- Email: [email protected]
Kinetics of sequential nitrification and denitrification processes
Enzyme and Microbial Technology 27 (2000) 37– 42
www.elsevier.com/locate/enzmictec
Kinetics of sequential nitrification and denitrification processes Ali R. Dinc¸er, Fikret Kargı* Department of Environmental Engineering, Dokuz Eylu¨l University, Buca, I˙zmir, Turkey Received 22 July 1999; received in revised form 6 January 2000; accepted 18 January 2000
Abstract Kinetics of nitrification and denitrification of synthetic wastewater was investigated by using two reactors in series. An activated sludge unit was used for nitrification followed by a downflow biofilter for denitrification. Glucose solution was fed to the denitrification column to supply carbon source. Reactors were operated at different operating conditions and data were collected for determination of kinetic constants. Experimental data indicated that nitrification and denitrification kinetics followed Monod kinetics. By using the experimental data, kinetic constants for nitrification were determined as k ⫽ 1.15 d⫺1, KN ⫽ 5.14 mg/l, Y ⫽ 0.34 mgX/mgN and b ⫽ ⫺0.021 d⫺1. Similarly, kinetic constants for denitrification were determined as k ⫽ 0.23 d⫺1 and KDN ⫽ 0.27 mg/l. Rates of nitrification and denitrification increased with increasing nitrogen loading rate. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Nitrification; Denitrification; Kinetic constants
1. Introduction Nomenclature Nitrification and denitrification processes are advanced biological treatment processes used for removal of excess nitrogen present in the effluent of BOD removal processes. Nitrification is an aerobic, autotrophic process used for conversion of ammonium to nitrate that entails two steps. Ammonium is converted to nitrite by Nitrosomonas species in the first step, and nitrite is converted to nitrate by Nitrobacter species in the second. The fraction of nitrifying organisms in activated sludge culture increases with increasing N/COD ratio in the wastewater. Denitrification is an anoxic, heterotrophic process used for conversion of nitrate to nitrogen gas by denitrifying organisms. Denitrifying organisms require external carbon source for biosynthesis and energy generation. These two processes are usually realized in two separate stages, as the environmental conditions required for each process are different. Composition of the feed wastewater (i.e. C/N ratio and types of nitrogen compounds) determines the order of nitrification and denitrification processes. Numerous studies are reported in literature for nitrification and denitrification of wastewaters [1–28]. Nitrification * Corresponding author. Tel.: ⫹0090-232-4531143; fax: ⫹0090-2324531153. E-mail address: [email protected] (F. Kargı).
KN KDN LCOD LN No, N NH4-N NO3-N NO2-N NOx-N Q RN RDN Rm U V X Y b k ⌰c ⌰H
0141-0229/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 0 ) 0 0 1 4 5 - 9
saturation constant for nitrification saturation constant for denitrification COD loading rate (Q 䡠 CODo, mgCOD/h) nitrogen loading rate (QNo, mgN/h) feed and effluent nitrogen concentrations (mg/l) ammonium nitrogen concentration (mg/l) nitrate nitrogen concentration (mg/l) nitrite nitrogen concentration (mg/l) nitrate ⫹ nitrite nitrogen concentrations flow rate of wastewater (l/h) rate of nitrification (mgN/l 䡠 h) rate of denitrification (mgN/l 䡠 h) maximum rate of denitrification (kX, mgN/l 䡠 h) specific rate of nitrification (mgN/mgX 䡠 d) volume of liquid in reactors (l) biomass concentration (mg/l) growth yield coefficient (mgX/mgN) death rate constant (d⫺1) nitrification rate constant (d⫺1) sludge age or solids retention time (d) hydraulic residence time (V/Q, h) specific rate of growth (d⫺1)
38
A.R. Dinc¸er, F. Kargı / Enzyme and Microbial Technology 27 (2000) 37– 42
Fig. 1. A schematic of the experimental set-up for nitrification and denitrification.
kinetics was found to be zeroth order for effluent ammonium concentration above 10 mg/l [5]. Adverse effects of high C/N ratio such as C/N ⬎ 1.5 on performance of nitrifying bacteria in biofilms was observed by Okabe [6]. Carbon to nitrogen ratio in wastewater should be less than 0.25 for effective nitrification. Inhibition of nitrification by organic compounds [7] and by the substrate NH⫹ 4 [8] were investigated. Inhibition by free ammonia and nitrous acid was observed by Anthonisen et al [10]. Effects of temperature and pH on nitrification process was investigated by Shammas [17]. Optimal pH for nitrification was reported as pH ⫽ 8 ⫾ 0.5. However, pH of the media decreases as a result of H⫹ ion release to the media requiring alkalinity addition to the media. Theoretically, 7.14 mg of alkalinity is consumed per 1 mg of NH4-N oxidized [13]. Dissolved oxygen limitations were reported to be important in nitrification when oxygen supply is below 2 g O2/g NH4-N [19]. Denitrification organisms were also reported to be inhibited at high concentrations of nitrate and nitrite [20,21]. The rate of denitrification was reported to be zeroth order for NO3-N concentrations above 5 mg/L [25]. Use of various carbon sources for denitrification was tested by several investigators [22,23]. The optimal C/N ratio for denitrification was reported to be C/N ⫽ 1.1 when glycerol was used as carbon source [22]. Denitrifying organisms can tolerate pH between 6 and 9. However, the optimal pH was reported as pH ⫽ 7– 8 [26]. pH increases as a result of H⫹ ion removal from the media during denitrification. Usually no external addition of acid is required for pH control during denitrification, since pH variations can be tolerated by the nitrifying bacteria. Despite reported studies on separate nitrification and denitrification processes in the literature, kinetics of sequential nitrification and denitrification processes were not systematically investigated. For this reason, this study was designed to investigate the kinetic behavior of such processes and to determine the kinetic constants. Synthetic
wastewater containing ammonium and bicarbonate was subjected to nitrification in an activated sludge unit. The effluent of nitrification was fed to a downflow biofilter for denitrification with external addition of glucose solution. The reactors were operated at different operating conditions (e.g. hydraulic residence time and sludge age), and experimental data were used for determination of kinetic constants.
