The present study reports the inhibition kinetics and granular sludge in an anaerobic ammonium oxidation (ANAMMOX) – up-flow anaerobic sludge blanket reactor fed with diluted mature landfill leachate. The activity of ANAMMOX bacteria was inhibited by addition of mature landfill leachate, but gradually adapted to the leachate. The system achieved efficient nitrogen removal during 65–75 d and the average removal efficiencies for NH4+-N, NO2-N and total nitrogen (TN) were 96%, 95% and 87%, respectively. ANAMMOX was the main pathway of nitrogen removal in the system, and heterotrophic denitrification occurred simultaneously. In addition, aerobic ammonia oxidation and aerobic nitrite oxidation were active in this system. Inhibition kinetic experiments showed that the NH4+-N and NO2-N inhibition concentration threshold of ANAMMOX were 489.03 mg/L and 192.36 mg/L, respectively. ANAMMOX was significantly inhibited by mature landfill leachate, and was completely inhibited when the leachate concentration was 1,450.69 mg/L (calculated in chemical oxygen demand). Thus, the inhibition concentration of substrate and landfill leachate should be considered when applying the ANAMMOX process to landfill leachate. The color of granular sludge ANAMMOX changed from brick-red into a reddish-brown. The particle size increased from small to large, with evident granulation of the ANAMMOX sludge.

INTRODUCTION

Landfill leachate has a high concentration of organic matter, ammonia nitrogen, salinity, and various heavy metals (Bodzek et al. 2006; Farah & Christopher 2012). Thus, the untreated leachate would cause harm to the surrounding surface and ground water. According to the time it spent in the landfill, landfill leachate is classified as early, middle and mature. The chemical oxygen demand (COD)/ammonia nitrogen (C/N) of early landfill leachate is higher, and the majority of the organic matter is biodegradable. The C/N of mature landfill leachate is lower, and most organic matter is not biodegradable. Technologies meant for leachate treatment can be classified as biological methods and chemical and physical methods, and the biological methods are the more efficient and cheaper processes to eliminate nitrogen from leachate (Wiszniowski et al. 2006). A lot of significant carbon source is required for nitrogen removal processes of conventional nitrification and denitrification, which results in cost increases (Iaconi et al. 2006). In addition, conventional nitrification and denitrification processes produce a large amount of sludge, which results in increase in the burden of sewage treatment plant. Thus, there is a current need for efficient and low consumption nitrogen removal for mature landfill leachate.

Anaerobic ammonia oxidation (ANAMMOX) is an emerging efficient energy-saving nitrogen removal process. ANAMMOX bacteria use ammonia nitrogen and nitrite nitrogen as the substrate and inorganic carbon as carbon source for autotrophic nitrogen removal (van de Graaf et al. 1995). At present, autotrophic nitrogen removal technology of nitritation combined with an aerobic ammonia oxidation is of great interest in high ammonia nitrogen and low carbon nitrogen ratio wastewater treatment. There are two combination methods used for nitritation and an aerobic ammonia oxidation. In the first method, NO2-N accumulation is first performed and then is mixed with landfill leachate as ρ(NO2-N)/ρ(NH4+-N) at 1:1.32 for an aerobic ammonia oxidation. The second method, the control effluent ρ(NO2-N)/ρ(NH4+-N) is about 1:1.32 in the nitritation stage for an aerobic ammonia oxidation. ANAMMOX process would be appropriate for use in the nitrogen removal of mature landfill leachate (Shen et al. 2012). However, the growth of ANAMMOX bacteria is very slow (Tang et al. 2011; Gilbert et al. 2014). Sludge intercept is essential for the process to function in a stable operation, and granular sludge is the most commonly used method. Most studies have only focused on the effects of factors when landfill leachate was treated (Zhang & Zhou 2006; Liu et al. 2010), and have not studied the granular sludge properties that are essential for effective application of ANAMMOX in engineering applications. Moreover, an additional difficulty in application of this method is that ANAMMOX bacteria are inhibited by organic matter (Liang & Liu 2008; Ni et al. 2012; Alyne et al. 2014; Carlos et al. 2015), salinity (Dapena-Mora et al. 2010; Jin et al. 2013; Liu et al. 2014) and heavy metal (Bi et al. 2014; Zhang et al. 2016) in wastewater. The ANAMMOX bacterial activity is also inhibited by a high concentration of substrate (Tang et al. 2010a; Lotti et al. 2012; José et al. 2013; Li et al. 2016). However, little is known about the in inhibition of the anammox activity by mature landfill leachate.

