Middle tank Feeding pump
Feeding tank Feeding pump
Outlet Middle tank
Air pump
Air pump
Pretreat SBR
Aerators
Nitritation SBR
Feeding Aerators pump
ASBR
ANAMMOX
Fig. 1. Schematic diagram of the three-stage SBR process treating mature landfill leachate.
Table 1 Operational conditions for the three SBRs in each period. Characteristics
SBRpre
SBRni
SBRana
Volumetric application rate (%) Feed flow-rate (L/period) HRT (h) pH DO (mg/L) T (°C) Exchange volumetric rate (%)
83.3 5 10 7.7 ± 0.2 0.2–0.5 mg/L 25 ± 1 50
83.3 3 16.7 7.5 ± 0.2 0.2–0.5 mg/L 25 ± 1 30
86.7 5 18.4 8.0 ± 0.2 0 32 ± 1 38
Table 2 Major characteristics of the landfill leachate. Compound
Concentration
COD (mg/L) BOD5 (mg/L) NHþ 4 —N (mg/L) NO 3 —N (mg/L) NO 2 —N (mg/L) TN (mg/L) Alkalinity (mg/L) pH
2200 ± 100 100 ± 20 2000 ± 100 0.5 ± 0.1 0.4 ± 0.1 2030 ± 120 7000 ± 100 8.0 ± 0.5
contained 10 PCR buffer (5 lL), dNTPs (4 lL, 2.5 mmol/L), Ex Taq polymerase (0.5 lL, 2.5U, Takara, Dalian, China), each primer (1 lL, 10 mmol/L), DNA template (1.25 lL, 1–10 ng), and ddH2O (37.25 lL) (Zhu et al., 2011). 2.6. Quantitative PCR The abundances of all the bacterial and Anammox DNAs were determined by real-time PCR using an MX3000P Real-Time PCR system (Stratagene, La Jolla, CA) equipped with the fluorescent dye SYBR-Green approach. The primers for all the bacteria and Anammox in the real-time PCR were 341f-543r and Amx368fAmx820r. The amplification was performed in the 20 lL reaction mixtures, consisting of SYBR Green exTaq (10 lL, Takara, Dalian, China), ROX Reference Dye 50 (0.3 lL), each primer (0.3 lL, 10 mmol/L), and DNA template (2 lL, 1–10 ng). The program consisted of the following steps: 3 min at 95 °C followed by 40 cycles of 30 s at 95 °C, 30 s at 59 °C, and 30 s at 72 °C. 3. Results and discussion 3.1. Performance of SBRpre
Germany). The MLSS, NHþ 4 —N, NO3 —N, NO2–N, and COD concentrations were measured according to the Standard Methods (APHA, 1995). The total nitrogen (TN) was analyzed using a TN/TOC analyzer (MultiN/C3000, Analtikjena AG, Germany).
2.5. DNA isolation and PCR Anammox bacterial population was quantified and correlated with nitrogen removal efficiency in the SBR systems. The activated sludge samples of the SBRana were collected during the periods A, B, C, and D. The DNA was extracted from 0.25 g of the sludge sample using a FastDNA SPIN Kit for soil (Bio 101, Vista, CA). A nested PCR assay was conducted to amplify the Anammox 16S rRNA gene. The all-bacterial amplification was carried out using a 341f-543r (Koike et al., 2007) with a thermal profile of 95 °C for 10 min, followed by 25 cycles of 30 s at 95 °C, 30 s at 55 °C, and 30 s at 72 °C. The Anammox amplification was carried out using an Amx368f-Amx820r primers31 (Schmid et al., 2005) with a thermal profile of 96 °C for 10 min followed by 25 cycles of 1 min at 96 °C, 1 min at 52 °C, and 1 min at 72 °C. The PCR reagents (50 lL)
The SBRpre was used for pretreating the biodegradable organic substances in mature landfill leachate, and reducing the adverse effects of the organic substances on the subsequent Anammox reactions. The start-up and stabilization of the SBRpre took approximately 100 days and consist of two periods (Fig. 2). The first period (marked as pre-A) lasted for 30 days, and the inocula were acclimated to the landfill leachate. The influent of the SBRpre was a mixture of the raw mature landfill leachate and tap water during the acclimation period; therefore, the aeration was started after the filling to remove the biodegradable organics. The average biodegradable organics removal reached 88 mg/L after the acclimation for 30 days (pre-A) (Fig. 2). The real-time controls of pH and ORP were used to determine the terminal points of the degradation of the organic substances; only 90 mg/L ammonia (10% of the influent) was oxidized into nitrite because of the high ammonia concentration in the influent. The second period (pre-B) lasted for 70 days during which the SBRpre was connected to the SBRni and SBRana. The influent of the SBRpre contained NO 3 , because it was a mixture of the mature landfill leachate and the effluent of the SBRana. The operational mode of the SBRpre was changed from the filling-aeration-settling-drain to
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150
2200
pre-B
pre-A
140
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-
NO2-N(eff)
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100
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NO2-N,NO3-N/(mg/L)
1800
10 0 0
10
20
30
40
50
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70
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Time(Day) Fig. 2. Startup and acclimation periods of SBRpre treating mature landfill leachate.
2010
NO 2-N
1980 1970
805
1960 1950
800
COD (mg/L)
+
NH4-N (mg/L)
30
1990
COD
810
1940
795
1920
785
1910
0
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90 120 Time(minutes)
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ORP
8.1
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b
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NO 3-N
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NOx-N (mg/L)
a 825
50 0 -50 -100
A
7.9
-150
ORP (mv)
8.0
pH (-)
the filling-stir-aeration-settling-drain to utilize the carbon sources in landfill leachate. The effluent NO 3 decreased to less than 1 mg/L after the stirring stage, indicating that the carbon sources in landfill leachate were fully used and the nitrogen removal efficiency was improved. The influent COD of the SBRpre and organic removal were maintained at 2000 mg/L (raw mature landfill leachate) and 500 mg, respectively (Fig. 2). The COD and nitrogen concentrations varied during a typical cycle (3 h) in the SBRpre (Fig. 3a). All the NO 3 was removed, and COD decreased slightly (30 mg/L) during the stirring period (denitrification). The COD dropped by 70 mg/L and the NO 2 increased by 30 mg/L during the aeration period (nitrification). Trace NO 3 (0.5 mg/L) was present because of the inhibition of the high fatty acid (FA). The removal of the TN and COD were 35 and 110 mg/L, respectively, after the denitrification and aeration, which were judged via the ORP and pH profiles. It is important to determine the terminal point of the COD degradation and denitrification to save the energy and improve the nitrogen removal efficiency. The variations in the pH and ORP in a typical SBRpre cycle indicated that the terminal point of denitrification right before the aeration could be judged via the ORP profile (Fig. 3b) (Peng et al., 2008). A significant point (Point A), referred to ‘‘nitrate knee’’, was observed in the ORP profile after 80 min of the cycle. The NO 3 was removed at Point A, indicating the completion of denitrification. Then, aeration was started to remove the organic substances. Point B was observed in the pH profile after 155 min of the cycle, indicating that the COD was degraded and the oxidation of ammonia started. The appearance of Point B indicated the end of a cycle. Therefore, the terminal points of the COD degradation and denitrification can be determined precisely using the real-time control, significantly improving the system efficiency.
