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Inhibition kinetics of nitritation and half-nitritation of old landfill leachate in a membrane bioreactor
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e7, 2016 www.elsevier.com/locate/jbiosc
Inhibition kinetics of nitritation and half-nitritation of old landfill leachate in a membrane bioreactor Li Yun,1 Li Jun,1, * Wang Zhaozhao,2 Wei Jia,1 Zhang Yanzhuo,1 and Zhao Baihang1 College of Architecture and Civil Engineering, Beijing University of Technology, Beijing 1000124, China1 and College of Energy and Environmental Engineering, Hebei University of Engineering, Handan, 056038 Hebei, China2 Received 5 July 2016; accepted 18 November 2016 Available online xxx
Nitritation can be used as a pretreatment for anaerobic ammonia oxidation (anammox). Various control strategies for nitritation and half-nitritation of old landfill leachate in a membrane bioreactor were investigated in this study and the inhibition kinetics of substrate, product and old landfill leachate on nitritation were analyzed via batch tests. The results demonstrated that old landfill leachate nitritation in the membrane bioreactor can be achieved by adjusting the influent loading and dissolved oxygen (DO). From days 105e126 of the observation period, the average effluent concentration was 871.3 mg/L and the accumulation rate of NO2 L LN was 97.2%. Half-nitritation was realized quickly by adjusting hydraulic retention time and DO. A low-DO control strategy appeared to best facilitate long-term and stable operation. Nitritation inhibition kinetic experiments showed that the inhibition of old landfill leachate was stronger than that of the substrate ðNH4 D LNÞ or product ðNO2 L LNÞ. The ammonia oxidation rate dropped by 22.2% when the concentration of old landfill leachate (calculated in chemical oxygen demand) was 1600.2 mg/L; further, when only free ammonia or free nitrous acid were used as a single inhibition factor, the ammonia oxidation rate dropped by 4.7e6.5% or 14.5e15.9%, respectively. Haldane, Aiba, and a revised inhibition kinetic model were adopted to separately fit the experimental data. The R2 correlation coefficient values for these three models were 0.982, 0.996, and 0.992, respectively. Ó 2016, The Society for Biotechnology, Japan. All rights reserved. [Key words: Old landfill leachate; Membrane bioreactor; Nitritation; Half-nitritation; Inhibition kinetics]
Landfill leachate is a complex composition containing high levels of ammonia nitrogen in addition to toxic substances such as toxic organic matter and heavy metals (1,2). The organic matter of fresh landfill leachate shows good biodegradability and can be used in denitrification, which makes it well-suited to nitrogen removal. The biodegradability of organic matter in old landfill leachate is very poor, however, and may require expensive additional organic carbon sources for a conventional biological nitrogen removal process. In short: There is high demand for a cost-effective and energy-efficient nitrogen removal method. Autotrophic nitrogen removal methods combining nitritation with anaerobic ammonia oxidation have garnered substantial research interest in high ammonia nitrogen and low carbon nitrogen ratio wastewater treatment (3,4). Existing combination methods can be split into two categories. In the first, NO2 N accumulation is performed before old landfill leachate as CðNO2 NÞ=CðNH4 þ NÞ is mixed in at 1:1.32 for anaerobic ammonia oxidation. In the second category, the control effluent CðNO2 NÞ=CðNH4 þ NÞ is about 1:1.32 in the nitritation stage for anaerobic ammonia oxidation. Both methods necessitate stable nitritation, which requires the interception of ammonia oxidizing bacteria (AOB) and the elimination of nitrite oxidizing bacteria (NOB). AOB are autotrophic and slow-growing (5). Membrane bioreactor (MBR) can efficiently intercept sludge and rapid concentrate AOB to truncate the amount of time necessary for * Corresponding author. Tel./fax: þ86 01067391726. E-mail address: [email protected] (L. Jun).
nitritation. There have been several studies on MBR nitritation, but only in regards to treating wastewater with low concentrations of ammonia nitrogen (6,7), while the concentration of ammonia nitrogen is generally greater than 1000 mg/L in old landfill leachate. There have been few studies on the nitritation of old landfill leachate using MBR reactors. Nitritation can be realized quickly if the appropriate temperature, pH, and dissolved oxygen (DO) are controlled (8,9). Halfnitritation can be controlled by hydraulic retention time (HRT), DO, or alkalinity if nitritation has been effectively realized (10,11). To achieve nitritation and allow stable operation of the MBR reactor, the operating conditions must be controlled and the influent loading gradually increased. HRT and DO should be separately adjusted to allow half-nitritation and stable operation. Old landfill leachate exhibits high ammonia nitrogen and toxic effects on microbes. High NH4 þ N and NO2 N accumulation causes a high concentration of free ammonia (FA) and free nitrous acid (FNA) that inhibits the activity of AOB and NOB (12,13). The organic matter and heavy metals in old landfill leachate also are toxic to AOB and NOB (14,15). There have been several studies on substrate inhibition kinetics (16), but few on product or old landfill leachate inhibition kinetics for nitritation. In an effort to fill this research gap, we determined the nitritation inhibition kinetics characteristics of substrates ðNH4 þ NÞ, product ðNO2 NÞ, and old landfill leachate through batch experiments. Our primary goal is to provide guidance for the practical application of old landfill leachate nitritation in MBRs.
1389-1723/$ e see front matter Ó 2016, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2016.11.007
Please cite this article in press as: Yun, L., et al., Inhibition kinetics of nitritation and half-nitritation of old landfill leachate in a membrane bioreactor, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.007
2
YUN ET AL.
J. BIOSCI. BIOENG.,
FIG. 1. Schematic diagram of MBR reactor. FIG. 2. Performance of nitritation during start-up.
MATERIALS AND METHODS Experimental set-up and operational conditions An MBR reactor made from organic glass with an effective volume of 25 L was used for these experiments (as shown in Fig. 1). The membrane component is a hollow fiber membrane made from polyvinylidene fluoride with a pore size and area of 0.1 mm and 0.5 m2, respectively. The influent and effluent pumps were controlled using a programmable logic controller. The membrane was operated at a flux of 2.28 L/ (m2$h) with intermittent suction (8 min suction, 2 min relaxation). The aeration rates were 40e160 L/h and controlled via rotor flowmeter throughout the experiment. The transmembrane pressure (TMP) was measured with a vacuum gauge (JZ 00000578, Tianjin Ji Xing Company, China). The temperature was controlled at (30 1) C using a heater. The HRT was 22 h, and there was no sludge discharge during the experiment. Characteristics of old landfill leachate and seed sludge The seed sludge (nitrification sludge) was taken from Gao Bei Dian wastewater treatment plant in Beijing, which employs a typical anoxic-aerobic process for treating municipal wastewater and achieves satisfactory biological nitrogen removal. Initial inoculation sludge concentration mixed liquor suspended solids (MLSS) was 3104 mg/L and mixed liquor volatile suspended solids (MLVSS) was 2540 mg/L. The old (over 5 years) landfill leachate was taken from Gao An Tun municipal landfill, stored in sealed in plastic drums, and renewed once per month during the experiment. The specific water quality is as follows: NH4 þ N, 900e1500 mg/L; NO2 N, 0e2 mg/L; NO3 N, 0e8 mg/L; chemical oxygen demand (COD), 2000e4000 mg/L; pH, 7.5e8.5; alkalinity, 6000e10,000 mg/L. Inhibition kinetics experiments The nitritation-activated sludge samples were separately cleaned 3e5 times with deionized water and phosphate buffered saline (PBS) after removal from the MBR at 71 d. The level of MLSS was about 5.04 g/L after concentration. For each batch experiment, 150 mL of concentrated sludge was transferred to a beaker and diluted to 1 L. The concentration of NH4 þ N was 60e1000 mg/L when NH4 þ N was the single inhibition factor, and the concentration of NO2 N was 50e700 mg/L but the NH4 þ N concentration was adjusted to about 180 mg/L when NO2 N was the single inhibition factor. In order to eliminate the influence of substrate inhibition, ammonium chloride was used to adjust the NH4 þ N concentration to match the concentration during the old landfill leachate inhibition experiments. Sodium bicarbonate and hydrochloric acid were used to adjust the alkalinity and keep the pH constant. All experiments were conducted in a constant temperature incubator which was controlled at (30 1) C temperature. Sampling intervals were 30 min. Ammonia oxidation rate, NO2 N generation rate, and NO3 N generation rate were measured and calculated in triplicate. Inhibition kinetics models The inhibition kinetics of the substrate is described by the Haldane model as follows (17):
n ¼
nmax S ks þ S þ kS
2
(1)
h
where n is the substrate conversion rate, g/(g$d); nmax is the maximum conversion rate, g/(g$d); S is the substrate concentration, mg/L; kS is half-saturation constant, mg/L; kh is Haldane inhibiting kinetics constant, mg/L. The inhibition kinetics of the product is described by the Aiba model (18), which was originally used to describe ethanol fermentation product inhibition. The equation as the follows: S n ¼ exp ks þ S ka
nmax S
(2)
where n is the substrate conversion rate, g/(g$d); nmax is the maximum conversion rate, g/(g$d); S is the substrate concentration, mg/L; kS is half-saturation constant, mg/L; ka is Aiba inhibiting kinetics constant, mg/L. The inhibition kinetics of old landfill leachate is described by chlorophenol inhibition kinetics model when acetic acid was degraded (19). The equation as the follows:
n ¼
nmax ½k0 Sx
(3)
ks ½k1 þ S
where n is the substrate conversion rate, g/(g$d); nmax is the maximum conversion rate, g/(g$d); S is the substrate concentration, mg/L; kS is half-saturation constant, mg/L; k0 and k1 are inhibiting constant. k0 and k1 are calculated according to the following formula: k0 ¼ ½1 a=b
m
k1 ¼ ½1 a=b
(4)
n
(5)
where a is toxic substance concentration, mg/L; b is toxic substance fully inhibition concentration, mg/L; m and n are constant. The above formula was revised by introducing the speed ratio (l) as follows:
l ¼
1 ½a=b m 1 þ ½a=b n
(6)
where l ¼ n/n0, l is speed ratio, n is the conversion rate under different old landfill leachate concentration, g/(g$d); n0 is the conversion rate of no old landfill leachate, g/(g$d). Chemical analysis and calculation procedures NH4 þ N, NO2 N, NO3 N, chemical oxygen demand (COD), MLSS and MLVSS were analyzed according to standard methods (20). pH and temperature were determined by WTW/ Multi3420 tester. NO2 N accumulation rate (R), free ammonia (FA) and free nitrous acid (FNA) were calculated according to the following equations: R ¼
C½NO C½NO
2
FA ¼
FNA ¼
N
2 N þ C½NO
3
N
100%
(7)
C½NH4 þ N 10pH 17 ½6344=ð273þTÞ 14 e þ10pH
(8)
C½NO N 47 2 14 e½2300=ð273þTÞ 10pH
(9)
where C½NH4 þ N, C½NO2 N and C½NO3 N are the effluent concentration of NH4 þ N, NO2 N and NO3 N, respectively, mg/L; T is temperature, C.
