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Nitrification Activity of Immobilized Activated Sludge Evaluated by Respiration Rate
280
R.H. Wijffels, R.M. Buitelaar, C. Bucke and J. Tramper (Eds) Immobilized Cells: Basics and Applications © 1996 Elsevier Science B.V. All rights reserved.
Nitrification Activity of Immobilized Activated Sludge Evaluated by Respiration Rate H.Nakamura, S.Miyabayashi, K.Noto and T.Sumino Hitachi Plant Engineering & Construction Co.,Ltd., Research Laboratory, 537 Kamihongo, Matsudo, Chiba, 271 Japan
Introduction As a method for removal of nitrogen in wastewater, the high rate nitrification process using entrapped immobilized nitrifiers (nitrifier pellets) has reached the stage of practical use^\ When this process is applied to the actual treatment, however, in order to ensure stable treatment in spite of probable fluctuation in load, water temperature and other factors, it is important to establish the control system for the process performance. Above all, in order to control the nitrification performance of immobilized nitrifiers, it is necessary to select its indicators and grasp the nitrification characteristics in detail under various conditions. For the activity of immobilized nitrifiers, if it can be measured from the respiration rate on a real time basis^^'^\ it is expected that the nitrification process can be controlled to the optimal condition, thus leading to the prediction of treated water quality. With a view to utilizing the respiration rate as a control indicator for the nitrification performance of immobilized nitrifiers, some investigations were made on the relation between the nitrification characteristics and respiration rate under various operational conditions.
Methods Apparatus and Method for Continuous TreatmentTest Cubic pellets 3mm in size were prepared by immobilizing the activated sludge in a sewage treatment plant using a pre-polymer constituted mainly of polyethylene glycol. While these pellets were being subjected to aeration in a 1.5 Utter continuous treatment tank as shown in Fig.l, continuous treatment of ammonia containing synthetic wastewater (Table 1) was started. The NH4-N concentration of the synthetic wastewater was set at 20 mg/L, being nearly same as that of actual sewage. The operational conditions are shown in Table 2. The retention time of tiie wastewater was set at tiiree hours (NH4-N loading 66.7 mg-N/L-pellet/h), being nearly
281 same as that in a nitrification tank for the high rate nitrification process. The pellet volumetric ratio in tank was set at 10%, and the water temperature was kept continuously at 20 °C. For the treated water quality, the daily changes in NH4-N, N03-N, and NO2-N concentrations were measured, respectively. Treated water
-(P)-
Continuous treatment tank(1.5L)
Synthetic wastewater (NH4-N 20nlg/L)
Nitrifier pellet
Air T
Fig.l Apparatus for continuous operation Table 2 Operational condition Retention time (h) Aeration rate (NL/L-tank/min) Water temperature ("C) Pellet volumetric ratio in tank (%)
Table 1 Composition of synthetic wastewater (mg/L- tap water) NH4C1 76.4 NaHCOs 234 Na2HP04 • 12H20 23.2 NaCl 10.2 KCl 4.8 CaCl2 • 2H20 4.8 MgS04 • 7H20 16.8 7.6-7.8 pH
3 1
DO sensor Flask (100m:
20 10
Pellet (lOmL)
Recorder 0 0
Fig.2 Apparatus for off-line measurement of pellets respiration rate
Measuring Method for Various Resptation Rates of ImmobilizedNitrifiers Various respiration rates as shown in Table 3 were measured by the methods as described below^'": [1] Respiration rate in continuous treatment tank (a) Method for calculation using tank DO and KLa The steady-state respiration rate of immobilized nitrifiers in a continuous treatment tank was calculated using the tank DO concentration and the KLa value by the formula as given below : Krs = (Cs - C) • KLa • (100/P) (1) Where, Cs : Saturated DO concentration (8.84mg/L at 20°C) C : DO concentration in continuous treatment tank (mg/L) KLa: Overall oxygen transfer coefficient (Measured value, 21.0 1/h) P : Pellet volumetric ratio in tank (10%)
282 Table 3 Respiration rates (Kr) of immobilized nitrifiers (pellets) [1] Respiration rate in the continuous treatment tank (on-line measurement) Total Kr Krs N-Krs (=Krs-KrO) Kr due to N-Krs theoretical value (calculated by formula (2)) nitrification 2] Respiration rate at various NH4-N concentrations (off-line measureme Kr20 (NH4-N: 20mg/L) Total Kr Kr2 (NH4-N: 2mg/L) Krl (NH4-N: Img/L) Kr due to N-Kr20 (= Kr20 - KrO) nitrification Endogenous Kr KrO By subtracting endogenous respiration rate (KrO) of pellets from Krs, the respiration rate (N-Krs) caused by nitrification reaction was determined. (b) Method for calculation of theoretical value from nitrification rate From the balance of NH4-N nitrified by continuous treatment, the respiration rate (Theoretical value of N-Krs) as atiieoreticaloxygen amount required for nitrification was determined by formula (2). N-Krs = (3.43[N02]/t + 4.57[N03]/t) • (100/P) Where, [NO2]: NO2-N concentration in treated water (mg/L) [NO3]: NO3-N concentration in treated water (mg/L) t: Retention time (3h)
(2)