-200
7.8
-250 7.7
-300 0
20
40
60
80
100
120
140
160
180
Time(minutes) Fig. 3. Variations in the COD, nitrogen, pH, and ORP in a typical cycle of the SBRpre cycle treating the mature landfill leachate (a) variations in the COD and nitrogen. (b) variations in the pH and ORP.
3.2. Performance of SBRni Nitrite is a critical substrate for Anammox that requires a stable nitritation before Anammox. The results showed that stable nitritation was achieved in the SBRni after three periods (period ni-A, period ni-B, and period ni-C) (Fig. 4). The inocula were acclimated to the landfill leachate gradually in ni-A (Fig. 4a). The ammonia concentration in the influent of the SBRni was 60 mg/L. At the beginning of ni-A, the inocula did not adapt to the mature landfill leachate, and the nitrification duration and ammonia oxidation rate (AOR) were 4.5 h and 0.16 kg N/ (m3 d), respectively. On 24th day, the nitrification duration was
shortened to 2 h, and the AOR increased to 0.32 kg N/(m3 d), indicating that the sludge adapted to the mature landfill leachate. The ratio of NO 2 /NOx was only 30%, and the low FA (1.3–1.5) resulted in the nitrification not nitritation, since the high FA could inhibit the activity of nitrite oxidizing bacteria (NOB) (Peng et al., 2007), but low FA did not inhibit NOB and make nitrification proceed completely. On 25th day (ni-B), the ammonia concentration and FA in the SBRni increased to 105 and 0.955 mg/L (Fig. 4a), respectively. The nitrification duration was prolonged to 4.5 h because of high
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110 -
100
NO2-N(inf)
90
NO3-N(inf)
-
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12 15 18 21 24 27 30 33 36 39 42 45 48 51 Time (Day)
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400
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NH4-N(eff), NO2-N(inf), NO3-N(inf) , NO2-N(eff) (mg/L)
NH4-N(inf), NO2-N(eff), NO3-N(eff) (mg/L)
ni-B
ni-A
nitrification time (h)
120
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a
NH4-N(eff), NO2-N(inf), NO3-N(inf) (mg/L)
262
0 55
60
65
70
75 80 time (Day)
85
90
95
100
Fig. 4. Performance of SBRni (a) Period ni-A and ni-B, (b) Period ni-C.
ammonia concentration, and the AOR decreased to 0.28 kg N/(m3 d). The nitrification duration steadily decreased, and the ratio of NO 2 /NOx increased over the time. On 50th day, the AOR increased to 0.32 kg N/(m3 d), and the ratio of NO 2 /NOx was 90%, indicating that nitritation was achieved. On 52nd day (ni-C), the ammonia concentration in the SBRni further increased after every ten days, making the sludge adapt to the high ammonia concentrations and enhance the nitrite concentration in the effluent (Fig. 4b). On 82th day, the ammonia concentration in the influent increased to 500 mg/L, and the ratio of NO 2 /NOx reached 95%, setting a good foundation for the subsequent Anammox reactions. The MLSS was maintained at 3500 mg/L in the SBR system because of the low contents of the organic substances, high contents of ammonia, and the presence of poisonous matters in the landfill leachate. The fast start-up of the SBRni was achieved because of the inhibition of NOB by high FA. Moreover, proper (not excessive) aeration could prevent the transformation of nitrite to nitrate. The terminal point of nitritation was determined by the real-time pH control (Peng et al., 2008). The high FA and the prevention of the excessive aeration were critical for achieving nitritation in SBR systems. 3.3. The start-up and acclimation of the SBRana The start-up and stabilization of the SBRana took 150 days (Fig. 5), comprising ana-A, ana-B, and ana-C periods. Ana-A Period
lasted 36 days, and the influent of the SBRana was the synthetic wastewater with a low TN content (200 mg/L). At the beginning of the start-up, the inocula were not adapted, and the treatment performance was unstable. From 25th day, the TN removal of the SBRana was stabilized at 85%, indicating that the start-up and acclimation was achieved. From 37th day (ana-B), the nitrogen load of SBRana increased to enrich the Anammox bacteria. During the following 53 days, the TN concentration of the influent gradually increased, and the NLR increased from 0.5 to 1.2 kg N/(m3 d) from 37th to 90th day. The NRR of the SBRana finally reached 1.06 kg N/(m3 d). From 91st day (ana-C), the influent of the SBRana was changed to a mixture of tap water and the effluent of the SBRni to make the Anammox adapt to the natural landfill leachate condition. About 20% nitrite of the SBRana influent came from the effluent of SBRni, and the rest 80% came from the synthetic wastewater. The proportion of the effluent in the SBRni increased at every ten days interval, and all the nitrite in the SBRana influent came from the effluent of the SBRni on 113th day. Throughout ana-C, the TN removal efficiency of the SBRana was stabilized at 90%. The effluent ammonia and nitrite concentrations were below 3 mg/L at an NLR of 1.2 kg N/(m3 d). Notably, the nitrate in the effluent of the SBRana decreased from 80 to 60 mg/L with increasing proportion of the effluent of the SBRni, which increased the NRR of the SBRana from 1.063 to 1.10 kg N/(m3 d). The decrease in the nitrate in the effluent indicated the presence of biodegradable organic substances,
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200 ana-C
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0 100 110 120 130 140 150
Fig. 5. Performance of SBRana in periods ana-A, ana-B, and ana-C.