2. Materials and methods 2.1. Experimental set-up A schematic diagram of the experimental set-up is depicted in Fig. 1. The system consisted of an activated sludge unit used for nitrification and a downflow packed column for denitrification. The sedimentation tank was separated from the aeration tank by an inclined plate in the activated sludge unit. Wastewater passage from the aeration tank to the sedimentation tank was through holes on the inclined plate and the sludge recycle was through the gap (2 cm) underneath the plate. The packed column was filled with pieces of plastic tubes of 1 cm in length. Glucose solution was fed to the denitrification column to provide carbon source for the organisms. The liquid volumes in the aeration and sedimentation tanks were VAT ⫽ 7.2 l and VST ⫽ 2.2 l, respectively. The denitrification column had a liquid volume of VDC ⫽ 5.1 l and support particle surface area of Ap ⫽ 2.41 m2, resulting in total support surface area per unit liquid volume of a ⫽ 472 m2/m3. 2.2. Wastewater composition The synthetic wastewater used for nitrification was composed of 382 mg 䡠 L⫺1 NH4Cl (NH4-N ⬵ 100 mg 䡠 L⫺1);
A.R. Dinc¸er, F. Kargı / Enzyme and Microbial Technology 27 (2000) 37– 42
39
1510 mg 䡠 L⫺1 NaHCO3 (900 mg CaCO3 䡠 L⫺1 alkalinity); 100 mg 䡠 L⫺1 COD (93 mg 䡠 L⫺1 glucose); 20 mg 䡠 L⫺1 CaCl2 䡠 2H2O; 40 mg 䡠 L⫺1 MgSO4 䡠 7H2O; 85 mg 䡠 L⫺1 KH2PO4. pH of the media was adjusted to pH ⫽ 7.5. The effluent of the nitrification stage containing different nitrate nitrogen (NO3-N) concentrations was fed to the denitrification column. Sugar solution containing 744 mg 䡠 L⫺1 glucose resulting in 800 mg 䡠 L⫺1 COD; 50 mg 䡠 L⫺1 KH2PO4; 20 mg 䡠 L⫺1 MgSO4; 10 mg 䡠 L⫺1 Na2S2O3 with a pH of 7 was fed to the denitrification column continuously to obtain COD/N ⫽ 4.8 in the feed.
under the same experimental conditions were negligible resulting in less than 5% deviations from the average.
2.3. Organisms
3.1. Nitrification kinetics
Nitrification organisms (Nitrosomonas and Nitrobacter) were obtained from Clemson University, SC, USA in mixed culture form and cultivated in our laboratory on a shaker by using the aforementioned media. The denitrification stage was inoculated by a heterotrophic culture obtained from activated sludge unit of Pak-maya Baker’s yeast company, Izmir, Turkey. Denitrifiers were concentrated by slow continuous operation of the packed column for about 2 weeks. Continuous experiments were started after dense population of organisms were obtained by batch operation for 10 days.
Activated sludge unit used for nitrification of synthetic wastewater with NH4-No ⬵ 100 mg/l was operated at different sludge ages (solids retention time) between c ⫽ 3–20 days and hydraulic residence times between H ⫽ 3–30 hours. Nitrogen balance around the aeration tank of activated sludge unit results in the following equation:
2.4. Experimental procedure Inoculum culture cultivated and adapted to the synthetic media on a shaker was used for inoculation of reactors. The reactors were operated batchwise for 10 days until dense culture of organisms were obtained in both stages before continuous operation. Sludge age in the activated sludge unit was adjusted by daily removal of sludge from the aeration tank. The effluent of the nitrification step was fed to the denitrification column with the same flow rate. Hydraulic residence time was varied by changing the feed flow rate. Glucose solution was fed to the denitrification column with a known flow rate to obtain a COD to nitrogen loading ratio of LCOD/LN ⬵ 4.8. Dissolved oxygen and pH measurements were made twice per day. Experiments were performed for at least 7 days after the system reached steady-state. 2.5. Analytical methods
3. Results and discussion Experimental data obtained at different sludge ages for nitrification and at different hydraulic residence times for denitrification were used for determination of kinetic constants.
U⫽
kN Q(No⫺N) ⫽ VX KN ⫹ N
(1)
where, U is specific rate of nitrification (mgN/mgX 䡠 d); Q is flow rate of feed wastewater (L/d); V is volume of the aeration tank (L); No and N are the feed and effluent NH4-N concentrations (mg/L); X is the biomass concentration in the aeration tank (mg/L); k (d⫺1) and K N (mg/L) are the maximum nitrification rate and saturation constants, respectively. In double reciprocal form eqn. 1 takes the following form: 1 1 KN 1 HX ⫽ ⫽ ⫹ U (No ⫺ N) k k N
(2)
A plot of 1/U versus 1/N results in a line with a slope of KN/k and an intercept of 1/k. Experimental data obtained at different sludge and hydraulic residence times were plotted as 1/U versus 1/N as shown in Fig. 2. From the slope and intercept of the best-fit line the following values were found: k ⫽ 1.15 d⫺1 and K N ⫽ 5.14 mg/L
Samples were withdrawn daily from the reactor effluents and were centrifuged to remove cells from the wastewater. Ammonium, nitrate, nitrite, and COD analyses were performed on clear samples. Merck-Spectroquant analytical kits were used for ammonium (Kit No: 1.14559.0001), nitrate (Kit No: 1.14773.0001), and nitrite (Kit No: 14776) analyses. COD analyses were carried out by using the Standard Methods [29]. Dissolved oxygen (DO) measurements were carried out by using a Solomat 520C, DO analyzer, and a DO probe. Nitrogen measurements were done at least three times after the system reached steady-state. Differences among the three nitrogen concentration measurements
By using the definition of sludge age, the following equation can be written,
⫽
1 YkN ⫽ ⫺ b ⫽ YU ⫺ b c KN ⫹ N
(3)
where, is specific growth rate of organisms (d⫺1); c is sludge age (d); Y is the growth yield coefficient (gX/gN), and b is the death rate constant (d⫺1). A plot of 1/c versus U results in a line with a slope of Y and intercept of ⫺b. Experimental data obtained at different sludge ages were plotted as 1/c versus U as shown in Fig. 3. From the slope
40
A.R. Dinc¸er, F. Kargı / Enzyme and Microbial Technology 27 (2000) 37– 42
Fig. 2. Double reciprocal plot of 1/U versus 1/N for determination of k and KN coefficients.