[2] Respiration rates at various NH4-N concentrations In order to examine the influence of NH4-N concentration on the nitrification performance of pellets, the respiration rates at various NH4-N concentrations were off-line measured. lOmL of pellets was sampled from the continuous treatment tank. Measurement of DO consumption was done together with the ammonia containing synthetic wastewater (NH4-N:20 mg/L, DO saturated water) as given Table 1 in the flask as shown in Fig.2. From the DO concentration decline curve thus obtained, the gradient at each DO concentration was calculated to determine tiie respiration rate (Kr20). Measurement was made also on the cases (Krl, Kr2) where ammonia containing wastewater diluted with t^^ water to tiie NH4-N concentration of 1 mg/L and 2 mg/L (pH7.2 to 7.5) was added, respectively. In parallel witii these measurements, tiie endogenous respiration rate (KrO) of pellets was measured by addition of tap water (pH 7.2 to 7.5) only. By subtracting KrO from Kr20, tiie
283 respiration rate (N-Kr20) due to nitrification was determined. The water temperature during the measurement was kept at 20 °C o Method for Examining the Influence of Dissolved Oxygen Concentration on the Respiration Rate The nitrification reaction by activated sludge containing nitrifying bacteria is influenced by operational conditions such as DO concentration. The respiration rate (Kr) is generally expressed by the Monod's Formula as given below : Kr = ko • [DO]/(Ko+[DO]) Where, [DO]: DO concentration ko: Maximum respiration rate as to [DO] (mg/L-pellet/h) Ko: Saturation constant as to [DO] (mg/L)
(3)
At various stages of acclimation of immobilized nitrifiers, respiration rates at various NH4-N concentration were off-line measured. Ko and ko were determined based on these respiration rates. Method for Counting the Number ofBacteriain ImmobilizedNitrifier Pellets ( Pellets ) (5mL/45mL distilled water) Homogenizing
(10,000rpm, lOmin)
Ultrasonic treatment (300W, 400|LiA, Imin) Cultivation
Bacteria
Nitrifiers Ammonoa oxidizing Nitrite oxidizing bacteria bacteria
Culture media (mg/L)
K2HPO4 200 MgS04 • 7H20 50 CaCl2 • 2H20 20 FeS04 • 7H20 1 (NH4)2S04 100 NaN02
Conditions Enumeration
400 100 40 2 60
30°C,56days
(MPN for nitrifiers) Fig.3 Procedure for bacteria enumeration
Heterotrophes M-TGE medium (DIFCO) Bacto-beef 6,000 Bacto-tryptone 10,000 Bacto-dextrose 2,000 30°C, lOdays
284 The nitrifiers are generally constituted of ammonia oxidizing bacteria (Nitrosomonas, etc.) and nitrite oxidizing bacteria (Nitrobacter, etc.). For these two species of nitrifiers and other bacteria grown on standard medium (heterotrophs), the number of bacteria was counted in the procedure as shown in Fig.3.
Results and discussion Change of Respiration Rate in Continuous Treatment [ 1 ] Change of treated water quality The daily change of treated water quality obtained by the continuous treatment of the ammonia containing synthetic wastewater is given in Fig.4. Immediately after the commencement of treatment, NH4-N concentration began to lower. Ten days after, the NH4-N removal ratio has reached nearly 90%. The NO2-N and NO3-N concentrations showed an increasing tendency and fast increase of nitrification activity was observed. Although 5 to 6 mg/L of NO2-N was left unnitrified at an early stage, its concentration lowered gradually, NO2-N became nitrified almost completely after the 50th day onward . The DO concentration in a continuous treatment tank lowered gradually after the commencement of experiment, and showed an almost constant value between 7.2 and 7.5 mg/L after the 10th day onward. ^20 p o|l6
IT
^ ^ ^ - - •
0
D
§ ? 4001 ^
DO
•000
S S 200 100
L'T ^^-^^..NO2-N
h V ^^5:^.^—NH4-N 0
N-Kr20 ..a
11500!
-"°N03-N
r\ ' 1 \ / rw ^ d 4
600r
-•
20
40
60 Day
affli
80
1
1
100 120
Fig.4 Change of treated water quality
0
o
-r V - ^ N-Krs
N-Krs theoretical value 40
60 80 100 120 Day Fig.5 Change of N-Krs and N-Kr20
[2] Change of respiration rate The respiration rate (N-Krs) due to nitrification was determined by subtracting the endogenous respiration rate (KrO) from the total respiration rate (Krs) of pellets in the continuous treatment tank. Its daily change is given in Fig.5. N-Krs increased linearly up to around the 10th day, and then reached the maximum value of 250 to 300 mg-N/L-pellet/h and
285 was kept constant at this value. This shows a good coincidence with the fact that the NH4-N removal ratio reached nearly 90% on the 10th day. In addition, the daily change of N-Krs theoretical value determined as a theoretical oxygen amount required for actual nitrification in continuous treatment is given in Fig.5. As a result, N-Krs showed a tendency to coincide nearly with the N-Krs theoretical value. From this fact, it was found that the nitrification rate of immobilized nitrifiers could be assumed from N-Krs, and that N-Krs might be a good indicator of nitrification activity. Furthermore, the change of the respiration rate (N-Kr20) due to nitrification reaction determined by subtracting the endogenous respiration rate (KrO) from the respiration rate (Kr20) measured by adding 20mg/L NH4-N wastewater is plotted in Fig.5. These values were determined from the gradient of DO decline curve in DO concentration almost equal to that in the continuous treatment tank. N-Kr20 continued to increase even after the 10th day when N-Krs reached practicaUy the maximum value, and became constant at the maximum value of 450 to 500 mg-N/L-pellet/h after the 30th day onward.