In this work, we use ANAMMOX granular sludge that was cultivated in the laboratory under an inorganic environment to treat mature landfill leachate. The performance of nitrogen removal, the pathway of nitrogen transformation, and the sludge characteristics were determined. The inhibition of ANAMMOX granular sludge by substrate and landfill leachate were determined and inhibition kinetic models were established. These findings will serve as reference for future application of ANAMMOX to mature landfill leachate treatment.

MATERIALS AND METHODS

Experimental set-up and solutions

  • (1) Continuous-flow experiment: up-flow anaerobic sludge bed (UASB) reactor was adopted as shown in Figure 1(a). The effective volume was 10 L, black soft material was used to reduce light, one third of the upside reactor had spherical polyvinyl chloride filler added to reduce the loss of the sludge, the diameter of the spherical filler was 10 cm, and a fibrous annular braided belt was in the spherical filler. The control temperature was 25 ± 1 °C, the hydraulic retention time (HRT) was 1.2 h, and the pH was 7.5–8.0. The concentration of influent ammonia nitrogen was unchanged during reactor operation, and the concentration of COD was adjusted by addition of the mature landfill leachate. The nitrogen removal performance and the characteristics of ANAMMOX granular sludge were tested in the different stages of the system. The process parameters of continuous-flow in different stages are shown in Table 1.

  • (2) Sequencing batch measurements: 500 mL serum bottle was used as shown in Figure 1(b) to investigate the performance of ANAMMOX, nitrification, and denitrification. Sequencing batch experiments were carried out using ANAMMOX granular sludge collected from UASB at the stable operation period (70 d). Deionized water and phosphate buffer solutions were used to wash the ANAMMOX granular sludge three times to remove residual ammonium. The control temperature was 25 ± 1 °C, the pH was 7.5–7.8, and the rotor speed of the magnetic stirring apparatus was 200 ± 10 r/min. High purity nitrogen (99.999%) was used to remove the dissolved oxygen (DO) from water and maintain an anaerobic environment when the activity tests of ANAMMOX and denitrification were performed. Sufficient oxygen was provided during the activity tests of nitrification.

  • (3) Inhibition kinetics measurements: the tests set-up is also shown in Figure 1(b). The ANAMMOX granular sludge was collected from UASB at 75 d and was cleaned as described above. The concentrations of NH4+-N were 60–1,000 mg/L but the NO2-N concentrations were adjusted to about 100 mg/L when NH4+-N was the single inhibition factor, and the concentrations of NO2-N were 80–500 mg/L but the NH4+-N concentrations were adjusted to about 150 mg/L when NO2-N was the single inhibition factor. The concentrations of mature landfill leachate (calculated in COD) were 0–1,300 mg/L in the landfill leachate inhibition kinetics experiment, and the concentrations of NH4+-N were adjusted with ammonium chloride to about 460 mg/L to eliminate the effect of substrate concentration, and the NO2-N concentrations were adjusted with sodium nitrite to about 100 mg/L. The alkalinity and pH were adjusted by addition of sodium bicarbonate and hydrochloric acid. The experiment was performed in the constant temperature incubator. Samples were removed every 1 h to calculate the NH4+-N and NO2-N removal efficiencies. All tests were repeated three times.

Figure 1

Schematic diagram of tester. (a) ANAMMOX UASB reactor system; (b) sequencing batch test system.