and intercept of the best-fit line the following values were determined: Y ⫽ 0.34 gX/gN,
b ⫽ 0.021 d⫺1
These constants are in good agreement with the literature values [30]. Small differences between our results and the literature values may be because of lower fraction of nitrifying organisms in microbial population and suboptimal environmental conditions. Therefore, the design equation for nitrification in activated sludge unit takes the following form:
⫽
1 0.34 (1.15)N ⫺ 0.021 ⫽ c 5.14 ⫹ N
Fig. 4. Variation of the rate of nitrification (R) with effluent ammonium concentration.
tration. Nitrification rate increased with steady-state NH4-N concentration up to 50 mg/l and remained constant for larger NH4-N levels as expected by the Monod equation. The maximum rate of nitrification was Rm ⫽ 25 mgN/l 䡠 h and the saturation constant (ammonium nitrogen concentration at 1⁄2 of Rm) was approximately KN ⫽ 5 mg/l. Variation of the rate of nitrification with ammoniumnitrogen loading rate (LN ⫽ Q 䡠 No) is depicted in Fig. 5. The rate increased more steeply at low loading rates and reached a constant (Rm ⫽ 25 mgN/l 䡠 h) level at high nitrogen loading rates between LN ⫽ 250 –300 mgN/h. This trend is in agreement with Fig. 4.
(4)
where c is in days and N is in mg/L. Fig. 4 depicts variation of nitrification rate (R ⫽ Q (No-N)/V) with effluent ammonium-nitrogen concen-
Fig. 3. A plot of U versus 1/C for determination of kinetics constants.
3.2. Denitrification kinetics The effluent of the nitrification reactor was fed to the denitrification column operating in down-flow mode. Deni-
Fig. 5. Variation of NH4 ⫺ N removal rate (R) with NH4 ⫺ N loading rate (LN) in nitrification (c ⫽ 15 d, H ⫽ 12 h).
A.R. Dinc¸er, F. Kargı / Enzyme and Microbial Technology 27 (2000) 37– 42
41
Fig. 6. A plot of 1/H 䡠 Ln No/N versus (No ⫺ N)/H for determination of kinetic constants of denitrification.
Fig. 7. Variation of the denitrification rate RDN with NOx ⫺ N loading rate, LN 䡠 (H ⫽ 6 h).
trification organisms were immobilized on the surfaces and within the interstitial spaces of support particles. Denitrification column was operated at different hydraulic residence times and the experimental data were used for determination of the kinetic constants. Nitrogen balance on differential volume of the packed column yields the following design equation.
d⫺1 ⫽ 0.0096 h⫺1. These constants are in good agreement with the literature values [30]. Therefore, the design equation for denitrification column takes the following form,
⫺QdN ⫽ RDN 䡠 dV ⫽
Rm 䡠 N kX 䡠 N dV ⫽ dV K DN ⫹ N K DN ⫹ N
(5)
Rearrangement of Eqn. 5 results in the following equation
冉
冊
冉
冊
dV 1 K DN ⫹ N 1 K DN ⫽⫺ ⫹ 1 dN dN ⫽ ⫺ Q Rm N Rm N
(6)
Integration of Eqn. 6 from V ⫽ 0 and N ⫽ No to V ⫽ V and N ⫽ N and rearrangement of the resulting equation yields, Rm 1 (No ⫺ N) 1 No ⫺ 䡠 ⫽ Ln K DN K DN H H N
(7)
where, Rm (⫽kX) is the maximum rate of denitrification (mgN/l 䡠 h); KDN is the saturation constant of denitrification (mg/l); No and N are the feed and effluent NOx-N (nitrate ⫹ nitrite nitrogen) concentrations (mg/l); H (⫽V/Q) is the hydraulic residence time (h). A plot of 1/H Ln (No/N) versus (No ⫺ N)/H yields a line with a slope of ⫺1/KDN and intercept of Rm/KDN. Experimental data obtained at different hydraulic residence times (feed flow rates) were plotted as 1/H Ln (No/N) versus (No-N)/H as shown in Fig. 6. From the slope and the intercept of the best-fit line the following values were found. Rm ⫽ 280 mgN/L 䡠 d ⫽ 11.7 mgN/l 䡠 h,
KDN ⫽ 0.27 mg/l
By using the definition of Rm ⫽ kX and average biomass concentration in the column as X ⫽ 1200 mg/l, the maximum denitrification rate constant was found as k ⫽ 0.23
0.23 X 1 No 1 (No ⫺ N) ⫽ Ln ⫺ 䡠 0.27 0.27 H H N where X, No, N are in mg/L and H is in days. Fig. 7 depicts variation of rate of denitrification with NOx-N (nitrate ⫹ nitrite nitrogen) loading rate (LN ⫽ Q 䡠 No). The rate increased sharply up to loading rate of L ⫽ 250 mgN/h and remained constant (R ⫽ 30 mgN/L 䡠 h) for loading levels between 250 –300 mg N/h.