Since N-Kr20 is regarded, as will be
described afterward, as a potential maximum respiration rate, it was confirmed that nearly complete nitrification was achieved at the nitrification capacity of about 60% of that of pellets in the continuous treatment of NH4-N loading 66.7 mg-N/L-pellet/h. As a breakdown of total respiration rate Kr20, the change in the N-Kr20 and KrO, as well as the ratio N-Kr20/Kr20 is given in Fig.6. The N-Kr20/Kr20 ratio was around 10% at the start of continuous treatment, however, it increased significantly in a few days and became stable at the level of 90 to 95% on the 10th day onward. This result suggests that among all bacteria in the pellets, the nitrifiers rapidly increased in numbers during this period. The KrO was consistently kept at 20 to 50 mg/L-pellet/h after the commencement of continuous treatment, showing no remarkable change.
s 60or
Formula (5) Kr20
I 30*
* ^KrO N-Kr20
^ I § 20*
•a lod- / 0
,K ^
Kr2 /
^Kil
KrO
0 1 2 3 4 5 6 7 8 9 60 80 100 120 10 Day DO concentration (mg/L) Fig.7 Relation between DO concentration Fig.6 Change of respiration rate (N-K20) due to nitrification and endogenous and respiration rate in the off-line respiration rate (KrO) of pellets measurement (28th day)
286 Influence of Dissolved Oxygen Concentration and Ammonia Nitrogen Concentration on the Respiration Rate [1] Relation between DO concentration and respiration rate The respiration rates (Krl, Kr2 and Kr20) were measured for tiie cases where each of EX) saturated synthetic wastewater with an NH4-N concentration of 1 mg/L, 2 mg/L and 20 mg/L was added, respectively. Using the DO concentration decline curves obtained for respective cases, the Kr values were determined from the decUne rates of DO concentration at various points of time, and its relation with the DO concentration was plotted. The example in the case when the pellets at 28 days after the commencement of continuous treatment were used is given in Fig.7. As is clear from Fig.7, every Kr value showed the tendency to lower with the decline in the DO concentration. In measurement of each Kr value, NH4-N is nitrified simultaneously witii the consumption of DO in tiie flask. Namely, when saturated DO of 8.84mg/L at 20°C is totally consumed, about 2 mg/L of NH4-N is nitrified as is assumed from formula (2). For Kr20, the influence by the decline in NH4-N concentration is regarded practically negligible because an excessive amount of NH4-N (20 mg/L) was added. Therefore, the parameters ko, Ko relating to the DO concentration in formula (3) were determined for Kr20 as follows. When [DO]/Kr20 is plotted against [DO], a nearly straight line is obtained (Fig.8). As a regression straight line,tiiefollowing formula of Hofstee type was determined. [DO]/Kr20 = 0.000620[DO] + 0.0109
(4)
13th day 28th day 86th day Kr20 = 980 - [DO] Kr20 = ^^10 • [DO] Kr20 = 873 - [DO] 13.9 + [DO] 17.5 + [DO] 5.77 + [DO]
_ 0.02
a 60or
C 0.016h
^
I 0.0121
206th day 800th day Kr2n=765-[DO] K r 2 0 = ^ 8 1 l i 5 O ] 3.29 +[DO] 1.23 +I DO]
500
Formula (4)
\ 0.0081
o a 0.004
0 0 1 2 3 4 5 6 7 8 9
DO concentration (mg/L)
10
Fig.8 Relation between [DO] and [DO]/Kr20
2 3 4 5 6 7 8 9 DO concentration (mg/L) Fig.9 Relation between DO concentration and Kr20
287
Kr20 was expressed by the formula given below : Kr20 = 1610[DO]/(17.5+[DO])
(5)
Likewise, the relation between the DO concentration and respiration rate (Kr20) for the pellets on 13th day, 86th day, 206th day, and 800th day are given collectively in Fig.9. It was found that Ko became smaller with the lapse of days and the value closer to the maximum respiration rate was obtained at lower DO concentration. This is assumed due to the following reason. The nitrifiers immobiUzed almost evenly in the pellet grow wholly at first to enhance the nitrification activity rapidly. Then, growth of bacteria becomes significant at the parts near to the pellet surface through which DO is easily permeable with the result that higher activity can be obtained even at lower DO concentration. [2] Relation between NH4-N Concentration and Respiration Rate From Fig.7, respiration rate measured with the NH4-N concentration of 20mg/L (Kr20) shows almost the same value as that measured with the NH4-N concentration of 2mg/L. The similar results were obtained in the respiration rates measured on the other days. It was confirmed that the respiration rates (Kr20, N-Kr-20) measured by adding 20mg/L of NH4-N might be a good indicator for the potential maximum nitrification activity of immobiUzed nitrifiers. Relation between Number of Bacteria and Resptation Rate [1] Change in number of bacteria Some investigation were made on the change in number of bacteria in the pellets along with the continuous treatment of ammonia containing synthetic wastewater. The number of bacteria was measured for the nitrifiers, ammonia oxidizing