Figure 1

Schematic diagram of tester. (a) ANAMMOX UASB reactor system; (b) sequencing batch test system.

Table 1

The run parameters of ANAMMOX UASB reactor system in different stages

Items time/d ρ(NH4+-N)/mg/L ρ(NO2-N)/ mg/L ρ(NO3-N)/ mg/L ρ(NO2-N)/ρ (NH4+-N) TN volume loading kg /(m3 d) COD volume loading kg /(m3 d) 
1–7 50–60 50–70 4–8 1.23 2.1–2.7 
8–24 50–60 50–70 3–8 1.11 2.1–2.7 0.63 
25–47 50–60 50–70 3–9 1.13 2.1–2.7 1.60 
48–75 50–60 70–90 3–13 1.42 2.7–3.2 1.60 
Items time/d ρ(NH4+-N)/mg/L ρ(NO2-N)/ mg/L ρ(NO3-N)/ mg/L ρ(NO2-N)/ρ (NH4+-N) TN volume loading kg /(m3 d) COD volume loading kg /(m3 d) 
1–7 50–60 50–70 4–8 1.23 2.1–2.7 
8–24 50–60 50–70 3–8 1.11 2.1–2.7 0.63 
25–47 50–60 50–70 3–9 1.13 2.1–2.7 1.60 
48–75 50–60 70–90 3–13 1.42 2.7–3.2 1.60 

Inoculation sludge

The inoculation sludge was ANAMMOX granular sludge that was cultivated under an inorganic environment in a 50 L UASB reactor that had been in stable operation for 2–3 years. The color of the ANAMMOX granular sludge was brick-red. The main species of ANAMMOX bacteria present in the reactor was Candidatus Brocadia fulgida (JX852965-JX8529 69). The inoculation sludge concentration (mixed liquor volatile suspended solids, MLVSS) was about 5 g/L.

Water quality of mature landfill leachate

The mature landfill leachate (more than 5 years old) was taken from Gao An Tun municipal landfill, which was sealed in a plastic drum after retrieval from the landfill and renewed approximately once a month. The water quality was as follows: NH4+-N, 900–1,500 mg/L; NO2-N, 0–2 mg/L; NO3-N, 0–8 mg/L; COD, 2,000–4,000 mg/L; pH, 7.5–8.5; and alkalinity, 6,000–10,000 mg/L. The mature landfill leachate was diluted to the required concentration of ammonia nitrogen and moderate amounts of sodium nitrite were added to serve as an electron acceptor for anaerobic ammonia oxidation.

Analytical methods

NH4+-N, NO2-N, NO3-N, total nitrogen (TN), COD, mixed liquor suspended solids (MLSS) and MLVSS were analyzed according to Standard Methods (APHA 2005). Temperature, DO, and pH values were determined using a WTW/Multi 3420 multiparameter device. For scanning electron microscopy (SEM), a Hitachi S-4300 instrument was used.

Inhibition kinetics of substrate would be described by the Haldane model, the equation as the following (Sheintuch et al. 1995; Surmacz-Gorska et al. 1996): 
formula
1
where ν is the substrate conversion rate, mg/(mg d); νmax is the maximum conversion rate, mg/(mg d); S is the substrate concentration, mg/L; kS is half-saturation constant, mg/L; kh is Haldane inhibiting kinetics constant, mg/L.
Inhibition kinetics by maturel and fill leachate would be described by the model of chlorophenol inhibition kinetics model when acetic acid was degraded (Kim et al. 1997). The equation used was as follows: 
formula
2
where ν is the substrate conversion rate, mg/(mg d); νmax is the maximum conversion rate, mg/(mg d); S is the substrate concentration, mg/L; kS is half-saturation constant, mg/L; k0 and k1 are inhibiting constant.
k0 and k1 are calculated according to the following formula: 
formula
3
 
formula
4
where α is toxic substance concentration, mg/L; β is toxic substance fully inhibition concentration, mg/L; m and n are constant.
The above formula was revised by introduced speed ratio (λ), the revised equation as the follows: 
formula
5
where λ = ν/ν0, λ is speed ratio, ν is the conversion rate under different maturel and fill leachate concentration, mg/(mg d); ν0 is the conversion rate of no maturel and fill leachate, mg/(mg d).