RESULTS AND DISCUSSION Nitritation start-up under low DO conditions It is generally accepted that NOB are inhibited and AOB are concentrated when the appropriate temperature, pH, and DO are controlled to realize
Please cite this article in press as: Yun, L., et al., Inhibition kinetics of nitritation and half-nitritation of old landfill leachate in a membrane bioreactor, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.007
HALF-NITRITATION OF OLD LANDFILL LEACHATE IN MBR
HRT
DO
3
100 60 40 20 0 6 4 2
130
140 +
inf.NH4 -N -
eff.NO2 -N
150
160
t (d) + eff.NH4 -N -
eff.NO3 -N
170
180
190
+
NH4 -N removal -
NO2 -N conversion
Conversion (%)
80
DO (mg/L)
1200 1000 800 600 400 200 0 20 15 10 5 0
HRT (h)
Nitrogen Concentration (mg/L)
VOL. xx, 2016
0 200 HRT DO
FIG. 3. Performance of nitritation during load increase period. FIG. 4. Performance of half-nitritation.
nitritation. The optimal conditions are 30e35 C (8), pH value close to 8.0 (12), and DO less than 1 mg/L (21). To quickly begin nitritation in this experiment, the conditions were controlled at (30 1) C, pH 7.8e8.2, and DO of 0.5e1 mg/L. The start-up period was divided into two phases: The synthetic wastewater was used to begin nitrification in the first phase (0e18 d) when the influent concentration of NH4 þ N was 280 20 mg/L, and the old landfill leachate was used in the second phase (19e32 d) at influent concentration of NH4 þ N diluted to 280 20 mg/L. The performance of nitritation during the start-up period in the MBR system is shown in Fig. 2. In the first phase, the effluent concentration of NO3 N was reduced and the concentration of NO2 N increased gradually. These observations suggested that NOB activity was significantly inhibited and AOB activity gradually improved when the experimental temperature, DO, and pH value were controlled appropriately as the nitrification process shifted towards nitritation. At 15 and 17 d, the NO2 N accumulation rates were 50.7% and 53.5%, respectively; this suggests that AOB was the dominant bacteria in the system. Shortcut nitrification was achieved with NO2 N accumulation rate greater than 50%. The diluted old landfill leachate was applied from 19 d, and microbial activity was inhibited at the beginning of the experiment due to the many toxic substances in old landfill leachate. The effluent NH4 þ N concentration was raised from 5.2 mg/L to 74.2 mg/L and the removal rate of NH4 þ N fell from 98.2% to 75.5%. Subsequently, microbial activity was gradually restored and the removal rate of NH4 þ N increased to 93.6% at 32 d, suggesting that the microorganisms gradually adapted to the presence of old landfill leachate in the system. The NO2 N accumulation rate also gradually increased throughout the start-up process, with a value of 84.3% at 32 d, indicating that nitritation was successfully initiated. Performance of nitritation during loading increase stage AOB replaced NOB and became the dominant bacteria while NO2 N gradually accumulated throughout the successful nitritation start-up in the system. Inhibiting factors of NOB include temperature, pH, FA, FNA, and DO. Low DO not only inhibits NOB but also affects AOB activity (22), while high DO can still maintain nitritation in the presence of the other inhibiting factors. DO was raised accordingly to 2e3 mg/L and the loading continually increased to control the other conditions. The period in which old landfill leachate loading was gradually increased was divided into three stages. The first, second, and third stages were 33e58 d, 59e80 d, and 81e112 d, respectively; the
influent concentration of NH4 þ N at each stage was 550e600 mg/ L, 750e850 mg/L, and 950e1050 mg/L, respectively. The nitritation performance of the loading increase period is shown in Fig. 3. The increased loading led to increase in effluent NH4 þ N concentration, because microbial biomass did not increase corresponding to loading though the inhibition of microbial activity did. Effluent NH4 þ N concentration gradually decreased and NH4 þ N removal rate increased as the system operation progressed after the loading increased. The effluent NO2 N concentration showed the same variation trend as effluent NH4 þ N, gradually increasing over the operation period e the effluent NO2 N concentration was 866.2 mg/L and accumulated NO2 N was 98.2% at 105 d, which suggests that AOB gradually adapted to the presence of the old landfill leachate and the population increased as the system was continuously run. Although the DO was 2e3 mg/L during the whole period, NO3 N did not over-accumulate and the range of effluent NO3 N concentration was 30e50 mg/L, suggesting that DO did not play a key role in NO2 N accumulation. Half-nitritation control strategy and stable operation Three control strategies currently exist for halfnitritation when nitritation has been realized: HRT, DO, and alkalinity control (23,24). The alkalinity of old landfill leachate is generally high, so alkalinity control is infeasible for old landfill leachate. Old landfill leachate allowed nitritation in the MBR reactor. The effluent NO2 N average concentration was 871.3 mg/L during 105e126 d and the average accumulation rate was 97.2%. The influent and effluent were adjusted to control HRT while the other conditions were kept unchanged (as shown in Fig. 4). The effluent NH4 þ N increased and NO2 N and HRT decreased with increase in influent and effluent until half-nitritation was effectively realized. Effluent NH4 þ N concentration was 362.6 mg/L, NO2 N was 513.1 mg/L, HRT was 13.9 h, and CðNO2 NÞ=CðNH4 þ NÞ was 1.42 e close to the anaerobic ammonia oxidation theoretical ratio (1.32). The reactor was operated for 25 days while HRT was 13.9 h and the average CðNO2 NÞ=CðNH4 þ NÞ was 1.38. Effluent NH4 þ N increased and NO2 N decreased between 149 and 156 d, however, suggesting that half-nitritation could be quickly and easily realized but may be difficult to stably maintain over long-term operation under HRT control once nitritation has been realized. This may be because AOB increases quickly given adequate nutrients and appropriate conditions. To this effect, HRT control should be considered just one part of a coordinated control
Please cite this article in press as: Yun, L., et al., Inhibition kinetics of nitritation and half-nitritation of old landfill leachate in a membrane bioreactor, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.007
YUN ET AL.
J. BIOSCI. BIOENG., Load increase period
Set-up period
10 DO
HRT
TMP MLSS
MLSS (g/L)
8
TABLE 1. Effect of FA, FNA and toxicant on nitritation.