bacteria and nitrite oxidizing bacteria, and other bacteria grown on standard medium (heterotrophs), respectively. The results obtained are shown in Fig. 10. At the commencement of continuous treatment, both nitrifiers existed in the number amounting to about lO^mL-pellet. The ammonia oxidizing bacteria increased sharply in number to about 10/ml^pellet down to the 10th day and then become constant in number. This tendency is well corresponding to the fact that the treated water NH4-N concentration shows a decline immediately after the commencement of continuous treatment as is clear from Fig.4. The growth rate was determined to be 0.70 1/d from the increase curve down to the 10th day. The nitrite oxidizing bacteria also increased in number to about lOVmL-pellet with a growth rate as high as that of ammonia oxidizing bacteria in a few days after the commencement of
288 10001 100 k 10 1
Fig. 10 Change in number of bacteria
10^ 10^ 10^ 10^ 10^ lOio Number of nitrifiers (cells/ml-pellet) Fig. 11 Relation between the number of nitrifiers in pellets and respiration rate
continuous treatment. On the other hand, the heterotrophs were stably kept at the number of about 107mL-pellet after the commencement of continuous treatment. This is well corresponding to the fact that the endogenous respiration rate (KrO) showed a stable value as is shown in Fig.6. Since the synthetic wastewater contains no organic constituents, it is assumed that these heterotrophs grow through metabolization of soluble organic constituents produced by decomposition of autotrophic nitrifying bacteria^l [2] Relation between number of nitrifying bacteria and respiration rate The number of nitrifiers in pellets is considered to have a direct influence on the nitrification activity. However, it takes a long period of time amounting to about two months to count the number of nitrifiers. An attempt was made to estimate the number of nitrifiers from the respiration rate. The relation between the number of nitrifiers (total of ammonia oxidizing bacteria and nitrite oxidizing bacteria) measured at each stage of continuous treatment and the maximum respiration rate (N-Kr20) due to nitrification is given in Fig.l 1. It was found that the logarithm of the number of bacteria was in a proportional relation to the logarithm of N-Kr. This result shows that the number of nitrifiers can be estimated from the maximum respiration rate of immobilized nitrifiers.
CONCLUSIONS The respiration rate was taken up as an indicator for the nitrification activity of immobilized nitrifiers. Using the pellets of newly immobilized activated sludge, the change in their nitrification performance and characteristics was examined during the period of acclimation of
289 nitrifiers in continuous treatment (retention time: three hours) of ammonia containing synthetic wastewater. As a result, the following findings were obtained. [1] Saturation constant of the pellets respiration rate as to DO concentration became smaller with the lapse of days, and the value closer to the maximum respiration rate was obtained at lower DO concentration. This result coincide with tiie fact tiiat the growth of nitrifiers is significant at the parts near to the surface through which DO is easily permeable. [2] From the respiration rate measured by adding 20mg/L of ammonia nitrogen to the wastewater (Kr20), the potential maximum activity of immobilized nitrifiers could be assumed. [3] Kr20 had a high correlation with the number of nitrifiers in the pellets. [4] It was considered possible to use the respiration rate as an indicator of nitrification activity of the pellets.
References 1) T.Takeshima, et.al. : "PEGASUS" An Innovative High-rate BOD and Nitrogen Removal Process for Municipal Wastewater: 66th Annual Conerence of WEF, Anahaim, California (1993) 2) H.Spanjars, et.al. : Respirametry as a Tool for Rapid Characterization of Wastewater and Activated Sludge : Wat.Sci.Tech., Vol.31, No.2, pp.105-114 (1995) 3) D.Dochain, et.al. : Structural Identifiability of Biokinetic Modelds of Activated Sludge Respkation: Wat.Sci.Tech., Vol.29, No.ll, pp.2571-2578 (1995) 4) L. Novak, et.al. : Estimation of Maximum Specific Growth Rate of Heterotrophic and Autotrophic Biomass: A Combined Technique of Mathematical Modelling and Batch Cultivations : Wat.Sci.Tech., Vol.30, No.ll, pp.171-180 (1994) 5) G.H.Kristensen, et.al. : Characterization of Functional Microorganism Groups and Substrate in Activated Sludge and Wastewater by AUR, NUR and OUR : Wat.Sci.Tech., Vol.25, No.6, pp.43-57 (1992) 6) B.E.Rittmann, et.ai. : Nirification as a Source of Soluble Organic Substrate in Biological Treatment: Wat.Sci.Tech., Vol.30, No.6, pp.1-8 (1994)
R.H. Wijffels, R.M. Buitelaar, C. Bucke and J. Tramper (Eds) Immobilized Cells: Basics and Applications © 1996 Elsevier Science B.V. All rights reserved.