RESULTS AND DISCUSSION

Operation performance of ANAMMOX system

Characteristics of nitrogen removal in ANAMMOX system

Simulated inorganic wastewater was used during phase 1 (0–7 d). The removal efficiencies of NH4+-N, NO2-N and TN were 85%, 92%, and 75%, respectively, at 1 d and increased to 95%, 99%, and 80% respectively, at 7 d. These increased rates indicated that the activity of inoculating ANAMMOX bacteria was robust, and the bacteria were able to quickly adapt to the new environment. The mature landfill leachate was added during phase 2 (8–24 d). The removal efficiencies of NH4+-N, NO2-N and TN decreased to 72%, 79%, and 64%, respectively, at 11 d and returned to 86%, 99%, and 86%, respectively, at 18 d and then continuously operated for 7 d. The concentration of influent mature landfill leachate was increased at 25 d. The activity of the ANAMMOX bacteria was inhibited because of the increase of influent mature landfill leachate concentration, and the removal efficiencies of NH4+-N, NO2-N and TN decreased to 34%, 48%, and 38%, respectively, at 33 d. The ANAMMOX bacteria begin to adapt to the new environment and showed gradually recovery of activity. The removal efficiencies of NH4+-N, NO2-N, and TN reached 71%, 88%, and 73%, respectively, at 47 d. The average influent ratio ρ(NO2-N)/ρ(NH4+-N) was 1.23, 1.11, and 1.13 during the above three phase, smaller than the theoretical ratio (1.32) of the ANAMMOX reaction (Strous et al. 1998). There were parts of NO2-N that would be simultaneously dislodged due to denitrification in the system when landfill leachate was used as the influent, leading to insufficient NO2-N levels. For this reason, the concentration of NO2-N was increased to satisfy to provide sufficient ANAMMOX electron acceptor during phase 4. The average influent ratio ρ(NO2-N)/ρ(NH4+-N) was 1.42, higher than the theoretical ratio of the ANAMMOX reaction. The average removal efficiencies of NH4+-N, NO2-N and TN were 96%, 95% and 87%, respectively, during the 65–75 d period (Figure 2). This suggests that the ANAMMOX bacteria gradually adapted to the presence of the mature landfill leachate, and the system exhibited enhanced nitrogen removal.
Figure 2

The change of influent and effluent nitrogen (a) and their removal rates (b) during the operation phases.

Figure 2

The change of influent and effluent nitrogen (a) and their removal rates (b) during the operation phases.

Stoichiometry relationship of ANAMMOX reaction in system

Stoichiometry reflects the ratio relationship between substrate consumption and production in a system, and can be used to adjust the ratio of influent substrate concentration. The stoichiometry relationship of the ANAMMOX reaction in a system is shown in Figure 3. ρ(NO2-N)/ρ(NH4+-N) was the average ratio of NO2-N and NH4+-N consumed by the ANAMMOX reaction, ρ(NO3-N)/ρ(NH4+-N) was the average ratio of NO3-N that was produced and the NH4+-N that was consumed by ANAMMOX reaction. The ratio of ρ(NO2-N)/ρ(NH4+-N) was 1.30 and the ratio of ρ(NO3-N)/ρ(NH4+-N) was 0.27 in phase 1 (0–7 d), consistent with the theoretical values. The ratio of ρ(NO2-N)/ρ(NH4+-N) was 1.30 and the ratio of ρ(NO3-N)/ρ(NH4+-N) was 0.19 in phase 2 (8–24 d), suggesting the synchronousdenitrification reaction reduced NO3-N in this phase. The ratio of ρ(NO2-N)/ρ(NH4+-N) was 1.43 and the ratio of ρ(NO3-N)/ρ(NH4+-N) was 0.13 in phase 3 (25–47 d), suggesting that the effect of the denitrification reaction increased with the increase in organic matter concentration.
Figure 3

Chemometrics relationship of ANAMMOX reaction during the operation phases.