4. Conclusions Kinetics of sequential nitrification and denitrification processes were investigated by using constant feed NH4No ⫽ 100 mg/l at different hydraulic and sludge residence times. Experimental data was used for determination of kinetic constants of nitrification and denitrification and the following values were found: k ⫽ 1.15 d⫺1, KN ⫽ 5.14 mg/l, Y ⫽ 0.34 gX/gN, b ⫽ 0.021 d⫺1 Denitrification: k ⫽ 0.23 d⫺1, KDN ⫽ 0.27 mg/l Nitrification:
The constants for nitrification determined from our experiments are in good agreement with the literature values of k ⫽ 2–5 d⫺1, KN ⫽ 1–5 mg/l, Y ⫽ 0.2– 0.3 gX/gN and b ⫽ 0.02– 0.06 d⫺1 [30]. Somewhat lower k and higher KN values obtained in this study may be because of lower fraction of nitrifying organisms in the total population and sub optimal environmental conditions. Experimentally determined constants for denitrification
42
A.R. Dinc¸er, F. Kargı / Enzyme and Microbial Technology 27 (2000) 37– 42
are also in good agreement with the literature values of k ⫽ 0.2– 0.8 d⫺1 and KDN ⫽ 0.1– 0.3 mg/l [30], indicating accuracy of our experimental results. The rates of nitrification and denitrification indicated a hyperbolic variation with the steady-state concentrations of effluent nitrogen concentrations as suggested by the Monod kinetics (Saturation kinetics). Increasing nitrogen loading rates resulted in significant increases in nitrification and denitrification rates, especially at low levels of loading rates. However, the rates reached nearly constant levels at high loading rates. Developed design equations with experimentally determined kinetic constants can be used for design of sequential nitrification and denitrification processes.
Acknowledgment This study was supported in part by the research funds of the Dokuz Eylu¨l University.
References [1] Chen SK, Juaw CK, Cheng SS. Nitrification and denitrification of high strength ammonium and nitrite wastewater in biofilm reactors. Wat Sci Technol 1991;23:1417–25. [2] Cheng SS, Chen WC. Organic carbon supplement influencing performance of biological nitrification in a fluidized bed reactor. Wat Sci Technol 1994;30:131– 42. [3] C¸ec¸en F, Go¨nenc¸ I˙F. Criteria for nitrification and denitrification of high strength wastes in two upflow submerged filters. Wat Env Res 1995;67:132– 42. [4] Horan NJ, Azimi AA. The effects of transient nitrogen loadings on nitrifying activated sludges in completely mixed and plug-flow reactors. Wat Res 1992;26:279 – 84. [5] Hall ER, Murphy KL. Sludge age and substrate effects on nitrification kinetics. J WPCF 1985;57:413– 8. [6] Okabe S, Oozawa Y, Hirata K, Watanabe Y. Relationship between population dynamics of nitrifiers in biofilms and reactor performance at various C:N ratios. Wat Res 1996;30:1563–72. [7] Oslislo A, Lewandowski Z. Inhibition of nitrification in the packed bed reactors by selected organic compounds. Wat Res 1985;19:423– 6. [8] Gee CS, Suidan MT, Pfeffer JT. Modelling of nitrification under substrate inhibiting conditions. J Environ Eng 1990;116:18 –31. [9] Painter HA, Loveless JA. Effect of temperature and pH value on the growth rate constants of nitrifying bacteria in the activated sludge process. Wat Res 1983;17:237– 48.
[10] Anthonisen AC, Loehr RC, Prakasam TBS, Srinath, EG. Inhibition of nitrification by ammonia and nitrous acid. J WPCF 1976;48:835–52. [11] Gupta SK, Sharma R. Biological oxidation of high strength nitrogenous wastewater. Wat Res 1996;30:593– 600. [12] Matsumura M, Yamamoto T, Wang P, Shinabe K, Yasuda K. Rapid nitrification with immobilized cell using macroporous cellulose carrier. Wat Res 1997;31:1027–34. [13] Villaverde S, Encina PAG, Polanco FF. Influence of pH over nitrifying biofilm activity in submerged biofilters. Wat Res 1997;31: 1180 – 6. [14] Liu Y, Capdeville B. Specific activity of nitrifying biofilm in water nitrification process. Wat Res 1996;30:1645–50. [15] Hem LJ, Ruston B, Odegard H. Nitrification in a moving bed biofilm reactor. Wat Res 1994;28:1425–33. [16] Lee JS, Johnson WK. Carbon-slurry activated sludge for nitrificationdenitrification. J WPCF 1979;51:111–25. [17] Shammas NK. Interactions of temperature, pH, and biomass on the nitrification process. J WPCF 1986;58:52–9. [18] Lewandowski Z. Nitrification process in activated sludge with suspended marble particles. Wat Res 1985;19:535–9. [19] Nogueira R, Lazarova V, Manem J, Melo LF. Influence of dissolved oxygen on the nitrification kinetics in a circulating bed biofilm reactor. Bioprocess Eng 1998;19:441–9. [20] Glass C, Silverstein J, Oh J. Inhibition of denitrification in activated sludge by nitrite. Wat Environ Res 1997;69:1086 –93. [21] Glass C, Silverstein J. Denitrification kinetics of high nitrate concentration water: pH effect on inhibition and nitrite accumulation. Wat Res 1998;32:831–9. [22] Grabinska-Loniewska A, Slomezynska T, Kanska Z. Denitrification studies with glycerol as a carbon source. Wat Res 1985;19: 1471–7. [23] Lemmer H, Zaglauer A, Metzner G. Denitrification in a methanol fed fixed-bed reactor. Wat Res 1997;31:1897–1902. [24] Akunna JC, Bizeau C, Moletta R. Nitrate and nitrite reductions with anaerobic sludge using various carbon sources. Wat Res 1993;27: 1303–12. [25] Carucci A, Ramadori R, Rosetti S, Tomei MC. Kinetics of denitrification reactions in single sludge systems. Wat Res 1996;30:51– 6. [26] Gumaelius L, Smith ED, Dalhammer G. Potential biomarker for denitrification of wastewaters: effect of process variables and cadmium toxicity. Wat Res 1996;30:3025–31. [27] Soares MI, Braester C, Belkin S, Abeliovich A. Denitrification in laboratory sand columns: carbon regimes, gas accumulation and hydraulic properties. Wat Res 1991;25:325–32. [28] Balderston WL, Sieburth JM. Nitrate removal in a closed system aquaculture by columnar denitrification. Appl Environ Microbiol 1976;32:808 –18. [29] Standard Methods for the Examination of Water and Wastewater. 17th edn. APHA, Washington, D.C., 1989. [30] Metcalf, Eddy. Tchobanoglous G, Burton, FL. Wastewater Engineering: Treatment, Disposal, Reuse. 3rd edn., McGraw Hill, New York, 1991. pp. 695–725.