Half nitrosation
60
Inhibiting factors
50
FA
40
6
30 4
TMP (kPa)
4
20 2 0
10 0
40
80 t (d)
120
160
FNA
0
FIG. 5. Evolution of TMP and MLSS during the operation phases.
strategy to quickly realize half-nitritation rather than a single control strategy. The nitritation of old landfill leachate was gradually recovered between 157 and 164 d and HRT increased to 22 h. DO was employed as a substrate of AOB and NOB during aerobic nitritation; the effect of nitritation is affected by DO (9). To achieve halfnitritation, DO could be slowly reduced to control the effluent concentration of NH4 þ N and NO2 N in the system. The effluent concentration of NH4 þ N and NO2 N changed from 139.4 mg/L and 834.5 mg/L, respectively, to 382.2 mg/L and 546.7 mg/L, respectively, as DO was gradually reduced from 2.52 mg/L to 0.93 mg/L during 165e171 d. The effluent CðNO2 NÞ=CðNH4 þ NÞ was 1.43 at 171 d and DO was steady at 0.75e1.25 mg/L. With other conditions unchanged, the system was stably operated for 29 days. The range of effluent CðNO2 NÞ=CðNH4 þ NÞ was 1.14e1.57 with an average value of 1.35 during the stable operation stage (171e199 d); the NO2 N average accumulation rate was 94.8%. Half-nitritation was quickly and easily achieved while long-term and stable operation using DO control with nitritation was effectively realized. The activity and growth rate of AOB increases when DO is high and decreases when DO is low (21), so half-nitritation was operated stably under low DO. AOB shows a high affinity to oxygen compared to NOB (25). In our experiment, the DO saturation constants of AOB and NOB were 0.2e0.4 mg/L and 1.2e1.5 mg/L, respectively (26). AOB becomes the dominant bacteria under low DO conditions (22,27), however, NO3 N did not accumulate at high DO (2e3 mg/L) or at low DO (0.75e1.25 mg/L) during this experiment within an effluent NO3 N concentration range of 30e60 mg/L. This is likely because NOB was inhibited by factors other than DO, such as FA, FNA, temperature, or pH (28). Sludge and membrane fouling characteristics during the experiment The extent of pollution in the membrane module is usually characterized by transmembrane pressure (TMP) during operation (29). TMP increases as the pollution level increases in the membrane module operating at constant flux. As discussed above, the nitritation process was divided into a start-up period, loading increase period, and half-nitritation control period; the loading increase period was further divided into three sub-stages. The TMP overall during the whole period is shown in Fig. 5, where membrane fouling rate accelerates with increase in influent loading due to the presence of organic matter and metal ions in the old landfill leachate.
Old landfill leachate (COD)
Concentration (mg/L)
Ammonia oxidation rate [g/(g$d)]
NO2 N generation rate [g/(g$d)]
NO3 N generation rate [g/(g$d)]
2.30 5.73 9.45 11.75 16.71 21.10 24.55 29.78 34.12 38.66 42.38 0.0080 0.0160 0.0317 0.0506 0.0576 0.0779 0.0872 0.1036 0.00 133.35 266.70 533.40 800.10 1066.80 1250.00 1600.20 2133.60 2667.00
0.9039 1.3868 1.4296 1.4364 1.4187 1.3820 1.3684 1.3432 1.2854 1.3058 1.2480 1.4027 1.4112 1.3956 1.3222 1.2371 1.2310 1.2072 1.1875 1.2650 1.2528 1.2310 1.2365 1.2215 1.1752 1.0746 0.9848 0.7651 0.5611
0.7825 1.2618 1.2994 1.3228 1.3102 1.2811 1.2680 1.2605 1.2086 1.2292 1.1751 1.2774 1.2869 1.2683 1.2202 1.1350 1.1517 1.1217 1.1142 1.1609 1.1557 1.1437 1.1544 1.1374 1.0859 1.0061 0.9201 0.7103 0.5238
0.1252 0.1231 0.1273 0.1148 0.1024 0.1020 0.0970 0.0836 0.0768 0.0724 0.0687 0.1290 0.1263 0.1246 0.1070 0.1042 0.0819 0.0836 0.0699 0.1017 0.0971 0.0903 0.0818 0.0804 0.0857 0.0722 0.0614 0.0516 0.0448
Extracellular polymers (EPS) and soluble microbial products (SMP) are released by microbes when conditions change (30) and membrane fouling rate is accelerated by the accumulation of ESP and SMP. The TMP increased from 0 kPa to 48 kPa at 16 d of membrane module operation during the HRT control stage, and the TMP increased from 0 kPa to 14 kPa when membrane module was operated for 16 d during the DO control stage. In other words, the membrane fouling rate during the HRT control stage was significantly greater than that during the DO control stage in the halfnitritation experiment. Changes in the sludge concentration in the test system are depicted in Fig. 5. MLSS decreased gradually during the nitritation start-up period due to the elimination of heterotrophic bacteria and NOB caused by in organics in the influent, as well as the increasing death and lysing of bacteria due to old landfill leachate toxicity. MLSS gradually increased in the system during the loading increase period because the microorganisms had adapted to the old landfill leachate, allowing for growth of aerobic heterotrophic bacteria and AOB. Inhibition kinetic characteristics of nitritation Nitritation was affected by the substrate ðNH4 þ NÞ, product ðNO2 NÞ, and old landfill leachate, as shown in Table 1. The ammonia oxidation rate and NO2 N generation rate increased at first and then decreased with increasing FA concentration from 2.9 to 42.4 mg/ L, while NO3 N generation rate gradually decreased. The ammonia oxidation rate was 1.436 g/(g$d) when FA concentration was 11.8 mg/L, and decreased to 1.248 mg/L at 42.4 mg/L FA. NH4 þ N was oxidized to NO2 N and NO3 N by nitrifying the bacteria that use its energy for growth. NOB activity was inhibited by FA formed from high NH4 þ N, allowing AOB to gradually replace NOB as the dominant bacterial community (31). Excessive FA can also suppress AOB, but to a lesser extent. Vadivelu et al. (32) reported inhibition of NOB at FA levels of 0.1e1 mg/L, with complete inhibition of NOB at 6 mg/L FA, but only saw inhibition of AOB at 10e150 mg/L FA (33). In our
Please cite this article in press as: Yun, L., et al., Inhibition kinetics of nitritation and half-nitritation of old landfill leachate in a membrane bioreactor, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.007
VOL. xx, 2016
HALF-NITRITATION OF OLD LANDFILL LEACHATE IN MBR
TABLE 2. Corresponding relation of COD and NH4 þ N, FA and FNA in the old landfill leachate. NH4 þ N (mg/L)
FA (mg/L)
FNA (mg/L)
53.32 106.65 213.29 319.94 426.58 533.23 639.87 853.16 1066.45
2.31 4.63 9.26 13.89 18.52 23.15 27.78 37.03 46.29
0.00803 0.01606 0.03213 0.04819 0.06426 0.08032 0.09638 0.12851 0.16064
133.35 266.70 533.40 800.10 1066.80 1333.50 1600.20 2133.60 2667.00
Removel rate [g/(g·d)]
experiment, the FA concentration range was 9.5e11.8 mg/L at the highest ammonia oxidation and NO2 N generation rates. NO3 N was also still present at FA concentration of 42.4 mg/L, suggesting that NOB activity was not fully suppressed at high FA concentrations. This may be because the activated sludge was operated at high NH4 þ N concentration for a lengthy duration and had undergone adaptation over the course of the experiment. Villaverde et al. (34) and Fux et al. (35) observed similar phenomena. NH4 þ N is oxidized to NO2 N by AOB and the presence of NO2 N leads to the formation of FNA that is able to inhibit AOB and NOB. In this study, for the inhibitory factor FA, ammonia oxidation rate, NO2 N, and NO3 N generation rates behaved similarly as FNA concentration increased from 0.008 to 0.1036 mg/ L. At 0.1036 mg/L FNA, the ammonia oxidation rate, NO2 N generation rate, and NO3 N generation rate were 84.1%, 84.2%, and 54.2% of their values at 0.008 mg/L. Vadivelu et al. (36,37) found that NOB activity is suppressed at FNA concentration of 0.011 mg/L, and that NOB and AOB activity are fully suppressed at FNA concentrations of 0.023 mg/L and 0.40 mg/L, respectively. In our experiment, FNA concentration was less than 0.40 mg/L but
AOB began to be inhibited at FNA concentration of only 0.0317 mg/ L. NOB activity was not fully inhibited though FNA concentration reached 0.1036 mg/L, possibly because the NOB had adapted to the FNA. The composition of landfill leachate is highly complex, as it contains toxic organic matter, salt ions, and heavy metals that can inhibit AOB in the reactor (38). Here, nitritation performance was investigated at different landfill leachate concentrations from 0 to 2667 mg/L (calculated in COD). We found that the ammonia oxidation rate, NO2 N, and NO3 N generation rate decreased as old landfill leachate concentration increased to 44.3%, 45.1%, and 44.1% of their maximum values, respectively. The correlations between COD and NH4 þ N, FA, and FNA in the old landfill leachate are shown in Table 2. The ammonia oxidation rate was 0.9848 g/(g$d), which is 22.2% less than the maximum ammonia oxidation rate when COD was 1600.2 mg/L. The FA was 27.8 mg/L in the old landfill leachate samples we used. When FA was used as the single inhibiting factor, the ammonia oxidation rate ranged between 1.3432 and 1.3684 mg/L e a decrease of 4.7e6.5% relative to the maximum ammonia oxidation rate. If NH4 þ N was fully converted into NO2 N, FNA was 0.0964 mg/L when COD was 1600.2 mg/L. If FNA was the single inhibiting factor, the ammonia oxidation rate was between 1.1875 and 1.2072 mg/L e a decrease of 14.5e15.9% relative to the maximum rate. These observations indicated that the old landfill leachate showed a stronger nitritation inhibitory effect than FNA or FA. The amounts of substrate, product, and old landfill leachate inhibition were nonlinearly fitted using Origin8.0 software as shown in Fig. 6. The correlation coefficients (R2) were 0.9821, 0.9961, and 0.9924, respectively. All three models fit well to the observed inhibition behavior. The inhibition kinetics data showed a maximum ammonia oxidation rate of 2.087 g/(g$d), and the Haldane inhibition kinetics constant was 67.234 mg/L when FA was the sole inhibiting factor. The maximum ammonia oxidation rate was
1.6
1.6
1.2
1.2
experimental data fitting curve
0.8
2
y =2.087x/(3.185+x+x /67.234) 2 R =0.9821
0.4
(a) 0.0
0
10
20 30 FA (mg/L)
40
Removel rate [g/(g·d)]
COD (mg/L)
5
experimental data fitting curve
0.8
y =1.484xexp(-x/0.432)/(0.001+x) 2
R =0.9961
0.4
(b) 0.0
0.00
0.02
0.04 0.06 0.08 FNA (mg/L)
0.10
FIG. 6. The inhibition kinetics models of nitritation.