Figure 3

Chemometrics relationship of ANAMMOX reaction during the operation phases.

The activity of granular sludge and nitrogen transformation pathway

Heterotrophic denitrifying bacteria can grow in the presence of organic matters when the environment is anaerobic. Moreover, the DO of influent was 5–7 mg/L, and there were some aerobic nitrifying bacteria in the system at this time. The performance of ANAMMOX, nitrification, and denitrification were investigated during the stable operation period (65–75 d). The activity of ANAMMOX is shown in Figure 4(a). The degradation rates of NH4+-N and NO2-N were 0.128 and 0.184 g/(g d), and the production of NO3-N was 0.026 g/(g d); the measurements showed linear regression indicating that the rates were constant during the batch experiment, which reflect good data quality (R2=values were 0.987, 0.987 and 0.979). The activity of aerobic ammonia oxidation is shown in Figure 4(b), and the degradation rate of NH4+-N was 0.031 g/(g d). The activity of aerobic nitrite oxide is shown in Figure 4(c), and the degradation rate of NO2-N was 0.010 g/(g d). The activity of denitrification is shown in Figure 4(d) and the degradation rates of NO3-N and NO2-N were very similar, 0.026 g/(g d) and 0.028 g/(g d), respectively. The analyses of granular sludge activity during the stable operation period suggest that ANAMMOX was the major path way of nitrogen removal, but heterotrophic denitrification occurred concurrently. Nitrogen removal performance was analyzed from the nitrogen transformation pathway: ① there were two pathways of NH4+-N removal, one was the ANAMMOX process, and the other was oxidized into NO2-N and NO3-N. ② The main pathway of NO2-N removal was ANAMMOX, and some NO2-N was removed by denitrification and oxidization; ③ NO3-N was mainly produced by ANAMMOX, and influent added NO3-N as well and the main removal pathway was heterotrophic denitrification.
Figure 4

The activity of granular sludge at stable operation period. (a) ANAMMOX; (b) aerobic ammonia oxidation; (c) aerobic nitrite oxide; (d) denitrification.

Figure 4

The activity of granular sludge at stable operation period. (a) ANAMMOX; (b) aerobic ammonia oxidation; (c) aerobic nitrite oxide; (d) denitrification.

Inhibition kinetic characteristics of ANAMMOX

NH4+-N and NO2-N are used as substrate by ANAMMOX bacteria at low concentration, but can inhibit at high concentrations. Most organic matter and heavy metals also act as inhibitors of ANAMMOX bacteria. Mature landfill leachate has characteristics such as high NH4+-N, high amounts of organic matter, and lots of heavy metals. We next investigated the inhibition of ANAMMOX granular sludge by substrate and landfill leachate.

Substrate inhibition and its dynamics

The effect of ANAMMOX inhibition by substrate concentration is shown in Table 2. NH4+-N and NO2-N removal rate was increased at first and then decreased as the experimental substrate concentration increased. The highest NH4+-N removal rate was 0.1540 mg/(mg d) when NH4+-N concentration was 295.62 mg/L, and decreased to 0.1455 mg/(mg d) when the NH4+-N concentration was increased to 930.51 mg/L. The NO2-N removal rate was 0.1649 mg/(mg d) when NO2-N concentration was 151.10 mg/L, and decreased to 0.1395 mg/(mg d) when the NO2-N concentration was increased to 497.82 mg/L. The inhibition of ANAMMOX by NH4+-N and NO2-N is essentially by free ammonia (FA) and free nitrous acid (FNA) (Waki et al. 2007; Fernández et al. 2010). Ammonium has the equilibrium reaction in an aqueous solution: NH4+ + OH ⇋ NH3 + H2O. Similarly, nitrite has the equilibrium reaction in aqueous solution: NO2 + H+⇋HNO2. Both dynamic balances will change as pH value changes. Unprotonated FA and FNA can penetrate the lipid membrane, but NH4+ and NO2 penetrate less easily (Kadam & Boone 1996). Most reports have proposed that it is ammonia (NH3) that serves as a substrate for microorganisms (Tang et al. 2010b).