Nitrification Activity of Immobilized Activated Sludge Evaluated by Respiration Rate H.Nakamura, S.Miyabayashi, K.Noto and T.Sumino Hitachi Plant Engineering & Construction Co.,Ltd., Research Laboratory, 537 Kamihongo, Matsudo, Chiba, 271 Japan
Introduction As a method for removal of nitrogen in wastewater, the high rate nitrification process using entrapped immobilized nitrifiers (nitrifier pellets) has reached the stage of practical use^\ When this process is applied to the actual treatment, however, in order to ensure stable treatment in spite of probable fluctuation in load, water temperature and other factors, it is important to establish the control system for the process performance. Above all, in order to control the nitrification performance of immobilized nitrifiers, it is necessary to select its indicators and grasp the nitrification characteristics in detail under various conditions. For the activity of immobilized nitrifiers, if it can be measured from the respiration rate on a real time basis^^'^\ it is expected that the nitrification process can be controlled to the optimal condition, thus leading to the prediction of treated water quality. With a view to utilizing the respiration rate as a control indicator for the nitrification performance of immobilized nitrifiers, some investigations were made on the relation between the nitrification characteristics and respiration rate under various operational conditions.
Methods Apparatus and Method for Continuous TreatmentTest Cubic pellets 3mm in size were prepared by immobilizing the activated sludge in a sewage treatment plant using a pre-polymer constituted mainly of polyethylene glycol. While these pellets were being subjected to aeration in a 1.5 Utter continuous treatment tank as shown in Fig.l, continuous treatment of ammonia containing synthetic wastewater (Table 1) was started. The NH4-N concentration of the synthetic wastewater was set at 20 mg/L, being nearly same as that of actual sewage. The operational conditions are shown in Table 2. The retention time of tiie wastewater was set at tiiree hours (NH4-N loading 66.7 mg-N/L-pellet/h), being nearly
281 same as that in a nitrification tank for the high rate nitrification process. The pellet volumetric ratio in tank was set at 10%, and the water temperature was kept continuously at 20 °C. For the treated water quality, the daily changes in NH4-N, N03-N, and NO2-N concentrations were measured, respectively. Treated water
-(P)-
Continuous treatment tank(1.5L)
Synthetic wastewater (NH4-N 20nlg/L)
Nitrifier pellet
Air T
Fig.l Apparatus for continuous operation Table 2 Operational condition Retention time (h) Aeration rate (NL/L-tank/min) Water temperature ("C) Pellet volumetric ratio in tank (%)
Table 1 Composition of synthetic wastewater (mg/L- tap water) NH4C1 76.4 NaHCOs 234 Na2HP04 • 12H20 23.2 NaCl 10.2 KCl 4.8 CaCl2 • 2H20 4.8 MgS04 • 7H20 16.8 7.6-7.8 pH
3 1
DO sensor Flask (100m:
20 10
Pellet (lOmL)
Recorder 0 0
Fig.2 Apparatus for off-line measurement of pellets respiration rate
Measuring Method for Various Resptation Rates of ImmobilizedNitrifiers Various respiration rates as shown in Table 3 were measured by the methods as described below^'": [1] Respiration rate in continuous treatment tank (a) Method for calculation using tank DO and KLa The steady-state respiration rate of immobilized nitrifiers in a continuous treatment tank was calculated using the tank DO concentration and the KLa value by the formula as given below : Krs = (Cs - C) • KLa • (100/P) (1) Where, Cs : Saturated DO concentration (8.84mg/L at 20°C) C : DO concentration in continuous treatment tank (mg/L) KLa: Overall oxygen transfer coefficient (Measured value, 21.0 1/h) P : Pellet volumetric ratio in tank (10%)
282 Table 3 Respiration rates (Kr) of immobilized nitrifiers (pellets) [1] Respiration rate in the continuous treatment tank (on-line measurement) Total Kr Krs N-Krs (=Krs-KrO) Kr due to N-Krs theoretical value (calculated by formula (2)) nitrification 2] Respiration rate at various NH4-N concentrations (off-line measureme Kr20 (NH4-N: 20mg/L) Total Kr Kr2 (NH4-N: 2mg/L) Krl (NH4-N: Img/L) Kr due to N-Kr20 (= Kr20 - KrO) nitrification Endogenous Kr KrO By subtracting endogenous respiration rate (KrO) of pellets from Krs, the respiration rate (N-Krs) caused by nitrification reaction was determined. (b) Method for calculation of theoretical value from nitrification rate From the balance of NH4-N nitrified by continuous treatment, the respiration rate (Theoretical value of N-Krs) as atiieoreticaloxygen amount required for nitrification was determined by formula (2). N-Krs = (3.43[N02]/t + 4.57[N03]/t) • (100/P) Where, [NO2]: NO2-N concentration in treated water (mg/L) [NO3]: NO3-N concentration in treated water (mg/L) t: Retention time (3h)
(2)