SBRana. Compared with the existing Anammox systems, the major advantage of this three-stage SBR system was that the ratio of þ NO 2 :NH4 in the Anammox influent did not need to be adjusted by adding external chemicals, thus simplifying operational modes. Before being connected to the SBRpre and SBRni, the influent of the SBRana was a mixture of tap water and the effluent of SBRni, leading to the low COD concentration of the influent (900 mg/L). After being connected to the SBRpre and SBRni, the COD concentration of the SBRana influent steadily increased. On 20th day, the COD concentration reached 1900 mg/L (Fig. 6), and the COD removal of the SBRana was 50 mg/L. Based on the theoretical calculations, 17 mg/L NO 3 could be removed if all the COD (50 mg/L) was used for denitrification. However, in this SBRana system, the NO 3 —N produced during the Anammox process was 60 mg/L, while the theoretical value was 79 mg/L. This difference (19 mg/L) was close to 17 mg/L calculated from the removal of NO 3 , indicating that most of the biodegradable organic substances were used by denitrifying bacteria in the SBRana system. This study showed that the TN removal efficiency of the system reached above 90% (Fig. 6), which was close to the theoretical values (95%) calculated for the Anammox process in the three-stage SBR system. The Anammox process could be inhibited by high nitrite concentration in the traditional filling (5 min)-stir-settle-decant mode. To alleviate the nitrite inhibition effect on Anammox, the filling mode of the modified SBRana was changed to the continuous filling
3.4. Performance of SBRana in the three-stage system treating mature landfill leachate The performance of the SBRana in the three-stage SBR system after the combination of three SBRs (in pre-B, ni-D, and ana-D periods) was studied using the mature landfill leachate (Fig. 6). The biodegradable organic substances were effectively degraded in the SBRpre before reaching the SBRana. The ammonia and nitrite removal efficiency was stable and the TN removal efficiency reached above 90% in the SBRana over the 70-day operational period (Fig. 6). The NLR and NRR of the SBRana reached 0.81 and 0.76 kg N/(m3 d), respectively. The NO 3 content of the SBRana effluent was 60 mg/L, þ and the ratio of NO 3 eff to NH4 inf was maintained at 0.2:1, which was lower than the reported values (Strous et al., 1998). The aver þ þ age stoichiometric parameters of NO 2 =NH4 and NO3 =NH4 were 1.32 ± 0.03 and 0.22 ± 0.02, respectively (Fig. 6). The theoretic þ stoichiometric parameter of NO 3 =NH4 was 0.26, which was little higher than the value in this experiment. That because there were the remaining biodegradable organic substances in the effluents of the SBRpre and SBRni, which led to the slight denitrification in the
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Fig. 6. Removal of COD and nitrogen in the three-stage SBR system over 70 days.
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even though the landfill leachate was treated by the aeration. The proper concentration of the organic substances (COD) could improve the TN removal efficiency with a minimal inhibition effect on the Anammox process.
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mode (5 h). The effect of this modification on the SBR performance was clear (Table 3). The reaction period of the modified SBRana was 8 h. The ammonia and nitrite concentrations in the SBRana were 48 and 33 mg/L, respectively, after the continuous filling for 5 h. In the rest 3 h, both NHþ 4 —N and NO2 —N were removed, and the effluent NO3 —N was 70 mg/L. In contrast, during the traditional SBRana for 9 h, both NHþ 4 —N and NO2 —N were removed, and the effluent NO —N was 70 mg/L. The nitrogen removal efficiency improved 3 to 11% in the modified SBRana compared to the traditional SBRana because of the high nitrite content. Anammox process is based on the nitrite content, but high nitrite concentration (100 mg/L) could inhibit the Anammox process (Strous et al., 1999). Continuous filling solved the problem of high nitrite concentration in the treatment systems, and reduced the nitrite inhibition on the Anammox process. Therefore, the Anammox activity in the modified SBRana was higher than that in the traditional SBRana, which resulted in shorter reaction time. From the reaction equation of Anammox, during one SBRana cycle, H+ was consumed, which made pH increased. When Anammox was over, pH decreased slightly via hydrolytic acidification. So Anammox process could be monitored via pH profile. The variation in the pH during a typical cycle was different in the modified and traditional SBRana (Table 3). pH is an important factor for Anammox. The activity of the Anammox process was inhibited at a pH higher than 8.0 or lower than 7.5 (Strous et al., 1997). In the continuous filling mode (filling time 5 h), the alkalinity produced by Anammox was neutralized with acids in the influent in the modified SBRana to a relatively stable pH (7.65–7.80), which was suitable for Anammox processes. In the traditional mode (filling time 5 min), pH decreased to 7.4 at the beginning of the cycle, and high concentration nitrite (180 mg/L) started to appear, which could inhibit Anammox processes. The pH profile showed a slight decrease in the first hour of the reaction, but it kept increasing over time. The modified SBRana had two advantages over the traditional mode. First, the continuous filling mode did not result in high nitrite concentration in the SBRana, which decreased the inhibition effects on the Anammox process. Second, the alkalinity produced by Anammox neutralized acids in influent, producing a suitable pH in the modified SBRana for Anammox. Nitrogen removal efficiency improved by 11% in the modified filling mode (filling in 5 h) compared to that of the traditional mode (filling in 5 min), when the influent of the SBRana contained 500 and 350 mg/L of nitrite and ammonia. 3.5. Quantitative microbial analysis of Anammox
Table 4 Abundance of Anammox microorganisms in the SBRana. Samples
Anammox gene numbers (gene copiesg1 dry sludge)
All bacteria gene numbers (gene copiesg1 dry sludge)
Average Anammox gene ratio (%)
Period ana-A Period ana-B Period ana-D
3.18 107 1.56 108 8.19 109
1.40 1010 2.73 1010 1.72 1011
0.23 0.57 4.77
Anammox gene ratio increased from 0.23% in period ana-A to 0.57% in period ana-B. In the stable operation of the three-stage SBR system (ana-D), the Anammox gene numbers reached 8.19 109 gene copies/(g dry sludge), which was similar to previous findings (3.70 107 to 8.64 109 gene copies/(g dry sludge) (Hu et al., 2010). Notably, synthetic wastewater with additional carbon sources was used in previous studies, whereas real mature landfill leachate containing far more complex organic substances was used in this study. Moreover, average Anammox gene ratio also increased from 0.23% to 4.77% in the three-stage SBR system, whereas it was less than 3% in previous studies (Yapsakli et al., 2011).