Table 2

Comparison of kinetic characteristics parameters

  Km/(mg/L)
 
Kh/(mg/L)
 
  
Sludge NH4+-N NO2-N NH4+-N NO2-N References 
HAG 17 19 11,679 735 Tang et al. (2013)  
FAS 25 21 9,016 179 Tang et al. (2013)  
IG 22 21 10,138 933 Chen et al. (2016
ANAMMOX granular 39 43 3,482 701 This study 
  Km/(mg/L)
 
Kh/(mg/L)
 
  
Sludge NH4+-N NO2-N NH4+-N NO2-N References 
HAG 17 19 11,679 735 Tang et al. (2013)  
FAS 25 21 9,016 179 Tang et al. (2013)  
IG 22 21 10,138 933 Chen et al. (2016
ANAMMOX granular 39 43 3,482 701 This study 

The experimental results of substrate inhibition were nonlinearly fitted according to Equation (1) and using Origin 8.0 as shown in Figure 5. The correlation coefficients (R2) were 0.9901 and 0.9985. This high correlation indicated that the Haldane model well described the inhibition behavior of the two inhibitory factors well. The maximum ammonia oxidation rate was 0.1893 mg/(mg d) and the Haldane inhibiting kinetics constant was 3,482.27 mg/L (FA was 151.16 mg/L) when ammonium was the single inhibiting factor. The maximum NO2-N removal rate was 0.246 mg/(mg d) and Haldane inhibiting kinetics constant was 701.15 mg/L (FNA was 0.1056 mg/L) when nitrite was the single inhibiting factor. This indicates that ANAMMOX bacteria were more significantly inhibited by NO2-N. Tang et al. (2013) and Chen et al. (2016) have studied high-rate anammox granules (HAG), flocculent anammox sludge (FAS) and immobilized granules (IG) in a UASB reactor and tested the kinetic characteristics which are shown in Table 2. The different kinetic characteristics parameters have been obtained. The difference might be caused by the complicated composition of the bacteria in different incubator sludge that could result in a large number of denitrifying bacteria and anaerobic methane bacteria, which would affect kinetic characteristics tests.
Figure 5

The substrate inhibition kinetic characteristics.

Figure 5

The substrate inhibition kinetic characteristics.

Mature landfill leachate inhibition and its dynamics

The composition of landfill leachate is complex, and includes a lot of toxic organic matter, salt ions and heavy metals that could act to inhibit ANAMMOX bacteria. ANAMMOX performance was investigated over a range of maturel and fill leachate concentration of 0–1,262.37 mg/L (calculated in COD). The ammonia oxidation rate was reduced to 16.16% at the highest leachate concentration.

The experimental results of mature landfill leachate inhibition were nonlinearly fitted by Origin 8.0 as shown in Figure 6. The correlation coefficient (R2) for the fit was 0.9714, indicating that a good fit of the observed data to the inhibition model. The fully inhibitory concentration of mature landfill leachate was 1,450.69 mg/L (calculated in COD). Kinetic constants m and n were 2.49 and 0.99 respectively.
Figure 6

The landfill leachate inhibition kinetic characteristics.

Figure 6

The landfill leachate inhibition kinetic characteristics.

The equations and parameters of the inhibition kinetics are shown in Table 3.

