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Development of bioreactors for denitrification with immobilized cells
JOURNAL OPFERMENTATION ANDBIOENGINEERING Vol. 84, No. 2, 144-150. 1997
Development
of Bioreactors for Denitrification
MASATOSHI
MATSUMURA,‘*
Institute of Applied Biochemistry,
with Immobilized Cells
HIROSHI TSUBOTA,’ OSAFUMI ITO,’ PI-CHAO WANG,’ AND KIMIAKI YASUDAZ
University of Tsukuba, Tsukuba, Ibaraki 305’ and Biomaterial Co. Ltd., 1-21-I Junka, Fukui, Fukui 910,= Japan
Received 29 January 19971Accepted15 May 1997 A macro-porous cellulose carrier (AQUACELTM) was used for immobilization of denitrifying bacteria to develop a practical, high-performance nitrogen removal system. When the immobilized cells were used for denitriiication at a high nitrogen loading rate, flotation of carriers caused by the evolution of nitrogen gas occurred. For countering the problem of carrier flotation, new reactors in which hydrodynamic jet flow and centrifugal force are used. In these new reactors the floating carriers were distributed homogeneously and complete denitriiication was achieved even at high loading rate of 20 kg-N/m3-carrier/d. This AQUACEL system was used for efficient denitrification of wastewater discharged from an electroplating factory. [Key words: denitrification, porous carrier, immobilization, floating carrier, jet pump] rate in denitrification processes in which cells immobilized in alginate (5) or polyvinyl alcohol (8) are used. In our previous work (9), we used a positively charged macro-porous cellulose carrier for immobilization of nitrifying bacteria by an adsorption method. This carrier (AQUACELTM) promotes stable adhesion of bacteria onto the carrier walls, and allows transfer of nutrients into the carrier. This results in the formation of a high concentration of active bacteria necessary to achieve a high nitrification rate of 12 kg-N/m3-carrier/d. In this study, this macro-porous cellulose carrier was used for denitrification. During rapid denitrification, much nitrogen gas is produced from nitrate, and release of nitrogen gas from the carrier can be a serious problem, as any nitrogen gas retained in the carrier tends to buoy the carrier with a corresponding loss of denitrification efficiency (10). We developed reactors in which the floating carriers are distributed in the reactor homogeneously without excessive mechanical stress, which could otherwise lead to reduced carrier life expectancies. The reactors were tested with synthetic wastewater and actual wastewater discharged from an electroplating factory for determination of their operational efficiencies.
Nitrogenous compounds are major pollutants of water and occur in domestic waste, in agricultural waste and aqueous waste from key industries such as fertilizer production, paper manufacturing, metal finishing and food processing. Nitrate contamination of groundwater supplies is a growing problem throughout the world. Nitrates are proven precursors to methemoglobinemia (blue-baby syndrome). This syndrome is caused by bacterial reduction of nitrates to nitrites in the infant’s upper gastrointestinal tract (1). Once absorbed into the circulatory system, nitrites then convert the blood pigment hemoglobin to methemoglobin which is incapable of transporting oxygen from the lungs to the body’s tissues. Since the recognition of the significance of nitrogenous compounds as pollutans, intensive efforts have been devoted to the development of processes for nitrogen control and the modification of conventional waste treatment processes so as to effect nitrogen removal. The biological process which consists of successive nitrification and denitrification is one of the most economical for the removal of nitrogen in the form of ammonia, nitrite and nitrate from aqueous waste. In this biological nitrogen removal system, the nitrification generally is the ratelimiting step because of the extremely low growth rate of obligate autotrophic nitrifying bacteria. Use of immobilized cells in nitrification overcomes this disadvantage and shortens greatly the hydraulic retention time in continuous nitrification (2, 3). In response to the need for the development of a more compact nitrogen removal system for wastewater and of an efficient system for purification of high nitrate groundwater, immobilized cells have also been used for denitrification. These immobilized cells were prepared by entrapment using alginate (4, 5), a polyelectrolyte complex (6), photocrosslinkable resins (7) and polyvinyl alcohol (8). This entrapment method requires use of specialized equipment and sufficient time to prepare an adequate number of gel beads for large-scale wastewater treatment. Traditionally, mass transfer behavior in the gel matrix has often been recognized as a rate-limiting factor in an immobilized cell system. Diffusional restriction causes a remarkable drop in the nitrate reduction