www.elsevier.com/locate/enzmictec
Kinetics of sequential nitrification and denitrification processes Ali R. Dinc¸er, Fikret Kargı* Department of Environmental Engineering, Dokuz Eylu¨l University, Buca, I˙zmir, Turkey Received 22 July 1999; received in revised form 6 January 2000; accepted 18 January 2000
Abstract Kinetics of nitrification and denitrification of synthetic wastewater was investigated by using two reactors in series. An activated sludge unit was used for nitrification followed by a downflow biofilter for denitrification. Glucose solution was fed to the denitrification column to supply carbon source. Reactors were operated at different operating conditions and data were collected for determination of kinetic constants. Experimental data indicated that nitrification and denitrification kinetics followed Monod kinetics. By using the experimental data, kinetic constants for nitrification were determined as k ⫽ 1.15 d⫺1, KN ⫽ 5.14 mg/l, Y ⫽ 0.34 mgX/mgN and b ⫽ ⫺0.021 d⫺1. Similarly, kinetic constants for denitrification were determined as k ⫽ 0.23 d⫺1 and KDN ⫽ 0.27 mg/l. Rates of nitrification and denitrification increased with increasing nitrogen loading rate. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Nitrification; Denitrification; Kinetic constants
1. Introduction Nomenclature Nitrification and denitrification processes are advanced biological treatment processes used for removal of excess nitrogen present in the effluent of BOD removal processes. Nitrification is an aerobic, autotrophic process used for conversion of ammonium to nitrate that entails two steps. Ammonium is converted to nitrite by Nitrosomonas species in the first step, and nitrite is converted to nitrate by Nitrobacter species in the second. The fraction of nitrifying organisms in activated sludge culture increases with increasing N/COD ratio in the wastewater. Denitrification is an anoxic, heterotrophic process used for conversion of nitrate to nitrogen gas by denitrifying organisms. Denitrifying organisms require external carbon source for biosynthesis and energy generation. These two processes are usually realized in two separate stages, as the environmental conditions required for each process are different. Composition of the feed wastewater (i.e. C/N ratio and types of nitrogen compounds) determines the order of nitrification and denitrification processes. Numerous studies are reported in literature for nitrification and denitrification of wastewaters [1–28]. Nitrification * Corresponding author. Tel.: ⫹0090-232-4531143; fax: ⫹0090-2324531153. E-mail address: [email protected] (F. Kargı).
KN KDN LCOD LN No, N NH4-N NO3-N NO2-N NOx-N Q RN RDN Rm U V X Y b k ⌰c ⌰H
0141-0229/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 0 ) 0 0 1 4 5 - 9
saturation constant for nitrification saturation constant for denitrification COD loading rate (Q 䡠 CODo, mgCOD/h) nitrogen loading rate (QNo, mgN/h) feed and effluent nitrogen concentrations (mg/l) ammonium nitrogen concentration (mg/l) nitrate nitrogen concentration (mg/l) nitrite nitrogen concentration (mg/l) nitrate ⫹ nitrite nitrogen concentrations flow rate of wastewater (l/h) rate of nitrification (mgN/l 䡠 h) rate of denitrification (mgN/l 䡠 h) maximum rate of denitrification (kX, mgN/l 䡠 h) specific rate of nitrification (mgN/mgX 䡠 d) volume of liquid in reactors (l) biomass concentration (mg/l) growth yield coefficient (mgX/mgN) death rate constant (d⫺1) nitrification rate constant (d⫺1) sludge age or solids retention time (d) hydraulic residence time (V/Q, h) specific rate of growth (d⫺1)
38
A.R. Dinc¸er, F. Kargı / Enzyme and Microbial Technology 27 (2000) 37– 42
Fig. 1. A schematic of the experimental set-up for nitrification and denitrification.
kinetics was found to be zeroth order for effluent ammonium concentration above 10 mg/l [5]. Adverse effects of high C/N ratio such as C/N ⬎ 1.5 on performance of nitrifying bacteria in biofilms was observed by Okabe [6]. Carbon to nitrogen ratio in wastewater should be less than 0.25 for effective nitrification. Inhibition of nitrification by organic compounds [7] and by the substrate NH⫹ 4 [8] were investigated. Inhibition by free ammonia and nitrous acid was observed by Anthonisen et al [10]. Effects of temperature and pH on nitrification process was investigated by Shammas [17]. Optimal pH for nitrification was reported as pH ⫽ 8 ⫾ 0.5. However, pH of the media decreases as a result of H⫹ ion release to the media requiring alkalinity addition to the media. Theoretically, 7.14 mg of alkalinity is consumed per 1 mg of NH4-N oxidized [13]. Dissolved oxygen limitations were reported to be important in nitrification when oxygen supply is below 2 g O2/g NH4-N [19]. Denitrification organisms were also reported to be inhibited at high concentrations of nitrate and nitrite [20,21]. The rate of denitrification was reported to be zeroth order for NO3-N concentrations above 5 mg/L [25]. Use of various carbon sources for denitrification was tested by several investigators [22,23]. The optimal C/N ratio for denitrification was reported to be C/N ⫽ 1.1 when glycerol was used as carbon source [22]. Denitrifying organisms can tolerate pH between 6 and 9. However, the optimal pH was reported as pH ⫽ 7– 8 [26]. pH increases as a result of H⫹ ion removal from the media during denitrification. Usually no external addition of acid is required for pH control during denitrification, since pH variations can be tolerated by the nitrifying bacteria. Despite reported studies on separate nitrification and denitrification processes in the literature, kinetics of sequential nitrification and denitrification processes were not systematically investigated. For this reason, this study was designed to investigate the kinetic behavior of such processes and to determine the kinetic constants. Synthetic
wastewater containing ammonium and bicarbonate was subjected to nitrification in an activated sludge unit. The effluent of nitrification was fed to a downflow biofilter for denitrification with external addition of glucose solution. The reactors were operated at different operating conditions (e.g. hydraulic residence time and sludge age), and experimental data were used for determination of kinetic constants.