Table 3

Inhibition kinetics models and parameters

Inhibition type Fitted equation Parameter 1 Parameter 2 Parameter 3 R2 
NH4+-N y = 0.1893/(1 + 39.39/x + x/3,482.27) νmax = 0.1893 ks = 39.39 kh = 3,482.27 0.9901 
NO2-N y = 0.246/(1 + 43.19/x + x/701.15) νmax = 0.246 ks = 43.19 ka = 701.15 0.9985 
Mature landfill leachate y = [1-(x/1,450.69)]2.49/ [1 + (x/1,450.69)]0.99 β = 1,450.69 m = 0.99 n = 2.49 0.9714 
Inhibition type Fitted equation Parameter 1 Parameter 2 Parameter 3 R2 
NH4+-N y = 0.1893/(1 + 39.39/x + x/3,482.27) νmax = 0.1893 ks = 39.39 kh = 3,482.27 0.9901 
NO2-N y = 0.246/(1 + 43.19/x + x/701.15) νmax = 0.246 ks = 43.19 ka = 701.15 0.9985 
Mature landfill leachate y = [1-(x/1,450.69)]2.49/ [1 + (x/1,450.69)]0.99 β = 1,450.69 m = 0.99 n = 2.49 0.9714 

Characteristics of granular sludge

Apparent characteristics

The particle size was small and the color of ANAMMOX granular sludge was brick-red at the start of the experiment (0 d; Figure 7(a)) due to the high concentration of ferroheme in the bacteria cells (Schalk et al. 2000; Shimamura et al. 2007). The color of ANAMMOX granular sludge deepened to crimson and granular sludge particle size was increased at 35 d (Figure 7(b)). At 70 d, the color of ANAMMOX granular sludge was reddish-brown and there were filamentous bacteria on the external surface (Figure 7(c)). The explanation for the deepening color change maybe due to: ① the covering of ANAMMOX bacteria by heterotrophic bacteria that grew on granular sludge surface; or ② adsorption of dark impurities from the landfill leachate on the granular sludge surface. The SEM photograph shows the granular sludge at 70 d. There are many filamentous bacteria evident on the surface of the granular sludge as shown in Figure 8(a), and the leachate organic matter may have stimulated growth of the filamentous heterotrophic bacteria. Under magnification, rod-shaped bacteria and spherical bacteria are evident (Figure 8(b)), possibly nitrobacteria. Nitrobacteria on the ANAMMOX granular sludge surface can relieve DO inhibition when present in the influent (Vázquez-Padín et al. 2009; Cho et al. 2011). ANAMMOX bacteria are spherical bacteria and the surface structure is crateriform (van de Graaf et al. 1996). Most of the ANAMMOX bacteria are located within the granular sludge and heterotrophic bacteria and nitrobacteria were on the surface. In addition, there were many holes in the sludge that would act to increase the mass-transfer effect of the sludge (An et al. 2013). These holes may arise as N2 is released after being produced by ANAMMOX bacteria inside the sludge (Bhunia & Ghangrekar 2007; Batstone & Keller 2001).
Figure 7

Photograph of ANAMMOX granular sludge at different period. (a) 0 d; (b) 35 d; (c) 70 d.

Figure 7

Photograph of ANAMMOX granular sludge at different period. (a) 0 d; (b) 35 d; (c) 70 d.

Figure 8

SEM photograph of ANAMMOX granular sludge at 70 d. (a) ×2.0 k; (b) ×6.0 k.

Figure 8

SEM photograph of ANAMMOX granular sludge at 70 d. (a) ×2.0 k; (b) ×6.0 k.