Please cite this article in press as: Yun, L., et al., Inhibition kinetics of nitritation and half-nitritation of old landfill leachate in a membrane bioreactor, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.007
6
YUN ET AL.
J. BIOSCI. BIOENG., TABLE 3. Inhibition kinetics models and parameters.
Inhibition type Substrate Product Old landfill leachate
Fitted equation 2
y ¼ 2.087x/(3.185 þ x þ x /67.234) y ¼ 1.484x exp(x/0.432)/(0.001 þ x) y ¼ [1(x/4054.02)]2.32/[1 þ (x/4054.02)]2.19
1.484 g/(g$d), the Aiba inhibition constant was 0.432 mg/L, and the full inhibitory concentration of old landfill leachate was 4054 mg/L (calculated in COD). The relevant inhibition kinetics equations and parameters are shown in Table 3. Conclusions Old landfill leachate nitration in a typical MBR was successfully initialized when the controlled DO was 0.5e1 mg/ L, HRT was 22 h, pH was 7.8e8.2, temperature was (30 1) C, and influent NH4 þ N concentration was 280 20 mg/L. Influent loading was gradually increased and DO was improved accordingly to 2e3 mg/L during the subsequent loading increase period, at which point the nitritation of old landfill leachate was effectively realized. Half-nitritation was quickly achieved by adjusting the HRT and DO after achieving nitritation. It is not possible to achieve longterm stable operation of half-nitritation using HRT as a single control strategy, but the addition of a DO control strategy allows stable half-nitritation operation. AOB and NOB activity was inhibited by high substrate concentration, high product concentration, and old landfill leachate during nitritation in the MBR. The inhibition of old landfill leachate was stronger than that of the substrate or product. The experimental data were separately nonlinearly fitted by Haldane, Aiba, and a revised inhibition kinetics model yielding correlation coefficients (R2) of 0.982, 0.996, and 0.992, respectively. ACKNOWLEDGMENTS 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 Scientific Research Project of Beijing Educational Committee (KM201210005028). References 1. Michał, B., Ewa, q.-M., and Marlena, Z.: Removal of organic compounds from municipal landfill leachate in a membrane bioreactor, Desalination, 198, 16e23 (2006). 2. Farah, N. A. and Christopher, Q. L.: Treatment of landfill leachate using membrane bioreactors: a review, Desalination, 287, 41e54 (2012). 3. Zhu, L. and Liu, J. X.: Landfill leachate treatment with a novel process: anaerobic ammonium oxidation (Anammox) combined with soil infiltration system, J. Hazard. Mater., 151, 202e212 (2008). 4. Ruscalleda, M., López, H., Ganigué, R., Puig, S., Balaguer, M. D., and Colprim, J.: Heterotrophic denitrification on granular anammox SBR treating urban landfill leachate, Water Sci. Technol., 58, 1749e1755 (2008). 5. Strous, M., Heijnen, J. J., Kuenen, J. G., and Jetten, M. S. M.: The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms, Appl. Microbiol. Biotechnol., 50, 589e596 (1998). 6. Tadashi, N., Hiroaki, O., Yuko, I., Yuya, H., Ayako, T., and Kanji, M.: Partial nitrification in a continuous pre-denitrification submerged membrane bioreactor and its nitrifying bacterial activity and community dynamics, Biochem. Eng. J., 55, 101e107 (2011). 7. Yuan, X., Fenglin, Y., Sitong, L., and Zhimin, F.: The influence of controlling factors on the start-up and operation for partial nitrification in membrane bioreactor, Bioresour. Technol., 100, 1055e1060 (2009). 8. van Dongen, U., Jetten, M. S. M., and van Loosdrecht, M. C. M.: The SHARONANAMMOX process for treatment of ammonium rich wastewater, Water Sci. Technol., 44, 153e160 (2001). 9. Bae, W., Baek, S. C., Chung, J. W., and Lee, Y.: Optimal operational factors for nitrite accumulation in batch reactors, Biodegradation, 12, 359e366 (2001).
Parameter 1
Parameter 2
Parameter 3
R2
nmax ¼ 2.087 nmax ¼ 1.484 b ¼ 4054.02
ks ¼ 3.185 ks ¼ 0.001 m ¼ 2.19
kh ¼ 67.234 ka ¼ 0.432 n ¼ 2.32
0.9821 0.9961 0.9924
10. Jong, H. H., Nazim, C., and Jan, O.: Effect of loading rate and oxygen supply on nitrification in a non-porous membrane biofilm reactor, Water Res., 43, 3301e3307 (2002). 11. Kimura, Y., Isaka, K., and Kazama, F.: Effects of inorganic carbon limitation on anaerobic ammonium oxidation (anammox) activity, Bioresour. Technol., 102, 4390e4394 (2011). 12. Anthonisen, A. C., Loehr, R. C., Prakasam, T. B. S., and Srinath, E. G.: Inhibition of nitrification by ammonia and nitrous-acid, J. Water Pollut. Control Fed., 48, 835e852 (1976). 13. Park, S. and Bae, W.: Modeling kinetics of ammonium oxidation and nitrite oxidation under simultaneous inhibition by free ammonia and free nitrous acid, Process Biochem., 44, 631e640 (2009). 14. Stuczynski, T. I., Mccarty, G. W., and Siebielec, G.: Response of soil microbiological activities to cadmium, lead, and zinc salt amendments, J. Environ. Qual., 32, 1346e1355 (2003). 15. Kargi, F. and Konya, I.: COD, para-chlorophenol and toxicity removal from para-chlorophenol containing synthetic waste water in an activated sludge unit, J. Hazard. Mater., 132, 226e231 (2006). 16. Surmacz-Gorska, J., Gernaey, K., Demuynck, C., Vanrolleghem, Peter, and Verstraete, Willy: Nitrification monitoring in activated sludge by oxygen uptake rate (OUR) measurements, Water Res., 30, 1228e1236 (1996). 17. Sheintuch, M., Tartakovsky, B., Narkis, N., and Rebhun, M.: Substrate inhibition and multiple states in a continuous nitrification process, Water Res., 29, 953e963 (1995). 18. Aiba, S., Shoda, M., and Nagatani, M.: Kinetics of product inhibition in alcohol fermentation, Biotechnol. Bioeng., 10, 845e864 (1968). 19. Kim, I. S., Tabak, H. H., and Young, J. C.: Modeling of the fate and effect of chlorinated phenols in anaerobic treatment processes, Water Sci. Technol., 36(6/7), 287e294 (1997). 20. APHA-American Public Health Association: Standard methods for water and wastewater examination, 21st ed. American Public Health Association, Washington, DC, USA (2005). 21. Wang, J. L. and Yang, N.: Partial nitrification under limited dissolved oxygen conditions, Process Biochem., 39, 1223e1229 (2004). 22. Hanaki, K., Wantawin, C., and Ohgaki, S.: Nitrification at low levels of dissolved oxygen with and without organic loading in a suspended growth reactor, Water Res., 24(3), 297e302 (1990). 23. Zhu, L. and Liu, H. X.: Control factors of partial nitritation for landfill leachate treatment, J. Environ. Sci. China, 19, 523e529 (2007). 24. Lee, P.-H., Cotter, S. F., Prieri, S. C. R., Attalage, Dinu, and Sung, S.: pHgradient real-time aeration control for nitritation community selection in a non-porous hollow fiber membrane biofilm reactor (MBfR) with dilute wastewater, Chemosphere, 90, 2320e2325 (2013). 25. Ruiz, G., Jeison, D., and Chamy, R.: Nitrification with high nitrite accumulation for the treatment of wastewater with high ammonia concentration, Water Res., 37, 1371e1377 (2003). 26. Carrera, J., Jubany, I., Carvallo, L., Chamyb, R., and Lafuente, J.: Kinetic models for nitrification inhibition by ammonium and nitrite in a suspended and an immobilized biomass systems, Process Biochem., 39, 1159e1165 (2004). 27. Tokutomi, T.: Operation of a nitrite-type airlift reactor at low DO concentration, Water Sci. Technol., 49, 81e88 (2004). 28. Vadivelu, V. M., Keller, J., and Yuan, Z. G.: Effect of free ammonia and free nitrous acid concentration on the anabolic and catabolic processes of an enriched Nitrosomonas culture, Biotechnol. Bioeng., 95, 830e839 (2006). 29. Kumar, S. M., Madhu, G. M., and Roy, S.: Fouling behaviour, regeneration options and on-line control of biomass-based power plant effluents using microporous ceramic membranes, Sep. Purif. Technol., 57, 25e36 (2007). 30. Meng, F. G., Yang, F. L., Shi, B. Q., and Zhang, H. M.: A comprehensive study on membrane fouling in submerged membrane bioreactors operated under different aeration intensities, Sep. Purif. Technol., 59, 91e100 (2008). 31. Welander, U., Henrysson, T., and Welander, T.: Biological nitrogen removal from municipal landfill leachate in a pilot scale suspended carrier biofilm process, Water Res., 32, 1564e1570 (1998). 32. Vadivelu, V. M., Keller, J., and Yuan, Z. G.: Effect of free ammonia on the respiration and growth processes of an enriched Nitrobacter culture, Water Res., 41, 826e834 (2007). 33. Yun, H. J. and Kim, D. J.: Nitrite accumulation characteristics of high strength ammonia wastewater in an autotrophic nitrifying biofilm reactor, J. Chem. Technol. Biotechnol., 78, 377e383 (2003).