4. Conclusions A novel integrated three-stage SBR system, comprising SBRpre, SBRni, and SBRana with real-time control was successfully developed in this study for advanced nitrogen removal from mature landfill leachate. The real-time control of pH and ORP can achieve the effective removal of the biodegradable organic substances in SBRpre. Nitritation was achieved in SBRni, and the NO 2 /NOx ratio reached above 95%. The modified continuous filling mode (5 h) in SBRana significantly minimized the nitrite inhibition effects. Quantitative PCR analysis of Anammox showed that the average Anammox gene ratio increased from 0.23% in the startup period to 4.77% in the stable operational period. Acknowledgement The financial support from the Project NSFC (21177005) and scientific research base and scientific innovation platform of Beijing municipal education commission are gratefully acknowledged. References
The quantitative microbial analysis of the Anammox process was studied to determine the variation in functional Anammox microorganisms in the SBRana during the ana-A, ana-B, and ana-D periods. The Anammox gene numbers, all-bacteria gene numbers, and the average Anammox gene ratio were determined (Table 4). In ana-A (acclimation) and ana-B (stable operation of the SBRana), all-bacteria gene numbers increased slightly, whereas the Anammox gene numbers significantly increased. Therefore, average
Table 3 Comparison between the modified and traditional SBRana cycles. Comparisons
Traditional SBRana
Modified SBRana
Influent NHþ 4 —N (mg/L) Influent NO 2 —N (mg/L) Highest NHþ 4 —N in the reactor (mg/L) Highest NO 2 —N in the reactor (mg/L) Reaction period (h) Variation in the pH
350 500 120 180 8 7.65–7.80
350 500 33 48 9 7.37–7.79
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Advanced nitrogen removal from landfill leachate using real-time controlled three-stage sequence batch reactor (SBR) system Lei Miao, Kai Wang, Shuying Wang ⇑, Rulong Zhu, Baikun Li, Yongzhen Peng, Dongchen Weng Engineering Research Center of Beijing, Beijing University of Technology, Beijing, China
h i g h l i g h t s A novel three-stage SBR process was firstly developed to treat landfill leachate. The real landfill leachate without diluted by tap-water was used in this study.
þ
The ratio of NO2 :NH4 in the Anammox influent was not needed to be adjusted. The modified continuous filling mode minimized the nitrite inhibition effects. Quantitative PCR analysis of Anammox showed Anammox gene ratio was less than 5%.
a r t i c l e
i n f o
Article history: Received 7 January 2014 Received in revised form 12 February 2014 Accepted 14 February 2014 Available online 24 February 2014 Keywords: Landfill leachate SBR Anammox Nitrogen removal Real-time control
a b s t r a c t A three-stage sequencing batch reactor (SBR), comprising pretreating SBR (SBRpre), nitritation SBR (SBRni), and anaerobic ammonium oxidation (Anammox) SBR (SBRana), was developed for the nitrogen removal from mature landfill leachate. The concentrations of ammonia and chemical oxygen demand (COD) in the leachate were 2000 ± 100 and 2200 ± 200 mg/L, respectively. About 100 mg/L of organic substance was removed from SBRpre to reduce the negative effect on the Anammox process under real-time control. After acclimation for 40 days, the nitrite to nitrogen oxide ratio (NO 2 /NOx) in SBRni was above 0.95. The nitrogen removal efficiency reached 90% in SBRana, and nitrogen load rate and nitrogen removal rate were 0.81 and 0.76 kg N/(m3 d), respectively. The continuous filling process was used to avoid the nitrite inhibition on the Anammox activity. The quantitative PCR analysis of Anammox indicated the average Anammox gene ratio increased from 0.23% to 4.77% after 220 days operation. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Landfill leachate contains high concentrations of organic/inorganic contaminants and may cause severe environmental pollution if not treated properly. There are three classes of landfill leachate in terms of landfill age: the young, middle-age, and mature landfill leachate (Bernard et al., 1997). Mature landfill leachate contains a relatively high concentration of ammonia and low concentration of biodegradable organic substances. In general, the ammonia concentration is above 1000 mg/L, chemical oxygen demand (COD) is below 3000 mg/L, and a ratio of biological oxygen demand (BOD5) to COD is below 0.1 (Chen, 1996; Kulikowska and Klimiuk, 2008). The low C:N ratio and low biodegradability of landfill leachate poses high challenges for treatment. The biological treatment (e.g. nitrification/denitrification) is used as the main treatment ⇑ Corresponding author. Tel./fax: +86 10 67392627. E-mail addresses: [email protected] (L. Miao), [email protected] (S. Wang). http://dx.doi.org/10.1016/j.biortech.2014.02.058 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
for landfill leachate (Renou et al., 2008). However, for the mature landfill leachate with low C:N ratio, the conventional nitrification/denitrification process needs an external carbon source, which makes the treatment systems complicated and expensive. Anaerobic ammonium oxidation (Anammox) is a cost-effective process with a great potential system (Kalyuzhnyi et al., 2006; Mulder et al., 1995; Kartal et al., 2010). Compared to conventional nitrification/denitrification processes, Anammox does not need external carbon sources and the power consumption for nitritation-Anammox process can be reduced 50% substantially. Even though Anammox is suitable for the treatment of mature landfill leachate with a low carbon-to-nitrogen (C/N) ratio (Shen et al., 2012; Chamchoi et al., 2008; Ganigué et al., 2009), there are some obstacles for real-world application. First, biodegradable organic substances have an adverse effect on Anammox (Chamchoi et al., 2008; Dapena-Mora et al., 2007; Molinuevo et al., 2009), thus nitritation and COD removal should be achieved prior to Anammox. Second, the doubling time of Anammox bacteria is longer than 11 days (Schmidt et al., 2003), so that long sludge retention time
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(SRT) is needed to prevent the loss of Anammox sludge (Chamchoi and Nitisoravut, 2007). Third, a high concentration of nitrite inhibits Anammox (Strous et al., 1999; Lotti et al., 2012). The activity of Anammox decreases when the nitrite concentration in the landfill is higher than 100 mg/L (Strous et al., 1999), and operational modes should be modified to alleviate the adverse effects of nitrite. Anammox has been studied for nitrogen removal from mature landfill leachate (Liang and Liu, 2008), and was coupled with partial nitritation (PN) in an upflow anaerobic sludge blanket (UASB) (Liu et al., 2010; Sri Shalini and Joseph, 2012; Anfruns et al., 2013). Although the organic substances can be removed by PN, the ratio of nitrite to ammonia suitable for Anammox could not be maintained. Until now, the studies on the Anammox process for the treatment of mature landfill leachate either used diluted landfill leachate (Liu et al., 2010), or employed complicated operaþ tional modes to maintain the ratio of NO 2 :NH4 (Anfruns et al., 2013). Therefore, it is critical to investigate the advanced nitrogen removal process for the treatment of real mature landfill leachate without diluting or adding extra chemicals. Sequencing batch reactor (SBR) has distinct advantages of space-saving, flexible operational mode, and auto-control capability (Chamchoi and Nitisoravut, 2007). It has been extensively used for biological nutrient removal (BNR) in municipal and industrial wastewaters (Wang et al., 2013). However, only a few researches applied SBR for Anammox process. In fact, SBR is suitable for Anammox enrichment due to its simplicity, efficient biomass retention, high stability over a long period of operation, and good BNR efficiency under substrate-shortage condition (Chamchoi and Nitisoravut, 2007; Strous et al., 1998). Therefore, this study aimed at developing a novel three-stage SBR process, comprising pretreatment SBR (SBRpre), nitritation SBR (SBRni), and Anammox SBR (SBRana) for nitrogen removal from real mature landfill leachate. Specifically, the biodegradable organic substances were removed in the SBRpre to reduce the negative effect on the Anammox process. Ammonia was oxidized to nitrite in the SBRni, and the effluents of the SBRpre and SBRni were mixed together for the SBRana. In addition, the pH profiles and redox potential (ORP) profiles that could distinguish different operational stages (e.g. nitrification, dinitrification) in SBR cycles (Peng et al., 2008) were used for real-time control to achieve high Anammox efficiency and save operational energy. Finally, the Anammox microbial communities were determined using quantitative PCR and correlated with nitrogen removal efficiency and SBR operational conditions.