2. Materials and methods 2.1. Experimental set-up A schematic diagram of the experimental set-up is depicted in Fig. 1. The system consisted of an activated sludge unit used for nitrification and a downflow packed column for denitrification. The sedimentation tank was separated from the aeration tank by an inclined plate in the activated sludge unit. Wastewater passage from the aeration tank to the sedimentation tank was through holes on the inclined plate and the sludge recycle was through the gap (2 cm) underneath the plate. The packed column was filled with pieces of plastic tubes of 1 cm in length. Glucose solution was fed to the denitrification column to provide carbon source for the organisms. The liquid volumes in the aeration and sedimentation tanks were VAT ⫽ 7.2 l and VST ⫽ 2.2 l, respectively. The denitrification column had a liquid volume of VDC ⫽ 5.1 l and support particle surface area of Ap ⫽ 2.41 m2, resulting in total support surface area per unit liquid volume of a ⫽ 472 m2/m3. 2.2. Wastewater composition The synthetic wastewater used for nitrification was composed of 382 mg 䡠 L⫺1 NH4Cl (NH4-N ⬵ 100 mg 䡠 L⫺1);
A.R. Dinc¸er, F. Kargı / Enzyme and Microbial Technology 27 (2000) 37– 42
39
1510 mg 䡠 L⫺1 NaHCO3 (900 mg CaCO3 䡠 L⫺1 alkalinity); 100 mg 䡠 L⫺1 COD (93 mg 䡠 L⫺1 glucose); 20 mg 䡠 L⫺1 CaCl2 䡠 2H2O; 40 mg 䡠 L⫺1 MgSO4 䡠 7H2O; 85 mg 䡠 L⫺1 KH2PO4. pH of the media was adjusted to pH ⫽ 7.5. The effluent of the nitrification stage containing different nitrate nitrogen (NO3-N) concentrations was fed to the denitrification column. Sugar solution containing 744 mg 䡠 L⫺1 glucose resulting in 800 mg 䡠 L⫺1 COD; 50 mg 䡠 L⫺1 KH2PO4; 20 mg 䡠 L⫺1 MgSO4; 10 mg 䡠 L⫺1 Na2S2O3 with a pH of 7 was fed to the denitrification column continuously to obtain COD/N ⫽ 4.8 in the feed.
under the same experimental conditions were negligible resulting in less than 5% deviations from the average.
2.3. Organisms
3.1. Nitrification kinetics
Nitrification organisms (Nitrosomonas and Nitrobacter) were obtained from Clemson University, SC, USA in mixed culture form and cultivated in our laboratory on a shaker by using the aforementioned media. The denitrification stage was inoculated by a heterotrophic culture obtained from activated sludge unit of Pak-maya Baker’s yeast company, Izmir, Turkey. Denitrifiers were concentrated by slow continuous operation of the packed column for about 2 weeks. Continuous experiments were started after dense population of organisms were obtained by batch operation for 10 days.
Activated sludge unit used for nitrification of synthetic wastewater with NH4-No ⬵ 100 mg/l was operated at different sludge ages (solids retention time) between c ⫽ 3–20 days and hydraulic residence times between H ⫽ 3–30 hours. Nitrogen balance around the aeration tank of activated sludge unit results in the following equation:
2.4. Experimental procedure Inoculum culture cultivated and adapted to the synthetic media on a shaker was used for inoculation of reactors. The reactors were operated batchwise for 10 days until dense culture of organisms were obtained in both stages before continuous operation. Sludge age in the activated sludge unit was adjusted by daily removal of sludge from the aeration tank. The effluent of the nitrification step was fed to the denitrification column with the same flow rate. Hydraulic residence time was varied by changing the feed flow rate. Glucose solution was fed to the denitrification column with a known flow rate to obtain a COD to nitrogen loading ratio of LCOD/LN ⬵ 4.8. Dissolved oxygen and pH measurements were made twice per day. Experiments were performed for at least 7 days after the system reached steady-state. 2.5. Analytical methods
3. Results and discussion Experimental data obtained at different sludge ages for nitrification and at different hydraulic residence times for denitrification were used for determination of kinetic constants.
U⫽
kN Q(No⫺N) ⫽ VX KN ⫹ N
(1)
where, U is specific rate of nitrification (mgN/mgX 䡠 d); Q is flow rate of feed wastewater (L/d); V is volume of the aeration tank (L); No and N are the feed and effluent NH4-N concentrations (mg/L); X is the biomass concentration in the aeration tank (mg/L); k (d⫺1) and K N (mg/L) are the maximum nitrification rate and saturation constants, respectively. In double reciprocal form eqn. 1 takes the following form: 1 1 KN 1 HX ⫽ ⫽ ⫹ U (No ⫺ N) k k N
(2)
A plot of 1/U versus 1/N results in a line with a slope of KN/k and an intercept of 1/k. Experimental data obtained at different sludge and hydraulic residence times were plotted as 1/U versus 1/N as shown in Fig. 2. From the slope and intercept of the best-fit line the following values were found: k ⫽ 1.15 d⫺1 and K N ⫽ 5.14 mg/L
Samples were withdrawn daily from the reactor effluents and were centrifuged to remove cells from the wastewater. Ammonium, nitrate, nitrite, and COD analyses were performed on clear samples. Merck-Spectroquant analytical kits were used for ammonium (Kit No: 1.14559.0001), nitrate (Kit No: 1.14773.0001), and nitrite (Kit No: 14776) analyses. COD analyses were carried out by using the Standard Methods [29]. Dissolved oxygen (DO) measurements were carried out by using a Solomat 520C, DO analyzer, and a DO probe. Nitrogen measurements were done at least three times after the system reached steady-state. Differences among the three nitrogen concentration measurements
By using the definition of sludge age, the following equation can be written,
⫽
1 YkN ⫽ ⫺ b ⫽ YU ⫺ b c KN ⫹ N
(3)
where, is specific growth rate of organisms (d⫺1); c is sludge age (d); Y is the growth yield coefficient (gX/gN), and b is the death rate constant (d⫺1). A plot of 1/c versus U results in a line with a slope of Y and intercept of ⫺b. Experimental data obtained at different sludge ages were plotted as 1/c versus U as shown in Fig. 3. From the slope
40
A.R. Dinc¸er, F. Kargı / Enzyme and Microbial Technology 27 (2000) 37– 42
Fig. 2. Double reciprocal plot of 1/U versus 1/N for determination of k and KN coefficients.