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VOL. xx, 2016 34. Villaverde, S., Fdz-Polanco, F., and Garcia, P. A.: Nitrifying biofilm acclimation to free ammonia in submerged biofilters. Start-up influence, Water Res., 34, 602e610 (2000). 35. Fux, C., Huang, D., Monti, A., and Siegrist, H.: Difficulties in maintaining longterm partial nitritation of ammonium-rich sludge digester liquids in a movingbed biofilm reactor (MBBR), Water Sci. Technol., 49(11e12), 53e60 (2004). 36. Vadivelu, V. M., Yuan, Z. G., Fux, C., and Keller, J.: Stoichiometric and kinetic characterisation of Nitrobacter in mixed culture by decoupling the growth and energy generation processes, Biotechnol. Bioeng., 94, 1176e1188 (2006).
HALF-NITRITATION OF OLD LANDFILL LEACHATE IN MBR
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37. Vadivelu, V. M., Yuan, Z. G., Fux, C., and Keller, J.: The inhibitory effects of free nitrous acid on the energy generation and growth processes of an enriched Nitrobacter culture, Environ. Sci. Technol., 40, 4442e4448 (2006). 38. Mosquera-Corral, A., Gonzlez, F., Campos, J. L., and Méndez, R.: Partial nitrification in a SHARON reactor in the presence of salts and organic carbon compounds, Process Biochem., 40, 3109e3188 (2005).
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Inhibition kinetics of nitritation and half-nitritation of old landfill leachate in a membrane bioreactor Li Yun,1 Li Jun,1, * Wang Zhaozhao,2 Wei Jia,1 Zhang Yanzhuo,1 and Zhao Baihang1 College of Architecture and Civil Engineering, Beijing University of Technology, Beijing 1000124, China1 and College of Energy and Environmental Engineering, Hebei University of Engineering, Handan, 056038 Hebei, China2 Received 5 July 2016; accepted 18 November 2016 Available online xxx
Nitritation can be used as a pretreatment for anaerobic ammonia oxidation (anammox). Various control strategies for nitritation and half-nitritation of old landfill leachate in a membrane bioreactor were investigated in this study and the inhibition kinetics of substrate, product and old landfill leachate on nitritation were analyzed via batch tests. The results demonstrated that old landfill leachate nitritation in the membrane bioreactor can be achieved by adjusting the influent loading and dissolved oxygen (DO). From days 105e126 of the observation period, the average effluent concentration was 871.3 mg/L and the accumulation rate of NO2 L LN was 97.2%. Half-nitritation was realized quickly by adjusting hydraulic retention time and DO. A low-DO control strategy appeared to best facilitate long-term and stable operation. Nitritation inhibition kinetic experiments showed that the inhibition of old landfill leachate was stronger than that of the substrate ðNH4 D LNÞ or product ðNO2 L LNÞ. The ammonia oxidation rate dropped by 22.2% when the concentration of old landfill leachate (calculated in chemical oxygen demand) was 1600.2 mg/L; further, when only free ammonia or free nitrous acid were used as a single inhibition factor, the ammonia oxidation rate dropped by 4.7e6.5% or 14.5e15.9%, respectively. Haldane, Aiba, and a revised inhibition kinetic model were adopted to separately fit the experimental data. The R2 correlation coefficient values for these three models were 0.982, 0.996, and 0.992, respectively. Ó 2016, The Society for Biotechnology, Japan. All rights reserved. [Key words: Old landfill leachate; Membrane bioreactor; Nitritation; Half-nitritation; Inhibition kinetics]
Landfill leachate is a complex composition containing high levels of ammonia nitrogen in addition to toxic substances such as toxic organic matter and heavy metals (1,2). The organic matter of fresh landfill leachate shows good biodegradability and can be used in denitrification, which makes it well-suited to nitrogen removal. The biodegradability of organic matter in old landfill leachate is very poor, however, and may require expensive additional organic carbon sources for a conventional biological nitrogen removal process. In short: There is high demand for a cost-effective and energy-efficient nitrogen removal method. Autotrophic nitrogen removal methods combining nitritation with anaerobic ammonia oxidation have garnered substantial research interest in high ammonia nitrogen and low carbon nitrogen ratio wastewater treatment (3,4). Existing combination methods can be split into two categories. In the first, NO2 N accumulation is performed before old landfill leachate as CðNO2 NÞ=CðNH4 þ NÞ is mixed in at 1:1.32 for anaerobic ammonia oxidation. In the second category, the control effluent CðNO2 NÞ=CðNH4 þ NÞ is about 1:1.32 in the nitritation stage for anaerobic ammonia oxidation. Both methods necessitate stable nitritation, which requires the interception of ammonia oxidizing bacteria (AOB) and the elimination of nitrite oxidizing bacteria (NOB). AOB are autotrophic and slow-growing (5). Membrane bioreactor (MBR) can efficiently intercept sludge and rapid concentrate AOB to truncate the amount of time necessary for * Corresponding author. Tel./fax: þ86 01067391726. E-mail address: [email protected] (L. Jun).
nitritation. There have been several studies on MBR nitritation, but only in regards to treating wastewater with low concentrations of ammonia nitrogen (6,7), while the concentration of ammonia nitrogen is generally greater than 1000 mg/L in old landfill leachate. There have been few studies on the nitritation of old landfill leachate using MBR reactors. Nitritation can be realized quickly if the appropriate temperature, pH, and dissolved oxygen (DO) are controlled (8,9). Halfnitritation can be controlled by hydraulic retention time (HRT), DO, or alkalinity if nitritation has been effectively realized (10,11). To achieve nitritation and allow stable operation of the MBR reactor, the operating conditions must be controlled and the influent loading gradually increased. HRT and DO should be separately adjusted to allow half-nitritation and stable operation. Old landfill leachate exhibits high ammonia nitrogen and toxic effects on microbes. High NH4 þ N and NO2 N accumulation causes a high concentration of free ammonia (FA) and free nitrous acid (FNA) that inhibits the activity of AOB and NOB (12,13). The organic matter and heavy metals in old landfill leachate also are toxic to AOB and NOB (14,15). There have been several studies on substrate inhibition kinetics (16), but few on product or old landfill leachate inhibition kinetics for nitritation. In an effort to fill this research gap, we determined the nitritation inhibition kinetics characteristics of substrates ðNH4 þ NÞ, product ðNO2 NÞ, and old landfill leachate through batch experiments. Our primary goal is to provide guidance for the practical application of old landfill leachate nitritation in MBRs.
1389-1723/$ e see front matter Ó 2016, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2016.11.007
Please cite this article in press as: Yun, L., et al., Inhibition kinetics of nitritation and half-nitritation of old landfill leachate in a membrane bioreactor, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.007
2
YUN ET AL.
J. BIOSCI. BIOENG.,
FIG. 1. Schematic diagram of MBR reactor. FIG. 2. Performance of nitritation during start-up.