[2] Respiration rates at various NH4-N concentrations In order to examine the influence of NH4-N concentration on the nitrification performance of pellets, the respiration rates at various NH4-N concentrations were off-line measured. lOmL of pellets was sampled from the continuous treatment tank. Measurement of DO consumption was done together with the ammonia containing synthetic wastewater (NH4-N:20 mg/L, DO saturated water) as given Table 1 in the flask as shown in Fig.2. From the DO concentration decline curve thus obtained, the gradient at each DO concentration was calculated to determine tiie respiration rate (Kr20). Measurement was made also on the cases (Krl, Kr2) where ammonia containing wastewater diluted with t^^ water to tiie NH4-N concentration of 1 mg/L and 2 mg/L (pH7.2 to 7.5) was added, respectively. In parallel witii these measurements, tiie endogenous respiration rate (KrO) of pellets was measured by addition of tap water (pH 7.2 to 7.5) only. By subtracting KrO from Kr20, tiie
283 respiration rate (N-Kr20) due to nitrification was determined. The water temperature during the measurement was kept at 20 °C o Method for Examining the Influence of Dissolved Oxygen Concentration on the Respiration Rate The nitrification reaction by activated sludge containing nitrifying bacteria is influenced by operational conditions such as DO concentration. The respiration rate (Kr) is generally expressed by the Monod's Formula as given below : Kr = ko • [DO]/(Ko+[DO]) Where, [DO]: DO concentration ko: Maximum respiration rate as to [DO] (mg/L-pellet/h) Ko: Saturation constant as to [DO] (mg/L)
(3)
At various stages of acclimation of immobilized nitrifiers, respiration rates at various NH4-N concentration were off-line measured. Ko and ko were determined based on these respiration rates. Method for Counting the Number ofBacteriain ImmobilizedNitrifier Pellets ( Pellets ) (5mL/45mL distilled water) Homogenizing
(10,000rpm, lOmin)
Ultrasonic treatment (300W, 400|LiA, Imin) Cultivation
Bacteria
Nitrifiers Ammonoa oxidizing Nitrite oxidizing bacteria bacteria
Culture media (mg/L)
K2HPO4 200 MgS04 • 7H20 50 CaCl2 • 2H20 20 FeS04 • 7H20 1 (NH4)2S04 100 NaN02
Conditions Enumeration
400 100 40 2 60
30°C,56days
(MPN for nitrifiers) Fig.3 Procedure for bacteria enumeration
Heterotrophes M-TGE medium (DIFCO) Bacto-beef 6,000 Bacto-tryptone 10,000 Bacto-dextrose 2,000 30°C, lOdays
284 The nitrifiers are generally constituted of ammonia oxidizing bacteria (Nitrosomonas, etc.) and nitrite oxidizing bacteria (Nitrobacter, etc.). For these two species of nitrifiers and other bacteria grown on standard medium (heterotrophs), the number of bacteria was counted in the procedure as shown in Fig.3.
Results and discussion Change of Respiration Rate in Continuous Treatment [ 1 ] Change of treated water quality The daily change of treated water quality obtained by the continuous treatment of the ammonia containing synthetic wastewater is given in Fig.4. Immediately after the commencement of treatment, NH4-N concentration began to lower. Ten days after, the NH4-N removal ratio has reached nearly 90%. The NO2-N and NO3-N concentrations showed an increasing tendency and fast increase of nitrification activity was observed. Although 5 to 6 mg/L of NO2-N was left unnitrified at an early stage, its concentration lowered gradually, NO2-N became nitrified almost completely after the 50th day onward . The DO concentration in a continuous treatment tank lowered gradually after the commencement of experiment, and showed an almost constant value between 7.2 and 7.5 mg/L after the 10th day onward. ^20 p o|l6
IT
^ ^ ^ - - •
0
D
§ ? 4001 ^
DO
•000
S S 200 100
L'T ^^-^^..NO2-N
h V ^^5:^.^—NH4-N 0
N-Kr20 ..a
11500!
-"°N03-N
r\ ' 1 \ / rw ^ d 4
600r
-•
20
40
60 Day
affli
80
1
1
100 120
Fig.4 Change of treated water quality
0
o
-r V - ^ N-Krs
N-Krs theoretical value 40
60 80 100 120 Day Fig.5 Change of N-Krs and N-Kr20
[2] Change of respiration rate The respiration rate (N-Krs) due to nitrification was determined by subtracting the endogenous respiration rate (KrO) from the total respiration rate (Krs) of pellets in the continuous treatment tank. Its daily change is given in Fig.5. N-Krs increased linearly up to around the 10th day, and then reached the maximum value of 250 to 300 mg-N/L-pellet/h and
285 was kept constant at this value. This shows a good coincidence with the fact that the NH4-N removal ratio reached nearly 90% on the 10th day. In addition, the daily change of N-Krs theoretical value determined as a theoretical oxygen amount required for actual nitrification in continuous treatment is given in Fig.5. As a result, N-Krs showed a tendency to coincide nearly with the N-Krs theoretical value. From this fact, it was found that the nitrification rate of immobilized nitrifiers could be assumed from N-Krs, and that N-Krs might be a good indicator of nitrification activity. Furthermore, the change of the respiration rate (N-Kr20) due to nitrification reaction determined by subtracting the endogenous respiration rate (KrO) from the respiration rate (Kr20) measured by adding 20mg/L NH4-N wastewater is plotted in Fig.5. These values were determined from the gradient of DO decline curve in DO concentration almost equal to that in the continuous treatment tank. N-Kr20 continued to increase even after the 10th day when N-Krs reached practicaUy the maximum value, and became constant at the maximum value of 450 to 500 mg-N/L-pellet/h after the 30th day onward.