MATERIALS
AND METHODS
Macro-porous cellulose carrier The AQUACELTM carrier is produced from cellulose with a polyethyleneimine (PEI) coating to form a stable cross-linked structure. The result is a large, effective surface area of 3-7 m2/g (as measured by the BET absorption isotherm method), a porosity of 97%, an ion exchange capacity (IEC) of 0.8-1.3 meq/g and a density of about 1.05 kg/dm3 in water. The particle and mean pore sizes of the AQUACELTM carrier in cubic form are variable in the ranges of l-5 mm and lOO-1,260 pm, respectively. A scanning electron micrograph of the exterior of an AQUACELTM is shown in Fig. 1. Reactors used for continuous denitrification Four different reactors were used for continuous denitrification. They were a gas-lift reactor, an expanded bed reactor with downflow, a column reactor with a jet pump and a stirred tank reactor with a downcomer. The performance of these reactors was evaluated mainly from the
* Corresponding author. 144
BIOREACTORS FOR DENITRIFICATION
145
‘-_=
9 _
u
q532mm di9omn
FIG. 1.
SEM image of macro-porous cellulose carrier AQUACELr”.
viewpoint of the extent of homogeneous distribution of the carrier which strongly influenced the rate of performance for nitrate removal. Since gas-lift reactors are commonly used for biological processes in which the cells are immobilized in a gel matrix which is highly susceptible to damage by mechanical stress (1 l), such a reactor was also used in our first experiment. The working volume of this reactor was 1.3 dm3, and the head space gas was circulated at a volumetric flow rate of 1 dm3/ min for distribution of carrier particles. Flotation of lnorganicmediuml
IOrganicrnediurn
1.
5ffluent
@I+----J/ Water recirculation
FIG. 2. downflow.
’
Schematic configuration
L I of expanded bed reactor with
i
FIG. 3. Schematic configuration of column reactor with jet pump. 1. Ejector; 2. liquid of circulation pump; 3. water jacket; 4. wire screen; 5. inlet of carbon source; 6. inlet inorganic component; 7. DO sensor; 8. pH sensor; 9. overflow tube; 10. drain tube.
gel beads entrapping active cells is caused by vigorous evolution of gas and its accumulation inside the gel matrix. The homogeneous distribution of these floating gel beads could be realized by balance of the drag force caused by downward liquid flow with the buoyancy force of the gel beads in the reactor shown in Fig. 2 (10). Figure 3 shows a novel column bioreactor for denitrification with immobilized cells and a macroporous cellulose carrier. The column reactor with a jet pump has the ability to draw the carrier particles which float to the top of the reactor downward to be reintroduced tangentially at the bottom of the reactor. The downflow in a pipe set at the center of the reactor is induced by a jet pump with an inlet on the clear water side of the reactor so that the carrier particles do not pass through the circulation pump. The top of the downflow tube is funnel-shaped, and the diameter of the column reactor is smaller around the top of the downflow tube. This structure is useful for enhancing the suction of the floating carrier particles into the downflow tube. The carrier particles pass through the jet pump as shown in Fig. 4. Since the diameter of the suction tube is much larger than that of the carrier particles, they could pass through the jet pump without sustaining mechanical damage, but a moderate shear stress was exerted on the carrier particle surfaces in the mixing part where the driving liquid and the entrained liquid met. The tangential flow causes a turbulence in the liquid in the reactor, which promotes homogeneous distribution of the carrier particles. This column reactor with a working volume of 12.5 dm3 was mainly used for basic research to determine the optimum operating conditions
146
.I. FERMENT.BIOENG.,
MATSUMURA ET AL. TABLE 1. Qs
Suction
Qs+Qj ed liquid
liquid
Qi drive liquid
FIG. 4. Schematic configuration of ejector and carrier movement inside the ejector.