MATERIALS AND METHODS Experimental set-up and operational conditions An MBR reactor made from organic glass with an effective volume of 25 L was used for these experiments (as shown in Fig. 1). The membrane component is a hollow fiber membrane made from polyvinylidene fluoride with a pore size and area of 0.1 mm and 0.5 m2, respectively. The influent and effluent pumps were controlled using a programmable logic controller. The membrane was operated at a flux of 2.28 L/ (m2$h) with intermittent suction (8 min suction, 2 min relaxation). The aeration rates were 40e160 L/h and controlled via rotor flowmeter throughout the experiment. The transmembrane pressure (TMP) was measured with a vacuum gauge (JZ 00000578, Tianjin Ji Xing Company, China). The temperature was controlled at (30 1) C using a heater. The HRT was 22 h, and there was no sludge discharge during the experiment. Characteristics of old landfill leachate and seed sludge The seed sludge (nitrification sludge) was taken from Gao Bei Dian wastewater treatment plant in Beijing, which employs a typical anoxic-aerobic process for treating municipal wastewater and achieves satisfactory biological nitrogen removal. Initial inoculation sludge concentration mixed liquor suspended solids (MLSS) was 3104 mg/L and mixed liquor volatile suspended solids (MLVSS) was 2540 mg/L. The old (over 5 years) landfill leachate was taken from Gao An Tun municipal landfill, stored in sealed in plastic drums, and renewed once per month during the experiment. The specific water quality is as follows: NH4 þ N, 900e1500 mg/L; NO2 N, 0e2 mg/L; NO3 N, 0e8 mg/L; chemical oxygen demand (COD), 2000e4000 mg/L; pH, 7.5e8.5; alkalinity, 6000e10,000 mg/L. Inhibition kinetics experiments The nitritation-activated sludge samples were separately cleaned 3e5 times with deionized water and phosphate buffered saline (PBS) after removal from the MBR at 71 d. The level of MLSS was about 5.04 g/L after concentration. For each batch experiment, 150 mL of concentrated sludge was transferred to a beaker and diluted to 1 L. The concentration of NH4 þ N was 60e1000 mg/L when NH4 þ N was the single inhibition factor, and the concentration of NO2 N was 50e700 mg/L but the NH4 þ N concentration was adjusted to about 180 mg/L when NO2 N was the single inhibition factor. In order to eliminate the influence of substrate inhibition, ammonium chloride was used to adjust the NH4 þ N concentration to match the concentration during the old landfill leachate inhibition experiments. Sodium bicarbonate and hydrochloric acid were used to adjust the alkalinity and keep the pH constant. All experiments were conducted in a constant temperature incubator which was controlled at (30 1) C temperature. Sampling intervals were 30 min. Ammonia oxidation rate, NO2 N generation rate, and NO3 N generation rate were measured and calculated in triplicate. Inhibition kinetics models The inhibition kinetics of the substrate is described by the Haldane model as follows (17):
n ¼
nmax S ks þ S þ kS
2
(1)
h
where n is the substrate conversion rate, g/(g$d); nmax is the maximum conversion rate, g/(g$d); S is the substrate concentration, mg/L; kS is half-saturation constant, mg/L; kh is Haldane inhibiting kinetics constant, mg/L. The inhibition kinetics of the product is described by the Aiba model (18), which was originally used to describe ethanol fermentation product inhibition. The equation as the follows: S n ¼ exp ks þ S ka
nmax S
(2)
where n is the substrate conversion rate, g/(g$d); nmax is the maximum conversion rate, g/(g$d); S is the substrate concentration, mg/L; kS is half-saturation constant, mg/L; ka is Aiba inhibiting kinetics constant, mg/L. The inhibition kinetics of old landfill leachate is described by chlorophenol inhibition kinetics model when acetic acid was degraded (19). The equation as the follows:
n ¼
nmax ½k0 Sx
(3)
ks ½k1 þ S
where n is the substrate conversion rate, g/(g$d); nmax is the maximum conversion rate, g/(g$d); S is the substrate concentration, mg/L; kS is half-saturation constant, mg/L; k0 and k1 are inhibiting constant. k0 and k1 are calculated according to the following formula: k0 ¼ ½1 a=b
m
k1 ¼ ½1 a=b
(4)
n
(5)
where a is toxic substance concentration, mg/L; b is toxic substance fully inhibition concentration, mg/L; m and n are constant. The above formula was revised by introducing the speed ratio (l) as follows:
l ¼
1 ½a=b m 1 þ ½a=b n
(6)
where l ¼ n/n0, l is speed ratio, n is the conversion rate under different old landfill leachate concentration, g/(g$d); n0 is the conversion rate of no old landfill leachate, g/(g$d). Chemical analysis and calculation procedures NH4 þ N, NO2 N, NO3 N, chemical oxygen demand (COD), MLSS and MLVSS were analyzed according to standard methods (20). pH and temperature were determined by WTW/ Multi3420 tester. NO2 N accumulation rate (R), free ammonia (FA) and free nitrous acid (FNA) were calculated according to the following equations: R ¼
C½NO C½NO
2
FA ¼
FNA ¼
N
2 N þ C½NO
3
N
100%
(7)
C½NH4 þ N 10pH 17 ½6344=ð273þTÞ 14 e þ10pH
(8)
C½NO N 47 2 14 e½2300=ð273þTÞ 10pH
(9)
where C½NH4 þ N, C½NO2 N and C½NO3 N are the effluent concentration of NH4 þ N, NO2 N and NO3 N, respectively, mg/L; T is temperature, C.
RESULTS AND DISCUSSION Nitritation start-up under low DO conditions It is generally accepted that NOB are inhibited and AOB are concentrated when the appropriate temperature, pH, and DO are controlled to realize
Please cite this article in press as: Yun, L., et al., Inhibition kinetics of nitritation and half-nitritation of old landfill leachate in a membrane bioreactor, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.007
HALF-NITRITATION OF OLD LANDFILL LEACHATE IN MBR
HRT
DO
3
100 60 40 20 0 6 4 2
130
140 +
inf.NH4 -N -
eff.NO2 -N
150
160
t (d) + eff.NH4 -N -
eff.NO3 -N
170
180
190
+
NH4 -N removal -
NO2 -N conversion
Conversion (%)
80
DO (mg/L)
1200 1000 800 600 400 200 0 20 15 10 5 0
HRT (h)
Nitrogen Concentration (mg/L)
VOL. xx, 2016
0 200 HRT DO
FIG. 3. Performance of nitritation during load increase period. FIG. 4. Performance of half-nitritation.
nitritation. The optimal conditions are 30e35 C (8), pH value close to 8.0 (12), and DO less than 1 mg/L (21). To quickly begin nitritation in this experiment, the conditions were controlled at (30 1) C, pH 7.8e8.2, and DO of 0.5e1 mg/L. The start-up period was divided into two phases: The synthetic wastewater was used to begin nitrification in the first phase (0e18 d) when the influent concentration of NH4 þ N was 280 20 mg/L, and the old landfill leachate was used in the second phase (19e32 d) at influent concentration of NH4 þ N diluted to 280 20 mg/L. The performance of nitritation during the start-up period in the MBR system is shown in Fig. 2. In the first phase, the effluent concentration of NO3 N was reduced and the concentration of NO2 N increased gradually. These observations suggested that NOB activity was significantly inhibited and AOB activity gradually improved when the experimental temperature, DO, and pH value were controlled appropriately as the nitrification process shifted towards nitritation. At 15 and 17 d, the NO2 N accumulation rates were 50.7% and 53.5%, respectively; this suggests that AOB was the dominant bacteria in the system. Shortcut nitrification was achieved with NO2 N accumulation rate greater than 50%. The diluted old landfill leachate was applied from 19 d, and microbial activity was inhibited at the beginning of the experiment due to the many toxic substances in old landfill leachate. The effluent NH4 þ N concentration was raised from 5.2 mg/L to 74.2 mg/L and the removal rate of NH4 þ N fell from 98.2% to 75.5%. Subsequently, microbial activity was gradually restored and the removal rate of NH4 þ N increased to 93.6% at 32 d, suggesting that the microorganisms gradually adapted to the presence of old landfill leachate in the system. The NO2 N accumulation rate also gradually increased throughout the start-up process, with a value of 84.3% at 32 d, indicating that nitritation was successfully initiated. Performance of nitritation during loading increase stage AOB replaced NOB and became the dominant bacteria while NO2 N gradually accumulated throughout the successful nitritation start-up in the system. Inhibiting factors of NOB include temperature, pH, FA, FNA, and DO. Low DO not only inhibits NOB but also affects AOB activity (22), while high DO can still maintain nitritation in the presence of the other inhibiting factors. DO was raised accordingly to 2e3 mg/L and the loading continually increased to control the other conditions. The period in which old landfill leachate loading was gradually increased was divided into three stages. The first, second, and third stages were 33e58 d, 59e80 d, and 81e112 d, respectively; the
influent concentration of NH4 þ N at each stage was 550e600 mg/ L, 750e850 mg/L, and 950e1050 mg/L, respectively. The nitritation performance of the loading increase period is shown in Fig. 3. The increased loading led to increase in effluent NH4 þ N concentration, because microbial biomass did not increase corresponding to loading though the inhibition of microbial activity did. Effluent NH4 þ N concentration gradually decreased and NH4 þ N removal rate increased as the system operation progressed after the loading increased. The effluent NO2 N concentration showed the same variation trend as effluent NH4 þ N, gradually increasing over the operation period e the effluent NO2 N concentration was 866.2 mg/L and accumulated NO2 N was 98.2% at 105 d, which suggests that AOB gradually adapted to the presence of the old landfill leachate and the population increased as the system was continuously run. Although the DO was 2e3 mg/L during the whole period, NO3 N did not over-accumulate and the range of effluent NO3 N concentration was 30e50 mg/L, suggesting that DO did not play a key role in NO2 N accumulation. Half-nitritation control strategy and stable operation Three control strategies currently exist for halfnitritation when nitritation has been realized: HRT, DO, and alkalinity control (23,24). The alkalinity of old landfill leachate is generally high, so alkalinity control is infeasible for old landfill leachate. Old landfill leachate allowed nitritation in the MBR reactor. The effluent NO2 N average concentration was 871.3 mg/L during 105e126 d and the average accumulation rate was 97.2%. The influent and effluent were adjusted to control HRT while the other conditions were kept unchanged (as shown in Fig. 4). The effluent NH4 þ N increased and NO2 N and HRT decreased with increase in influent and effluent until half-nitritation was effectively realized. Effluent NH4 þ N concentration was 362.6 mg/L, NO2 N was 513.1 mg/L, HRT was 13.9 h, and CðNO2 NÞ=CðNH4 þ NÞ was 1.42 e close to the anaerobic ammonia oxidation theoretical ratio (1.32). The reactor was operated for 25 days while HRT was 13.9 h and the average CðNO2 NÞ=CðNH4 þ NÞ was 1.38. Effluent NH4 þ N increased and NO2 N decreased between 149 and 156 d, however, suggesting that half-nitritation could be quickly and easily realized but may be difficult to stably maintain over long-term operation under HRT control once nitritation has been realized. This may be because AOB increases quickly given adequate nutrients and appropriate conditions. To this effect, HRT control should be considered just one part of a coordinated control
Please cite this article in press as: Yun, L., et al., Inhibition kinetics of nitritation and half-nitritation of old landfill leachate in a membrane bioreactor, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.007
YUN ET AL.