Since N-Kr20 is regarded, as will be
described afterward, as a potential maximum respiration rate, it was confirmed that nearly complete nitrification was achieved at the nitrification capacity of about 60% of that of pellets in the continuous treatment of NH4-N loading 66.7 mg-N/L-pellet/h. As a breakdown of total respiration rate Kr20, the change in the N-Kr20 and KrO, as well as the ratio N-Kr20/Kr20 is given in Fig.6. The N-Kr20/Kr20 ratio was around 10% at the start of continuous treatment, however, it increased significantly in a few days and became stable at the level of 90 to 95% on the 10th day onward. This result suggests that among all bacteria in the pellets, the nitrifiers rapidly increased in numbers during this period. The KrO was consistently kept at 20 to 50 mg/L-pellet/h after the commencement of continuous treatment, showing no remarkable change.
s 60or
Formula (5) Kr20
I 30*
* ^KrO N-Kr20
^ I § 20*
•a lod- / 0
,K ^
Kr2 /
^Kil
KrO
0 1 2 3 4 5 6 7 8 9 60 80 100 120 10 Day DO concentration (mg/L) Fig.7 Relation between DO concentration Fig.6 Change of respiration rate (N-K20) due to nitrification and endogenous and respiration rate in the off-line respiration rate (KrO) of pellets measurement (28th day)
286 Influence of Dissolved Oxygen Concentration and Ammonia Nitrogen Concentration on the Respiration Rate [1] Relation between DO concentration and respiration rate The respiration rates (Krl, Kr2 and Kr20) were measured for tiie cases where each of EX) saturated synthetic wastewater with an NH4-N concentration of 1 mg/L, 2 mg/L and 20 mg/L was added, respectively. Using the DO concentration decline curves obtained for respective cases, the Kr values were determined from the decUne rates of DO concentration at various points of time, and its relation with the DO concentration was plotted. The example in the case when the pellets at 28 days after the commencement of continuous treatment were used is given in Fig.7. As is clear from Fig.7, every Kr value showed the tendency to lower with the decline in the DO concentration. In measurement of each Kr value, NH4-N is nitrified simultaneously witii the consumption of DO in tiie flask. Namely, when saturated DO of 8.84mg/L at 20°C is totally consumed, about 2 mg/L of NH4-N is nitrified as is assumed from formula (2). For Kr20, the influence by the decline in NH4-N concentration is regarded practically negligible because an excessive amount of NH4-N (20 mg/L) was added. Therefore, the parameters ko, Ko relating to the DO concentration in formula (3) were determined for Kr20 as follows. When [DO]/Kr20 is plotted against [DO], a nearly straight line is obtained (Fig.8). As a regression straight line,tiiefollowing formula of Hofstee type was determined. [DO]/Kr20 = 0.000620[DO] + 0.0109
(4)
13th day 28th day 86th day Kr20 = 980 - [DO] Kr20 = ^^10 • [DO] Kr20 = 873 - [DO] 13.9 + [DO] 17.5 + [DO] 5.77 + [DO]
_ 0.02
a 60or
C 0.016h
^
I 0.0121
206th day 800th day Kr2n=765-[DO] K r 2 0 = ^ 8 1 l i 5 O ] 3.29 +[DO] 1.23 +I DO]
500
Formula (4)
\ 0.0081
o a 0.004
0 0 1 2 3 4 5 6 7 8 9
DO concentration (mg/L)
10
Fig.8 Relation between [DO] and [DO]/Kr20
2 3 4 5 6 7 8 9 DO concentration (mg/L) Fig.9 Relation between DO concentration and Kr20
287
Kr20 was expressed by the formula given below : Kr20 = 1610[DO]/(17.5+[DO])
(5)
Likewise, the relation between the DO concentration and respiration rate (Kr20) for the pellets on 13th day, 86th day, 206th day, and 800th day are given collectively in Fig.9. It was found that Ko became smaller with the lapse of days and the value closer to the maximum respiration rate was obtained at lower DO concentration. This is assumed due to the following reason. The nitrifiers immobiUzed almost evenly in the pellet grow wholly at first to enhance the nitrification activity rapidly. Then, growth of bacteria becomes significant at the parts near to the pellet surface through which DO is easily permeable with the result that higher activity can be obtained even at lower DO concentration. [2] Relation between NH4-N Concentration and Respiration Rate From Fig.7, respiration rate measured with the NH4-N concentration of 20mg/L (Kr20) shows almost the same value as that measured with the NH4-N concentration of 2mg/L. The similar results were obtained in the respiration rates measured on the other days. It was confirmed that the respiration rates (Kr20, N-Kr-20) measured by adding 20mg/L of NH4-N might be a good indicator for the potential maximum nitrification activity of immobiUzed nitrifiers. Relation between Number of Bacteria and Resptation Rate [1] Change in number of bacteria Some investigation were made on the change in number of bacteria in the pellets along with the continuous treatment of ammonia containing synthetic