2. Methods 2.1. Experimental setup and operational procedure The three-stage SBR system, comprising SBRpre, SBRni, and SBRana, was made of polymethyl methacrylate with a total volume capacity of 39 L, distributed as 12, 12, and 15 L for the SBRpre, SBRni, and SBRana, respectively (Fig. 1). The working volume capacities of the SBRpre, SBRni, and SBRana were 10, 10, and 13 L, respectively (Fig. 1). The SBRpre was equipped with pH meters, mechanical stirrers, and air diffusers. The SBRni was equipped with pH meters and air diffusers. The SBRana was equipped with a pH meter and a mechanical stirrer. An oxidation–reduction potential (ORP) meter was installed in the SBRpre to monitor the aeration/anoxic status. During the aeration period, the aeration intensity was maintained at 100 L/h. The operational temperatures for SBRpre, SBRni and SBRana were maintained at 25 °C, 25 °C and 35 °C using a temperature controller. In the three-stage SBR system, the SBRpre and SBRni were operated under the traditional mode: filling-aeration-settle-decant (5 h), whereas the SBRana was operated under the modified mode
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with continuous filling (5 h). The influent of the SBRana was the mixed effluents from the SBRpre and SBRni based on a ratio of þ NO 2 —N=NH4 —N of 1.3 (Fig. 1). The SBRana was operated under the modified mode: continuous filling (5 h) and stirring until the completion of the reaction. The terminal point of the Anammox was determined using the pH profile. The exchange volumetric rate of the SBRana was found to be 38%. The main operational conditions of the three SBRs in each period are shown in Table 1. 2.2. Influent and seed sludge During the acclimation period, the SBR system was fed with a synthetic wastewater consisting of KH2PO4 (10 mg/L), CaCl22H2O (5.6 mg/L), MgSO47H2O (300 mg/L), KHCO3 (1250 mg/L), trace element solution I (EDTA 5000 mg/L and FeSO4 5000 mg/L), and trace element solution II (EDTA 1000 mg/L, H3BO4 14 mg/L, MnCl24H2O 990 mg/L, CuSO45H2O 250 mg/L, ZnSO47H2O 430 mg/L, NiCl26H2O 190 mg/L, NaSeO410H2O 210 mg/L, NaMoO42H2O 220 mg/L) (Sliekers et al., 2002). NaNO2 and NH4Cl solutions were added to supply nitrite and ammonium for Anammox activities. The pH of the influent was controlled at 7.5 ± 0.2. After the acclimation period, the landfill leachate collected from the Liulitun Municipal Solid Waste (MSW) Sanitation Landfill Site (Beijing, China) was used as the feeding solution (Table 2). The raw landfill leachate was stored at 4 °C to preserve the characteristics of the landfill leachate. The excess sludge taken from the existing lab-scale nitrification SBR and Anammox UASB systems treating domestic wastewater was used as the inocula for the aerobic SBR (SBRpre and SBRni) and Anammox SBR (SBRana), respectively. The sludge from the lab-scale SBR was flocculent, while the sludge from the Anammox UASB system was the mixture of granular and flocculent. After being put in the three-stage SBR system, the mixed liquor suspended solid (MLSS) of the aerobic SBR and the Anammox sludge SBR were 3500 and 4200 mg/L, respectively. 2.3. Operational strategy of three-stage SBR process The operational strategies for each unit in the three-stage SBR system were different. The SBRpre had two periods: pre-A period (acclimation period) and pre-B period (combination with SBRana). In pre-A, the influent of the SBRpre was a mixture of the raw mature landfill leachate and tap water. In pre-B, the influent of the SBRpre was a mixture of the mature landfill leachate and effluent of the SBRana. The SBRni had four periods: (i) ni-A period (acclimation period), (ii) ni-B period (stable operation period), (iii) ni-C period (increasing load period), and (iv) ni-D period (the combination with SBRana). In ni-A, ni-B, and ni-C periods, the influent of the SBRni was a mixture of the raw mature landfill leachate and tap water. In ni-D period, the influent of the SBRni was the effluent of the SBRpre. The SBRana had four periods: (i) ana-A period (acclimation for synthetic wastewater), (ii) ana-B period (increasing nitrogen load), (iii) ana-C period (adding the effluent of SBRni), and (iv) ana-D period (combination of the SBRpre and SBRni). In ana-A and ana-B periods, the SBRana was fed with synthetic wastewater (Sliekers et al., 2002). In ana-C period, the nitrite in the influent of the SBRana was supplied by the effluent of the SBRni, and the ammonia was added in the form of NH4Cl. In ana-D period, the influent of the SBRana was the mixed effluents from the SBRpre and SBRni. 2.4. Analytical methods The dissolved oxygen (DO), pH, ORP, and temperature were monitored using a pH/Oxi 340i analyzer (WTW Company,
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Temperature controller
Stirrer ORP -300
Stirrer ORP -300
pH 7.00
pH 7.00
pH 7.00
ORP pH meter meter
ORP pH meter meter
pH meter
Heater
Heater
Sample outlets
Heater
Middle tank Feeding pump
Feeding tank Feeding pump
Outlet Middle tank
Air pump
Air pump
Pretreat SBR
Aerators
Nitritation SBR
Feeding Aerators pump
ASBR
ANAMMOX
Fig. 1. Schematic diagram of the three-stage SBR process treating mature landfill leachate.