Particle size distribution

The granular sludge particle size distribution was characterized as the percentage of MLSS of the different particle size and total MLSS. As shown in Table 4, the difference in granular sludge particle size was smaller at 0 d than at 35 d and 70 d. At 0 d, 27.3% of the granular sludge particles were in the range of 0.5–1.0 mm, 24.4% were 1.5–2.0 mm (24.4%), and 14.2% were less than 0.5 mm. At 35 d, 43.4% were 1.5–2.0 mm in size and 25.2% were 2.0–2.5 mm. There was little change in the granular sludge particle size distribution at 70 d compared with that at 35 d, and the majority of the particles were in the range of 1.5–2.5 mm. Thus, the ANAMMOX granular sludge particle size increased and then stabilized under the experimental condition. This could be due to the following reasons: ① the granulation of sludge could be related to up-flow velocity (O'Flaherty et al. 1997; Alves et al. 2000) or ② the growth of heterotrophic bacteria on the granular sludge surface could alter the particle size, the removal efficiencies of COD was 10–25% which could give a better indication on the heterotrophs’ rate. Kindaichi et al. (2007) proposed that ANAMMOX activity occurs in the 1 mm sized granular sludge particles, and larger sizes will suffer from low substrate concentration in the interior, reducing nitrogen removal performance. The ANAMMOX granular sludge particle size increased in this study although the smaller sludge particle size have the better mass transfer efficiency and the bigger specific surface area. Generally speaking, the larger size granular sludge is better able to resist adverse conditions. The inhibitors may be trapped on the granular sludge surface and the impact of internal ANAMMOX bacteria would be reduced. In a word, ANAMMOX granular sludge were mainly concentrated in 0.5–2.0 mm particles in the early stage and were mainly concentrated in 1.5–2.5 mm particles after domestication with maturel and fill leachate.

Table 4

Granule size distribution in ANAMMOX UASB reactor during different period

Granule size (mm) 0 d 35 d 70 d 
<0.5 14.2 1.1 1.5 
0.5–1.0 27.3 7.8 7.1 
1.0–1.5 15.2 9.6 10.7 
1.5–2.0 24.4 43.4 40.8 
2.0–2.5 13.5 25.2 25.3 
>2.5 5.4 12.9 14.6 
Granule size (mm) 0 d 35 d 70 d 
<0.5 14.2 1.1 1.5 
0.5–1.0 27.3 7.8 7.1 
1.0–1.5 15.2 9.6 10.7 
1.5–2.0 24.4 43.4 40.8 
2.0–2.5 13.5 25.2 25.3 
>2.5 5.4 12.9 14.6 

CONCLUSIONS

The mature landfill leachate was used to domesticate the ANAMMOX bacteria cultivated under inorganic conditions. After 75 d of running, the system gradually adapted to implement efficient denitrification of landfill leachate. The system improved nitrogen removal during 65–75 d. During the stable operation period, the average removal efficiencies of NH4+-N, NO2-N and TN were 96%, 95%, and 87%, respectively. ANAMMOX was still the main pathway of nitrogen removal in the system with concurrent heterotrophic denitrification. Aerobic ammonia oxidation and aerobic nitrite oxide showed activity of 0.031 g/(g d) and 0.010 g/(g d) in the system. ANAMMOX inhibition kinetics experiments showed that the inhibition concentration thresholds for NH4+-N and NO2-N were 489.03 mg/L and 192.36 mg/L, respectively, the half-saturated constants were 39.39 mg/L and 43.19 mg/L, respectively, and the inhibiting kinetic constants were 3,482.27 mg/L and 701.15 mg/L, respectively. ANAMMOX was significantly inhibited by mature landfill leachate, and activity was almost completely inhibited at a leachate concentration of 1,450.69 mg/L (calculated in COD). The color of the granular sludge ANAMMOX changed from brick-red to a reddish-brown. The particle size grew from small to large and then stabilized. During the stable operation period, particles greater than 1.5 mm in size comprised 80.7% of the total, and there were large numbers of holes in the granular sludge that facilitate mass-transfer. Rod-shaped bacteria, spherical bacteria and filamentous bacteria were detectable on the surface. Thus, the inhibition concentration of substrate and landfill leachate must be considered in the design of strategies to use the ANAMMOX process for treatment of landfill leachate.

ACKNOWLEDGEMENTS

This work was financially supported by the Chinese Critical Patented Project of the Control and Management of National Polluted Water Bodies (2014ZX07201-011), the Building Water System Microcirculation Reconstruction Technology Research and Demonstration Project (2014ZX07406002), and the National Natural Science Foundation of China (51308010).

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