wastewater. The number of bacteria was measured for the nitrifiers, ammonia oxidizing bacteria and nitrite oxidizing bacteria, and other bacteria grown on standard medium (heterotrophs), respectively. The results obtained are shown in Fig. 10. At the commencement of continuous treatment, both nitrifiers existed in the number amounting to about lO^mL-pellet. The ammonia oxidizing bacteria increased sharply in number to about 10/ml^pellet down to the 10th day and then become constant in number. This tendency is well corresponding to the fact that the treated water NH4-N concentration shows a decline immediately after the commencement of continuous treatment as is clear from Fig.4. The growth rate was determined to be 0.70 1/d from the increase curve down to the 10th day. The nitrite oxidizing bacteria also increased in number to about lOVmL-pellet with a growth rate as high as that of ammonia oxidizing bacteria in a few days after the commencement of
288 10001 100 k 10 1
Fig. 10 Change in number of bacteria
10^ 10^ 10^ 10^ 10^ lOio Number of nitrifiers (cells/ml-pellet) Fig. 11 Relation between the number of nitrifiers in pellets and respiration rate
continuous treatment. On the other hand, the heterotrophs were stably kept at the number of about 107mL-pellet after the commencement of continuous treatment. This is well corresponding to the fact that the endogenous respiration rate (KrO) showed a stable value as is shown in Fig.6. Since the synthetic wastewater contains no organic constituents, it is assumed that these heterotrophs grow through metabolization of soluble organic constituents produced by decomposition of autotrophic nitrifying bacteria^l [2] Relation between number of nitrifying bacteria and respiration rate The number of nitrifiers in pellets is considered to have a direct influence on the nitrification activity. However, it takes a long period of time amounting to about two months to count the number of nitrifiers. An attempt was made to estimate the number of nitrifiers from the respiration rate. The relation between the number of nitrifiers (total of ammonia oxidizing bacteria and nitrite oxidizing bacteria) measured at each stage of continuous treatment and the maximum respiration rate (N-Kr20) due to nitrification is given in Fig.l 1. It was found that the logarithm of the number of bacteria was in a proportional relation to the logarithm of N-Kr. This result shows that the number of nitrifiers can be estimated from the maximum respiration rate of immobilized nitrifiers.
CONCLUSIONS The respiration rate was taken up as an indicator for the nitrification activity of immobilized nitrifiers. Using the pellets of newly immobilized activated sludge, the change in their nitrification performance and characteristics was examined during the period of acclimation of
289 nitrifiers in continuous treatment (retention time: three hours) of ammonia containing synthetic wastewater. As a result, the following findings were obtained. [1] Saturation constant of the pellets respiration rate as to DO concentration became smaller with the lapse of days, and the value closer to the maximum respiration rate was obtained at lower DO concentration. This result coincide with tiie fact tiiat the growth of nitrifiers is significant at the parts near to the surface through which DO is easily permeable. [2] From the respiration rate measured by adding 20mg/L of ammonia nitrogen to the wastewater (Kr20), the potential maximum activity of immobilized nitrifiers could be assumed. [3] Kr20 had a high correlation with the number of nitrifiers in the pellets. [4] It was considered possible to use the respiration rate as an indicator of nitrification activity of the pellets.
References 1) T.Takeshima, et.al. : "PEGASUS" An Innovative High-rate BOD and Nitrogen Removal Process for Municipal Wastewater: 66th Annual Conerence of WEF, Anahaim, California (1993) 2) H.Spanjars, et.al. : Respirametry as a Tool for Rapid Characterization of Wastewater and Activated Sludge : Wat.Sci.Tech., Vol.31, No.2, pp.105-114 (1995) 3) D.Dochain, et.al. : Structural Identifiability of Biokinetic Modelds of Activated Sludge Respkation: Wat.Sci.Tech., Vol.29, No.ll, pp.2571-2578 (1995) 4) L. Novak, et.al. : Estimation of Maximum Specific Growth Rate of Heterotrophic and Autotrophic Biomass: A Combined Technique of Mathematical Modelling and Batch Cultivations : Wat.Sci.Tech., Vol.30, No.ll, pp.171-180 (1994) 5) G.H.Kristensen, et.al. : Characterization of Functional Microorganism Groups and Substrate in Activated Sludge and Wastewater by AUR, NUR and OUR : Wat.Sci.Tech., Vol.25, No.6, pp.43-57 (1992) 6) B.E.Rittmann, et.ai. : Nirification as a Source of Soluble Organic Substrate in Biological Treatment: Wat.Sci.Tech., Vol.30, No.6, pp.1-8 (1994)