Table 1 Operational conditions for the three SBRs in each period. Characteristics
SBRpre
SBRni
SBRana
Volumetric application rate (%) Feed flow-rate (L/period) HRT (h) pH DO (mg/L) T (°C) Exchange volumetric rate (%)
83.3 5 10 7.7 ± 0.2 0.2–0.5 mg/L 25 ± 1 50
83.3 3 16.7 7.5 ± 0.2 0.2–0.5 mg/L 25 ± 1 30
86.7 5 18.4 8.0 ± 0.2 0 32 ± 1 38
Table 2 Major characteristics of the landfill leachate. Compound
Concentration
COD (mg/L) BOD5 (mg/L) NHþ 4 —N (mg/L) NO 3 —N (mg/L) NO 2 —N (mg/L) TN (mg/L) Alkalinity (mg/L) pH
2200 ± 100 100 ± 20 2000 ± 100 0.5 ± 0.1 0.4 ± 0.1 2030 ± 120 7000 ± 100 8.0 ± 0.5
contained 10 PCR buffer (5 lL), dNTPs (4 lL, 2.5 mmol/L), Ex Taq polymerase (0.5 lL, 2.5U, Takara, Dalian, China), each primer (1 lL, 10 mmol/L), DNA template (1.25 lL, 1–10 ng), and ddH2O (37.25 lL) (Zhu et al., 2011). 2.6. Quantitative PCR The abundances of all the bacterial and Anammox DNAs were determined by real-time PCR using an MX3000P Real-Time PCR system (Stratagene, La Jolla, CA) equipped with the fluorescent dye SYBR-Green approach. The primers for all the bacteria and Anammox in the real-time PCR were 341f-543r and Amx368fAmx820r. The amplification was performed in the 20 lL reaction mixtures, consisting of SYBR Green exTaq (10 lL, Takara, Dalian, China), ROX Reference Dye 50 (0.3 lL), each primer (0.3 lL, 10 mmol/L), and DNA template (2 lL, 1–10 ng). The program consisted of the following steps: 3 min at 95 °C followed by 40 cycles of 30 s at 95 °C, 30 s at 59 °C, and 30 s at 72 °C. 3. Results and discussion 3.1. Performance of SBRpre
Germany). The MLSS, NHþ 4 —N, NO3 —N, NO2–N, and COD concentrations were measured according to the Standard Methods (APHA, 1995). The total nitrogen (TN) was analyzed using a TN/TOC analyzer (MultiN/C3000, Analtikjena AG, Germany).
2.5. DNA isolation and PCR Anammox bacterial population was quantified and correlated with nitrogen removal efficiency in the SBR systems. The activated sludge samples of the SBRana were collected during the periods A, B, C, and D. The DNA was extracted from 0.25 g of the sludge sample using a FastDNA SPIN Kit for soil (Bio 101, Vista, CA). A nested PCR assay was conducted to amplify the Anammox 16S rRNA gene. The all-bacterial amplification was carried out using a 341f-543r (Koike et al., 2007) with a thermal profile of 95 °C for 10 min, followed by 25 cycles of 30 s at 95 °C, 30 s at 55 °C, and 30 s at 72 °C. The Anammox amplification was carried out using an Amx368f-Amx820r primers31 (Schmid et al., 2005) with a thermal profile of 96 °C for 10 min followed by 25 cycles of 1 min at 96 °C, 1 min at 52 °C, and 1 min at 72 °C. The PCR reagents (50 lL)
The SBRpre was used for pretreating the biodegradable organic substances in mature landfill leachate, and reducing the adverse effects of the organic substances on the subsequent Anammox reactions. The start-up and stabilization of the SBRpre took approximately 100 days and consist of two periods (Fig. 2). The first period (marked as pre-A) lasted for 30 days, and the inocula were acclimated to the landfill leachate. The influent of the SBRpre was a mixture of the raw mature landfill leachate and tap water during the acclimation period; therefore, the aeration was started after the filling to remove the biodegradable organics. The average biodegradable organics removal reached 88 mg/L after the acclimation for 30 days (pre-A) (Fig. 2). The real-time controls of pH and ORP were used to determine the terminal points of the degradation of the organic substances; only 90 mg/L ammonia (10% of the influent) was oxidized into nitrite because of the high ammonia concentration in the influent. The second period (pre-B) lasted for 70 days during which the SBRpre was connected to the SBRni and SBRana. The influent of the SBRpre contained NO 3 , because it was a mixture of the mature landfill leachate and the effluent of the SBRana. The operational mode of the SBRpre was changed from the filling-aeration-settling-drain to
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the filling-stir-aeration-settling-drain to utilize the carbon sources in landfill leachate. The effluent NO 3 decreased to less than 1 mg/L after the stirring stage, indicating that the carbon sources in landfill leachate were fully used and the nitrogen removal efficiency was improved. The influent COD of the SBRpre and organic removal were maintained at 2000 mg/L (raw mature landfill leachate) and 500 mg, respectively (Fig. 2). The COD and nitrogen concentrations varied during a typical cycle (3 h) in the SBRpre (Fig. 3a). All the NO 3 was removed, and COD decreased slightly (30 mg/L) during the stirring period (denitrification). The COD dropped by 70 mg/L and the NO 2 increased by 30 mg/L during the aeration period (nitrification). Trace NO 3 (0.5 mg/L) was present because of the inhibition of the high fatty acid (FA). The removal of the TN and COD were 35 and 110 mg/L, respectively, after the denitrification and aeration, which were judged via the ORP and pH profiles. It is important to determine the terminal point of the COD degradation and denitrification to save the energy and improve the nitrogen removal efficiency. The variations in the pH and ORP in a typical SBRpre cycle indicated that the terminal point of denitrification right before the aeration could be judged via the ORP profile (Fig. 3b) (Peng et al., 2008). A significant point (Point A), referred to ‘‘nitrate knee’’, was observed in the ORP profile after 80 min of the cycle. The NO 3 was removed at Point A, indicating the completion of denitrification. Then, aeration was started to remove the organic substances. Point B was observed in the pH profile after 155 min of the cycle, indicating that the COD was degraded and the oxidation of ammonia started. The appearance of Point B indicated the end of a cycle. Therefore, the terminal points of the COD degradation and denitrification can be determined precisely using the real-time control, significantly improving the system efficiency.