J. BIOSCI. BIOENG., Load increase period
Set-up period
10 DO
HRT
TMP MLSS
MLSS (g/L)
8
TABLE 1. Effect of FA, FNA and toxicant on nitritation.
Half nitrosation
60
Inhibiting factors
50
FA
40
6
30 4
TMP (kPa)
4
20 2 0
10 0
40
80 t (d)
120
160
FNA
0
FIG. 5. Evolution of TMP and MLSS during the operation phases.
strategy to quickly realize half-nitritation rather than a single control strategy. The nitritation of old landfill leachate was gradually recovered between 157 and 164 d and HRT increased to 22 h. DO was employed as a substrate of AOB and NOB during aerobic nitritation; the effect of nitritation is affected by DO (9). To achieve halfnitritation, DO could be slowly reduced to control the effluent concentration of NH4 þ N and NO2 N in the system. The effluent concentration of NH4 þ N and NO2 N changed from 139.4 mg/L and 834.5 mg/L, respectively, to 382.2 mg/L and 546.7 mg/L, respectively, as DO was gradually reduced from 2.52 mg/L to 0.93 mg/L during 165e171 d. The effluent CðNO2 NÞ=CðNH4 þ NÞ was 1.43 at 171 d and DO was steady at 0.75e1.25 mg/L. With other conditions unchanged, the system was stably operated for 29 days. The range of effluent CðNO2 NÞ=CðNH4 þ NÞ was 1.14e1.57 with an average value of 1.35 during the stable operation stage (171e199 d); the NO2 N average accumulation rate was 94.8%. Half-nitritation was quickly and easily achieved while long-term and stable operation using DO control with nitritation was effectively realized. The activity and growth rate of AOB increases when DO is high and decreases when DO is low (21), so half-nitritation was operated stably under low DO. AOB shows a high affinity to oxygen compared to NOB (25). In our experiment, the DO saturation constants of AOB and NOB were 0.2e0.4 mg/L and 1.2e1.5 mg/L, respectively (26). AOB becomes the dominant bacteria under low DO conditions (22,27), however, NO3 N did not accumulate at high DO (2e3 mg/L) or at low DO (0.75e1.25 mg/L) during this experiment within an effluent NO3 N concentration range of 30e60 mg/L. This is likely because NOB was inhibited by factors other than DO, such as FA, FNA, temperature, or pH (28). Sludge and membrane fouling characteristics during the experiment The extent of pollution in the membrane module is usually characterized by transmembrane pressure (TMP) during operation (29). TMP increases as the pollution level increases in the membrane module operating at constant flux. As discussed above, the nitritation process was divided into a start-up period, loading increase period, and half-nitritation control period; the loading increase period was further divided into three sub-stages. The TMP overall during the whole period is shown in Fig. 5, where membrane fouling rate accelerates with increase in influent loading due to the presence of organic matter and metal ions in the old landfill leachate.
Old landfill leachate (COD)
Concentration (mg/L)
Ammonia oxidation rate [g/(g$d)]
NO2 N generation rate [g/(g$d)]
NO3 N generation rate [g/(g$d)]
2.30 5.73 9.45 11.75 16.71 21.10 24.55 29.78 34.12 38.66 42.38 0.0080 0.0160 0.0317 0.0506 0.0576 0.0779 0.0872 0.1036 0.00 133.35 266.70 533.40 800.10 1066.80 1250.00 1600.20 2133.60 2667.00
0.9039 1.3868 1.4296 1.4364 1.4187 1.3820 1.3684 1.3432 1.2854 1.3058 1.2480 1.4027 1.4112 1.3956 1.3222 1.2371 1.2310 1.2072 1.1875 1.2650 1.2528 1.2310 1.2365 1.2215 1.1752 1.0746 0.9848 0.7651 0.5611
0.7825 1.2618 1.2994 1.3228 1.3102 1.2811 1.2680 1.2605 1.2086 1.2292 1.1751 1.2774 1.2869 1.2683 1.2202 1.1350 1.1517 1.1217 1.1142 1.1609 1.1557 1.1437 1.1544 1.1374 1.0859 1.0061 0.9201 0.7103 0.5238
0.1252 0.1231 0.1273 0.1148 0.1024 0.1020 0.0970 0.0836 0.0768 0.0724 0.0687 0.1290 0.1263 0.1246 0.1070 0.1042 0.0819 0.0836 0.0699 0.1017 0.0971 0.0903 0.0818 0.0804 0.0857 0.0722 0.0614 0.0516 0.0448
Extracellular polymers (EPS) and soluble microbial products (SMP) are released by microbes when conditions change (30) and membrane fouling rate is accelerated by the accumulation of ESP and SMP. The TMP increased from 0 kPa to 48 kPa at 16 d of membrane module operation during the HRT control stage, and the TMP increased from 0 kPa to 14 kPa when membrane module was operated for 16 d during the DO control stage. In other words, the membrane fouling rate during the HRT control stage was significantly greater than that during the DO control stage in the halfnitritation experiment. Changes in the sludge concentration in the test system are depicted in Fig. 5. MLSS decreased gradually during the nitritation start-up period due to the elimination of heterotrophic bacteria and NOB caused by in organics in the influent, as well as the increasing death and lysing of bacteria due to old landfill leachate toxicity. MLSS gradually increased in the system during the loading increase period because the microorganisms had adapted to the old landfill leachate, allowing for growth of aerobic heterotrophic bacteria and AOB. Inhibition kinetic characteristics of nitritation Nitritation was affected by the substrate ðNH4 þ NÞ, product ðNO2 NÞ, and old landfill leachate, as shown in Table 1. The ammonia oxidation rate and NO2 N generation rate increased at first and then decreased with increasing FA concentration from 2.9 to 42.4 mg/ L, while NO3 N generation rate gradually decreased. The ammonia oxidation rate was 1.436 g/(g$d) when FA concentration was 11.8 mg/L, and decreased to 1.248 mg/L at 42.4 mg/L FA. NH4 þ N was oxidized to NO2 N and NO3 N by nitrifying the bacteria that use its energy for growth. NOB activity was inhibited by FA formed from high NH4 þ N, allowing AOB to gradually replace NOB as the dominant bacterial community (31). Excessive FA can also suppress AOB, but to a lesser extent. Vadivelu et al. (32) reported inhibition of NOB at FA levels of 0.1e1 mg/L, with complete inhibition of NOB at 6 mg/L FA, but only saw inhibition of AOB at 10e150 mg/L FA (33). In our
Please cite this article in press as: Yun, L., et al., Inhibition kinetics of nitritation and half-nitritation of old landfill leachate in a membrane bioreactor, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.007
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HALF-NITRITATION OF OLD LANDFILL LEACHATE IN MBR
TABLE 2. Corresponding relation of COD and NH4 þ N, FA and FNA in the old landfill leachate. NH4 þ N (mg/L)
FA (mg/L)
FNA (mg/L)
53.32 106.65 213.29 319.94 426.58 533.23 639.87 853.16 1066.45
2.31 4.63 9.26 13.89 18.52 23.15 27.78 37.03 46.29
0.00803 0.01606 0.03213 0.04819 0.06426 0.08032 0.09638 0.12851 0.16064
133.35 266.70 533.40 800.10 1066.80 1333.50 1600.20 2133.60 2667.00
Removel rate [g/(g·d)]