and intercept of the best-fit line the following values were determined: Y ⫽ 0.34 gX/gN,
b ⫽ 0.021 d⫺1
These constants are in good agreement with the literature values [30]. Small differences between our results and the literature values may be because of lower fraction of nitrifying organisms in microbial population and suboptimal environmental conditions. Therefore, the design equation for nitrification in activated sludge unit takes the following form:
⫽
1 0.34 (1.15)N ⫺ 0.021 ⫽ c 5.14 ⫹ N
Fig. 4. Variation of the rate of nitrification (R) with effluent ammonium concentration.
tration. Nitrification rate increased with steady-state NH4-N concentration up to 50 mg/l and remained constant for larger NH4-N levels as expected by the Monod equation. The maximum rate of nitrification was Rm ⫽ 25 mgN/l 䡠 h and the saturation constant (ammonium nitrogen concentration at 1⁄2 of Rm) was approximately KN ⫽ 5 mg/l. Variation of the rate of nitrification with ammoniumnitrogen loading rate (LN ⫽ Q 䡠 No) is depicted in Fig. 5. The rate increased more steeply at low loading rates and reached a constant (Rm ⫽ 25 mgN/l 䡠 h) level at high nitrogen loading rates between LN ⫽ 250 –300 mgN/h. This trend is in agreement with Fig. 4.
(4)
where c is in days and N is in mg/L. Fig. 4 depicts variation of nitrification rate (R ⫽ Q (No-N)/V) with effluent ammonium-nitrogen concen-
Fig. 3. A plot of U versus 1/C for determination of kinetics constants.
3.2. Denitrification kinetics The effluent of the nitrification reactor was fed to the denitrification column operating in down-flow mode. Deni-
Fig. 5. Variation of NH4 ⫺ N removal rate (R) with NH4 ⫺ N loading rate (LN) in nitrification (c ⫽ 15 d, H ⫽ 12 h).
A.R. Dinc¸er, F. Kargı / Enzyme and Microbial Technology 27 (2000) 37– 42
41
Fig. 6. A plot of 1/H 䡠 Ln No/N versus (No ⫺ N)/H for determination of kinetic constants of denitrification.
Fig. 7. Variation of the denitrification rate RDN with NOx ⫺ N loading rate, LN 䡠 (H ⫽ 6 h).
trification organisms were immobilized on the surfaces and within the interstitial spaces of support particles. Denitrification column was operated at different hydraulic residence times and the experimental data were used for determination of the kinetic constants. Nitrogen balance on differential volume of the packed column yields the following design equation.
d⫺1 ⫽ 0.0096 h⫺1. These constants are in good agreement with the literature values [30]. Therefore, the design equation for denitrification column takes the following form,
⫺QdN ⫽ RDN 䡠 dV ⫽
Rm 䡠 N kX 䡠 N dV ⫽ dV K DN ⫹ N K DN ⫹ N
(5)
Rearrangement of Eqn. 5 results in the following equation
冉
冊
冉
冊
dV 1 K DN ⫹ N 1 K DN ⫽⫺ ⫹ 1 dN dN ⫽ ⫺ Q Rm N Rm N
(6)
Integration of Eqn. 6 from V ⫽ 0 and N ⫽ No to V ⫽ V and N ⫽ N and rearrangement of the resulting equation yields, Rm 1 (No ⫺ N) 1 No ⫺ 䡠 ⫽ Ln K DN K DN H H N
(7)
where, Rm (⫽kX) is the maximum rate of denitrification (mgN/l 䡠 h); KDN is the saturation constant of denitrification (mg/l); No and N are the feed and effluent NOx-N (nitrate ⫹ nitrite nitrogen) concentrations (mg/l); H (⫽V/Q) is the hydraulic residence time (h). A plot of 1/H Ln (No/N) versus (No ⫺ N)/H yields a line with a slope of ⫺1/KDN and intercept of Rm/KDN. Experimental data obtained at different hydraulic residence times (feed flow rates) were plotted as 1/H Ln (No/N) versus (No-N)/H as shown in Fig. 6. From the slope and the intercept of the best-fit line the following values were found. Rm ⫽ 280 mgN/L 䡠 d ⫽ 11.7 mgN/l 䡠 h,
KDN ⫽ 0.27 mg/l
By using the definition of Rm ⫽ kX and average biomass concentration in the column as X ⫽ 1200 mg/l, the maximum denitrification rate constant was found as k ⫽ 0.23
0.23 X 1 No 1 (No ⫺ N) ⫽ Ln ⫺ 䡠 0.27 0.27 H H N where X, No, N are in mg/L and H is in days. Fig. 7 depicts variation of rate of denitrification with NOx-N (nitrate ⫹ nitrite nitrogen) loading rate (LN ⫽ Q 䡠 No). The rate increased sharply up to loading rate of L ⫽ 250 mgN/h and remained constant (R ⫽ 30 mgN/L 䡠 h) for loading levels between 250 –300 mg N/h.