-200
7.8
-250 7.7
-300 0
20
40
60
80
100
120
140
160
180
Time(minutes) Fig. 3. Variations in the COD, nitrogen, pH, and ORP in a typical cycle of the SBRpre cycle treating the mature landfill leachate (a) variations in the COD and nitrogen. (b) variations in the pH and ORP.
3.2. Performance of SBRni Nitrite is a critical substrate for Anammox that requires a stable nitritation before Anammox. The results showed that stable nitritation was achieved in the SBRni after three periods (period ni-A, period ni-B, and period ni-C) (Fig. 4). The inocula were acclimated to the landfill leachate gradually in ni-A (Fig. 4a). The ammonia concentration in the influent of the SBRni was 60 mg/L. At the beginning of ni-A, the inocula did not adapt to the mature landfill leachate, and the nitrification duration and ammonia oxidation rate (AOR) were 4.5 h and 0.16 kg N/ (m3 d), respectively. On 24th day, the nitrification duration was
shortened to 2 h, and the AOR increased to 0.32 kg N/(m3 d), indicating that the sludge adapted to the mature landfill leachate. The ratio of NO 2 /NOx was only 30%, and the low FA (1.3–1.5) resulted in the nitrification not nitritation, since the high FA could inhibit the activity of nitrite oxidizing bacteria (NOB) (Peng et al., 2007), but low FA did not inhibit NOB and make nitrification proceed completely. On 25th day (ni-B), the ammonia concentration and FA in the SBRni increased to 105 and 0.955 mg/L (Fig. 4a), respectively. The nitrification duration was prolonged to 4.5 h because of high
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262
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Fig. 4. Performance of SBRni (a) Period ni-A and ni-B, (b) Period ni-C.
ammonia concentration, and the AOR decreased to 0.28 kg N/(m3 d). The nitrification duration steadily decreased, and the ratio of NO 2 /NOx increased over the time. On 50th day, the AOR increased to 0.32 kg N/(m3 d), and the ratio of NO 2 /NOx was 90%, indicating that nitritation was achieved. On 52nd day (ni-C), the ammonia concentration in the SBRni further increased after every ten days, making the sludge adapt to the high ammonia concentrations and enhance the nitrite concentration in the effluent (Fig. 4b). On 82th day, the ammonia concentration in the influent increased to 500 mg/L, and the ratio of NO 2 /NOx reached 95%, setting a good foundation for the subsequent Anammox reactions. The MLSS was maintained at 3500 mg/L in the SBR system because of the low contents of the organic substances, high contents of ammonia, and the presence of poisonous matters in the landfill leachate. The fast start-up of the SBRni was achieved because of the inhibition of NOB by high FA. Moreover, proper (not excessive) aeration could prevent the transformation of nitrite to nitrate. The terminal point of nitritation was determined by the real-time pH control (Peng et al., 2008). The high FA and the prevention of the excessive aeration were critical for achieving nitritation in SBR systems. 3.3. The start-up and acclimation of the SBRana The start-up and stabilization of the SBRana took 150 days (Fig. 5), comprising ana-A, ana-B, and ana-C periods. Ana-A Period
lasted 36 days, and the influent of the SBRana was the synthetic wastewater with a low TN content (200 mg/L). At the beginning of the start-up, the inocula were not adapted, and the treatment performance was unstable. From 25th day, the TN removal of the SBRana was stabilized at 85%, indicating that the start-up and acclimation was achieved. From 37th day (ana-B), the nitrogen load of SBRana increased to enrich the Anammox bacteria. During the following 53 days, the TN concentration of the influent gradually increased, and the NLR increased from 0.5 to 1.2 kg N/(m3 d) from 37th to 90th day. The NRR of the SBRana finally reached 1.06 kg N/(m3 d). From 91st day (ana-C), the influent of the SBRana was changed to a mixture of tap water and the effluent of the SBRni to make the Anammox adapt to the natural landfill leachate condition. About 20% nitrite of the SBRana influent came from the effluent of SBRni, and the rest 80% came from the synthetic wastewater. The proportion of the effluent in the SBRni increased at every ten days interval, and all the nitrite in the SBRana influent came from the effluent of the SBRni on 113th day. Throughout ana-C, the TN removal efficiency of the SBRana was stabilized at 90%. The effluent ammonia and nitrite concentrations were below 3 mg/L at an NLR of 1.2 kg N/(m3 d). Notably, the nitrate in the effluent of the SBRana decreased from 80 to 60 mg/L with increasing proportion of the effluent of the SBRni, which increased the NRR of the SBRana from 1.063 to 1.10 kg N/(m3 d). The decrease in the nitrate in the effluent indicated the presence of biodegradable organic substances,
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Fig. 5. Performance of SBRana in periods ana-A, ana-B, and ana-C.
SBRana. Compared with the existing Anammox systems, the major advantage of this three-stage SBR system was that the ratio of þ NO 2 :NH4 in the Anammox influent did not need to be adjusted by adding external chemicals, thus simplifying operational modes. Before being connected to the SBRpre and SBRni, the influent of the SBRana was a mixture of tap water and the effluent of SBRni, leading to the low COD concentration of the influent (900 mg/L). After being connected to the SBRpre and SBRni, the COD concentration of the SBRana influent steadily increased. On 20th day, the COD concentration reached 1900 mg/L (Fig. 6), and the COD removal of the SBRana was 50 mg/L. Based on the theoretical calculations, 17 mg/L NO 3 could be removed if all the COD (50 mg/L) was used for denitrification. However, in this SBRana system, the NO 3 —N produced during the Anammox process was 60 mg/L, while the theoretical value was 79 mg/L. This difference (19 mg/L) was close to 17 mg/L calculated from the removal of NO 3 , indicating that most of the biodegradable organic substances were used by denitrifying bacteria in the SBRana system. This study showed that the TN removal efficiency of the system reached above 90% (Fig. 6), which was close to the theoretical values (95%) calculated for the Anammox process in the three-stage SBR system. The Anammox process could be inhibited by high nitrite concentration in the traditional filling (5 min)-stir-settle-decant mode. To alleviate the nitrite inhibition effect on Anammox, the filling mode of the modified SBRana was changed to the continuous filling
3.4. Performance of SBRana in the three-stage system treating mature landfill leachate The performance of the SBRana in the three-stage SBR system after the combination of three SBRs (in pre-B, ni-D, and ana-D periods) was studied using the mature landfill leachate (Fig. 6). The biodegradable organic substances were effectively degraded in the SBRpre before reaching the SBRana. The ammonia and nitrite removal efficiency was stable and the TN removal efficiency reached above 90% in the SBRana over the 70-day operational period (Fig. 6). The NLR and NRR of the SBRana reached 0.81 and 0.76 kg N/(m3 d), respectively. The NO 3 content of the SBRana effluent was 60 mg/L, þ and the ratio of NO 3 eff to NH4 inf was maintained at 0.2:1, which was lower than the reported values (Strous et al., 1998). The aver þ þ age stoichiometric parameters of NO 2 =NH4 and NO3 =NH4 were 1.32 ± 0.03 and 0.22 ± 0.02, respectively (Fig. 6). The theoretic þ stoichiometric parameter of NO 3 =NH4 was 0.26, which was little higher than the value in this experiment. That because there were the remaining biodegradable organic substances in the effluents of the SBRpre and SBRni, which led to the slight denitrification in the
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even though the landfill leachate was treated by the aeration. The proper concentration of the organic substances (COD) could improve the TN removal efficiency with a minimal inhibition effect on the Anammox process.