experiment, the FA concentration range was 9.5e11.8 mg/L at the highest ammonia oxidation and NO2 N generation rates. NO3 N was also still present at FA concentration of 42.4 mg/L, suggesting that NOB activity was not fully suppressed at high FA concentrations. This may be because the activated sludge was operated at high NH4 þ N concentration for a lengthy duration and had undergone adaptation over the course of the experiment. Villaverde et al. (34) and Fux et al. (35) observed similar phenomena. NH4 þ N is oxidized to NO2 N by AOB and the presence of NO2 N leads to the formation of FNA that is able to inhibit AOB and NOB. In this study, for the inhibitory factor FA, ammonia oxidation rate, NO2 N, and NO3 N generation rates behaved similarly as FNA concentration increased from 0.008 to 0.1036 mg/ L. At 0.1036 mg/L FNA, the ammonia oxidation rate, NO2 N generation rate, and NO3 N generation rate were 84.1%, 84.2%, and 54.2% of their values at 0.008 mg/L. Vadivelu et al. (36,37) found that NOB activity is suppressed at FNA concentration of 0.011 mg/L, and that NOB and AOB activity are fully suppressed at FNA concentrations of 0.023 mg/L and 0.40 mg/L, respectively. In our experiment, FNA concentration was less than 0.40 mg/L but
AOB began to be inhibited at FNA concentration of only 0.0317 mg/ L. NOB activity was not fully inhibited though FNA concentration reached 0.1036 mg/L, possibly because the NOB had adapted to the FNA. The composition of landfill leachate is highly complex, as it contains toxic organic matter, salt ions, and heavy metals that can inhibit AOB in the reactor (38). Here, nitritation performance was investigated at different landfill leachate concentrations from 0 to 2667 mg/L (calculated in COD). We found that the ammonia oxidation rate, NO2 N, and NO3 N generation rate decreased as old landfill leachate concentration increased to 44.3%, 45.1%, and 44.1% of their maximum values, respectively. The correlations between COD and NH4 þ N, FA, and FNA in the old landfill leachate are shown in Table 2. The ammonia oxidation rate was 0.9848 g/(g$d), which is 22.2% less than the maximum ammonia oxidation rate when COD was 1600.2 mg/L. The FA was 27.8 mg/L in the old landfill leachate samples we used. When FA was used as the single inhibiting factor, the ammonia oxidation rate ranged between 1.3432 and 1.3684 mg/L e a decrease of 4.7e6.5% relative to the maximum ammonia oxidation rate. If NH4 þ N was fully converted into NO2 N, FNA was 0.0964 mg/L when COD was 1600.2 mg/L. If FNA was the single inhibiting factor, the ammonia oxidation rate was between 1.1875 and 1.2072 mg/L e a decrease of 14.5e15.9% relative to the maximum rate. These observations indicated that the old landfill leachate showed a stronger nitritation inhibitory effect than FNA or FA. The amounts of substrate, product, and old landfill leachate inhibition were nonlinearly fitted using Origin8.0 software as shown in Fig. 6. The correlation coefficients (R2) were 0.9821, 0.9961, and 0.9924, respectively. All three models fit well to the observed inhibition behavior. The inhibition kinetics data showed a maximum ammonia oxidation rate of 2.087 g/(g$d), and the Haldane inhibition kinetics constant was 67.234 mg/L when FA was the sole inhibiting factor. The maximum ammonia oxidation rate was
1.6
1.6
1.2
1.2
experimental data fitting curve
0.8
2
y =2.087x/(3.185+x+x /67.234) 2 R =0.9821
0.4
(a) 0.0
0
10
20 30 FA (mg/L)
40
Removel rate [g/(g·d)]
COD (mg/L)
5
experimental data fitting curve
0.8
y =1.484xexp(-x/0.432)/(0.001+x) 2
R =0.9961
0.4
(b) 0.0
0.00
0.02
0.04 0.06 0.08 FNA (mg/L)
0.10
FIG. 6. The inhibition kinetics models of nitritation.
Please cite this article in press as: Yun, L., et al., Inhibition kinetics of nitritation and half-nitritation of old landfill leachate in a membrane bioreactor, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.007
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YUN ET AL.
J. BIOSCI. BIOENG., TABLE 3. Inhibition kinetics models and parameters.
Inhibition type Substrate Product Old landfill leachate
Fitted equation 2
y ¼ 2.087x/(3.185 þ x þ x /67.234) y ¼ 1.484x exp(x/0.432)/(0.001 þ x) y ¼ [1(x/4054.02)]2.32/[1 þ (x/4054.02)]2.19
1.484 g/(g$d), the Aiba inhibition constant was 0.432 mg/L, and the full inhibitory concentration of old landfill leachate was 4054 mg/L (calculated in COD). The relevant inhibition kinetics equations and parameters are shown in Table 3. Conclusions Old landfill leachate nitration in a typical MBR was successfully initialized when the controlled DO was 0.5e1 mg/ L, HRT was 22 h, pH was 7.8e8.2, temperature was (30 1) C, and influent NH4 þ N concentration was 280 20 mg/L. Influent loading was gradually increased and DO was improved accordingly to 2e3 mg/L during the subsequent loading increase period, at which point the nitritation of old landfill leachate was effectively realized. Half-nitritation was quickly achieved by adjusting the HRT and DO after achieving nitritation. It is not possible to achieve longterm stable operation of half-nitritation using HRT as a single control strategy, but the addition of a DO control strategy allows stable half-nitritation operation. AOB and NOB activity was inhibited by high substrate concentration, high product concentration, and old landfill leachate during nitritation in the MBR. The inhibition of old landfill leachate was stronger than that of the substrate or product. The experimental data were separately nonlinearly fitted by Haldane, Aiba, and a revised inhibition kinetics model yielding correlation coefficients (R2) of 0.982, 0.996, and 0.992, respectively. ACKNOWLEDGMENTS 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 Scientific Research Project of Beijing Educational Committee (KM201210005028). References 1. Michał, B., Ewa, q.-M., and Marlena, Z.: Removal of organic compounds from municipal landfill leachate in a membrane bioreactor, Desalination, 198, 16e23 (2006). 2. Farah, N. A. and Christopher, Q. L.: Treatment of landfill leachate using membrane bioreactors: a review, Desalination, 287, 41e54 (2012). 3. Zhu, L. and Liu, J. X.: Landfill leachate treatment with a novel process: anaerobic ammonium oxidation (Anammox) combined with soil infiltration system, J. Hazard. Mater., 151, 202e212 (2008). 4. Ruscalleda, M., López, H., Ganigué, R., Puig, S., Balaguer, M. D., and Colprim, J.: Heterotrophic denitrification on granular anammox SBR treating urban landfill leachate, Water Sci. Technol., 58, 1749e1755 (2008). 5. Strous, M., Heijnen, J. J., Kuenen, J. G., and Jetten, M. S. M.: The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms, Appl. Microbiol. Biotechnol., 50, 589e596 (1998). 6. Tadashi, N., Hiroaki, O., Yuko, I., Yuya, H., Ayako, T., and Kanji, M.: Partial nitrification in a continuous pre-denitrification submerged membrane bioreactor and its nitrifying bacterial activity and community dynamics, Biochem. Eng. J., 55, 101e107 (2011). 7. Yuan, X., Fenglin, Y., Sitong, L., and Zhimin, F.: The influence of controlling factors on the start-up and operation for partial nitrification in membrane bioreactor, Bioresour. Technol., 100, 1055e1060 (2009). 8. van Dongen, U., Jetten, M. S. M., and van Loosdrecht, M. C. M.: The SHARONANAMMOX process for treatment of ammonium rich wastewater, Water Sci. Technol., 44, 153e160 (2001). 9. Bae, W., Baek, S. C., Chung, J. W., and Lee, Y.: Optimal operational factors for nitrite accumulation in batch reactors, Biodegradation, 12, 359e366 (2001).
Parameter 1
Parameter 2
Parameter 3
R2
nmax ¼ 2.087 nmax ¼ 1.484 b ¼ 4054.02
ks ¼ 3.185 ks ¼ 0.001 m ¼ 2.19
kh ¼ 67.234 ka ¼ 0.432 n ¼ 2.32
0.9821 0.9961 0.9924
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