4. Conclusions Kinetics of sequential nitrification and denitrification processes were investigated by using constant feed NH4No ⫽ 100 mg/l at different hydraulic and sludge residence times. Experimental data was used for determination of kinetic constants of nitrification and denitrification and the following values were found: k ⫽ 1.15 d⫺1, KN ⫽ 5.14 mg/l, Y ⫽ 0.34 gX/gN, b ⫽ 0.021 d⫺1 Denitrification: k ⫽ 0.23 d⫺1, KDN ⫽ 0.27 mg/l Nitrification:
The constants for nitrification determined from our experiments are in good agreement with the literature values of k ⫽ 2–5 d⫺1, KN ⫽ 1–5 mg/l, Y ⫽ 0.2– 0.3 gX/gN and b ⫽ 0.02– 0.06 d⫺1 [30]. Somewhat lower k and higher KN values obtained in this study may be because of lower fraction of nitrifying organisms in the total population and sub optimal environmental conditions. Experimentally determined constants for denitrification
42
A.R. Dinc¸er, F. Kargı / Enzyme and Microbial Technology 27 (2000) 37– 42
are also in good agreement with the literature values of k ⫽ 0.2– 0.8 d⫺1 and KDN ⫽ 0.1– 0.3 mg/l [30], indicating accuracy of our experimental results. The rates of nitrification and denitrification indicated a hyperbolic variation with the steady-state concentrations of effluent nitrogen concentrations as suggested by the Monod kinetics (Saturation kinetics). Increasing nitrogen loading rates resulted in significant increases in nitrification and denitrification rates, especially at low levels of loading rates. However, the rates reached nearly constant levels at high loading rates. Developed design equations with experimentally determined kinetic constants can be used for design of sequential nitrification and denitrification processes.
Acknowledgment This study was supported in part by the research funds of the Dokuz Eylu¨l University.
References [1] Chen SK, Juaw CK, Cheng SS. Nitrification and denitrification of high strength ammonium and nitrite wastewater in biofilm reactors. Wat Sci Technol 1991;23:1417–25. [2] Cheng SS, Chen WC. Organic carbon supplement influencing performance of biological nitrification in a fluidized bed reactor. Wat Sci Technol 1994;30:131– 42. [3] C¸ec¸en F, Go¨nenc¸ I˙F. Criteria for nitrification and denitrification of high strength wastes in two upflow submerged filters. Wat Env Res 1995;67:132– 42. [4] Horan NJ, Azimi AA. The effects of transient nitrogen loadings on nitrifying activated sludges in completely mixed and plug-flow reactors. Wat Res 1992;26:279 – 84. [5] Hall ER, Murphy KL. Sludge age and substrate effects on nitrification kinetics. J WPCF 1985;57:413– 8. [6] Okabe S, Oozawa Y, Hirata K, Watanabe Y. Relationship between population dynamics of nitrifiers in biofilms and reactor performance at various C:N ratios. Wat Res 1996;30:1563–72. [7] Oslislo A, Lewandowski Z. Inhibition of nitrification in the packed bed reactors by selected organic compounds. Wat Res 1985;19:423– 6. [8] Gee CS, Suidan MT, Pfeffer JT. Modelling of nitrification under substrate inhibiting conditions. J Environ Eng 1990;116:18 –31. [9] Painter HA, Loveless JA. Effect of temperature and pH value on the growth rate constants of nitrifying bacteria in the activated sludge process. Wat Res 1983;17:237– 48.
[10] Anthonisen AC, Loehr RC, Prakasam TBS, Srinath, EG. Inhibition of nitrification by ammonia and nitrous acid. J WPCF 1976;48:835–52. [11] Gupta SK, Sharma R. Biological oxidation of high strength nitrogenous wastewater. Wat Res 1996;30:593– 600. [12] Matsumura M, Yamamoto T, Wang P, Shinabe K, Yasuda K. Rapid nitrification with immobilized cell using macroporous cellulose carrier. Wat Res 1997;31:1027–34. [13] Villaverde S, Encina PAG, Polanco FF. Influence of pH over nitrifying biofilm activity in submerged biofilters. Wat Res 1997;31: 1180 – 6. [14] Liu Y, Capdeville B. Specific activity of nitrifying biofilm in water nitrification process. Wat Res 1996;30:1645–50. [15] Hem LJ, Ruston B, Odegard H. Nitrification in a moving bed biofilm reactor. Wat Res 1994;28:1425–33. [16] Lee JS, Johnson WK. Carbon-slurry activated sludge for nitrificationdenitrification. J WPCF 1979;51:111–25. [17] Shammas NK. Interactions of temperature, pH, and biomass on the nitrification process. J WPCF 1986;58:52–9. [18] Lewandowski Z. Nitrification process in activated sludge with suspended marble particles. Wat Res 1985;19:535–9. [19] Nogueira R, Lazarova V, Manem J, Melo LF. Influence of dissolved oxygen on the nitrification kinetics in a circulating bed biofilm reactor. Bioprocess Eng 1998;19:441–9. [20] Glass C, Silverstein J, Oh J. Inhibition of denitrification in activated sludge by nitrite. Wat Environ Res 1997;69:1086 –93. [21] Glass C, Silverstein J. Denitrification kinetics of high nitrate concentration water: pH effect on inhibition and nitrite accumulation. Wat Res 1998;32:831–9. [22] Grabinska-Loniewska A, Slomezynska T, Kanska Z. Denitrification studies with glycerol as a carbon source. Wat Res 1985;19: 1471–7. [23] Lemmer H, Zaglauer A, Metzner G. Denitrification in a methanol fed fixed-bed reactor. Wat Res 1997;31:1897–1902. [24] Akunna JC, Bizeau C, Moletta R. Nitrate and nitrite reductions with anaerobic sludge using various carbon sources. Wat Res 1993;27: 1303–12. [25] Carucci A, Ramadori R, Rosetti S, Tomei MC. Kinetics of denitrification reactions in single sludge systems. Wat Res 1996;30:51– 6. [26] Gumaelius L, Smith ED, Dalhammer G. Potential biomarker for denitrification of wastewaters: effect of process variables and cadmium toxicity. Wat Res 1996;30:3025–31. [27] Soares MI, Braester C, Belkin S, Abeliovich A. Denitrification in laboratory sand columns: carbon regimes, gas accumulation and hydraulic properties. Wat Res 1991;25:325–32. [28] Balderston WL, Sieburth JM. Nitrate removal in a closed system aquaculture by columnar denitrification. Appl Environ Microbiol 1976;32:808 –18. [29] Standard Methods for the Examination of Water and Wastewater. 17th edn. APHA, Washington, D.C., 1989. [30] Metcalf, Eddy. Tchobanoglous G, Burton, FL. Wastewater Engineering: Treatment, Disposal, Reuse. 3rd edn., McGraw Hill, New York, 1991. pp. 695–725.