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mode (5 h). The effect of this modification on the SBR performance was clear (Table 3). The reaction period of the modified SBRana was 8 h. The ammonia and nitrite concentrations in the SBRana were 48 and 33 mg/L, respectively, after the continuous filling for 5 h. In the rest 3 h, both NHþ 4 —N and NO2 —N were removed, and the effluent NO3 —N was 70 mg/L. In contrast, during the traditional SBRana for 9 h, both NHþ 4 —N and NO2 —N were removed, and the effluent NO —N was 70 mg/L. The nitrogen removal efficiency improved 3 to 11% in the modified SBRana compared to the traditional SBRana because of the high nitrite content. Anammox process is based on the nitrite content, but high nitrite concentration (100 mg/L) could inhibit the Anammox process (Strous et al., 1999). Continuous filling solved the problem of high nitrite concentration in the treatment systems, and reduced the nitrite inhibition on the Anammox process. Therefore, the Anammox activity in the modified SBRana was higher than that in the traditional SBRana, which resulted in shorter reaction time. From the reaction equation of Anammox, during one SBRana cycle, H+ was consumed, which made pH increased. When Anammox was over, pH decreased slightly via hydrolytic acidification. So Anammox process could be monitored via pH profile. The variation in the pH during a typical cycle was different in the modified and traditional SBRana (Table 3). pH is an important factor for Anammox. The activity of the Anammox process was inhibited at a pH higher than 8.0 or lower than 7.5 (Strous et al., 1997). In the continuous filling mode (filling time 5 h), the alkalinity produced by Anammox was neutralized with acids in the influent in the modified SBRana to a relatively stable pH (7.65–7.80), which was suitable for Anammox processes. In the traditional mode (filling time 5 min), pH decreased to 7.4 at the beginning of the cycle, and high concentration nitrite (180 mg/L) started to appear, which could inhibit Anammox processes. The pH profile showed a slight decrease in the first hour of the reaction, but it kept increasing over time. The modified SBRana had two advantages over the traditional mode. First, the continuous filling mode did not result in high nitrite concentration in the SBRana, which decreased the inhibition effects on the Anammox process. Second, the alkalinity produced by Anammox neutralized acids in influent, producing a suitable pH in the modified SBRana for Anammox. Nitrogen removal efficiency improved by 11% in the modified filling mode (filling in 5 h) compared to that of the traditional mode (filling in 5 min), when the influent of the SBRana contained 500 and 350 mg/L of nitrite and ammonia. 3.5. Quantitative microbial analysis of Anammox
Table 4 Abundance of Anammox microorganisms in the SBRana. Samples
Anammox gene numbers (gene copiesg1 dry sludge)
All bacteria gene numbers (gene copiesg1 dry sludge)
Average Anammox gene ratio (%)
Period ana-A Period ana-B Period ana-D
3.18 107 1.56 108 8.19 109
1.40 1010 2.73 1010 1.72 1011
0.23 0.57 4.77
Anammox gene ratio increased from 0.23% in period ana-A to 0.57% in period ana-B. In the stable operation of the three-stage SBR system (ana-D), the Anammox gene numbers reached 8.19 109 gene copies/(g dry sludge), which was similar to previous findings (3.70 107 to 8.64 109 gene copies/(g dry sludge) (Hu et al., 2010). Notably, synthetic wastewater with additional carbon sources was used in previous studies, whereas real mature landfill leachate containing far more complex organic substances was used in this study. Moreover, average Anammox gene ratio also increased from 0.23% to 4.77% in the three-stage SBR system, whereas it was less than 3% in previous studies (Yapsakli et al., 2011).
4. Conclusions A novel integrated three-stage SBR system, comprising SBRpre, SBRni, and SBRana with real-time control was successfully developed in this study for advanced nitrogen removal from mature landfill leachate. The real-time control of pH and ORP can achieve the effective removal of the biodegradable organic substances in SBRpre. Nitritation was achieved in SBRni, and the NO 2 /NOx ratio reached above 95%. The modified continuous filling mode (5 h) in SBRana significantly minimized the nitrite inhibition effects. Quantitative PCR analysis of Anammox showed that the average Anammox gene ratio increased from 0.23% in the startup period to 4.77% in the stable operational period. Acknowledgement The financial support from the Project NSFC (21177005) and scientific research base and scientific innovation platform of Beijing municipal education commission are gratefully acknowledged. References
The quantitative microbial analysis of the Anammox process was studied to determine the variation in functional Anammox microorganisms in the SBRana during the ana-A, ana-B, and ana-D periods. The Anammox gene numbers, all-bacteria gene numbers, and the average Anammox gene ratio were determined (Table 4). In ana-A (acclimation) and ana-B (stable operation of the SBRana), all-bacteria gene numbers increased slightly, whereas the Anammox gene numbers significantly increased. Therefore, average
Table 3 Comparison between the modified and traditional SBRana cycles. Comparisons
Traditional SBRana
Modified SBRana
Influent NHþ 4 —N (mg/L) Influent NO 2 —N (mg/L) Highest NHþ 4 —N in the reactor (mg/L) Highest NO 2 —N in the reactor (mg/L) Reaction period (h) Variation in the pH
350 500 120 180 8 7.65–7.80
350 500 33 48 9 7.37–7.79
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