for denitrification. The other reactor, with a working volume of 500 dm3, used for denitrification of actual wastewater discharged from an electroplating factory was a stirred tank reactor with a downcomer. A tubular centrifugal force generator shaped like an elbow tube was installed at the end of the downcomer (Fig. 5). The centrifugal force generator was enclosed in a hollow cylindrical casing to relieve fluid resistance, which results in reducing the mechanical damage to carriers and the power requirement for rotation. The hollow cylindrical casing has the same radius as the maximum radius of rotation of the centrifugal force generator. It also has an opening in its circumferential wall, which communicates with the outlet of the centrifugal force generator. The floating carrier particles in this reactor flow into the downcomer together with the downward liquid flow induced by the centrifugal force,
E
______
3
=I+-1
---
b 4 J
rr --
2
P $!
E
8
2
II
KNOP Na2HP0, KH2P04 MgSOd-7HsO CaC12.2H20 FeS04-7Hz0 MnS09-HZ0 Na2Mo0,,Hz0 Methanol Tap water
Composition of synthetic wastewater 2.02 or 4.64 g 5.31 g 1.36g 0.1 g 1.99 mg l.Omg 0.35 mg 0.5 mg 0.80or 1.6Og II
and a moderate shear stress is exerted on the carrier particle surfaces at the outlet of the downcomer. Experimental procedures for continuous denitrification The start-up procedure was simple. The denitrifying seed inoculum was prepared from activated sludge from an existing wastewater treatment plant. A desired amount of dried porous cellulose carrier was soaked in the sludge suspension for 3 h inside the reactors. During this time the carrier particles swelled, and the bacterial cells were trapped in the particles. The sludge suspension was replaced with fresh synthesized medium containing nitrate ion, and then batch cultivation was carried out for proliferation of denitrifying bacteria. When the residual concentration of nitrate became lower than 5 mol/m3, the continuous denitrification experiments were initiated by pumping of synthetic feed medium or actual wastewater containing nitrate ion into the respective reactors. The nitrogen loading rate was gradually increased by increase of the nitrogen feed concentration or shortening of the hydraulic retention time (HRT). The basic composition of the synthetic wastewater feed medium for denitrification is shown in Table 1. Methanol was used as an electron donor for denitrification. Its concentration was fixed at 1.5 times the theoretical requirement calculated from the stoichiometric relationship between reduction of nitrate and the hydrolysis of methanol (0.83 mol-methanol/l .O mol-nitrate). For avoiding contamination of the feed medium inside the storage tank, the carbon source and the salt solution were separately supplied. These solutions were fed into the reactor at the same flow rates. Analytical procedures Water analyses were performed according to the methods described in the Japanese Industrial Standards (JIS) K0102. Nitrate and nitrite concentrations were determined calorimetrically using the brucine and the sulfanilamide-naphthylethylenediamine methods, respectively. Metals were identified using a Jarrel ash inductively coupled argon plasma analyzer (Model 975, USA). Dissolved organic carbon was determined by Total Organic Carbon (TOC) Auto Analyzer (Shimadzu SOOOA) after filtration with a nitrocellulose membrane of 0.2 pm pore size. RESULTS AND DISCUSSION
FIG. 5. Schematic configuration of stirred tank reactor with downcomer. 1. Influent tube; 2. wire screen; 3. motor; 4. overflow tube; 5. hollow cylindrical casing; 6. drain tube; 7. downcomer; 8. suction port of carriers; 9. exhaust port of carriers.
Continuous denitrification with gas-lift reactor and expanded bed reactor with downflow A 220-d continuous denitrification experiment with the AQUACELTM carrier (particle size 3 mm cubic, pore size 300 pm, IEC 1.3 meq/g) was conducted using the gas-lift reactor and the expanded bed reactor with downflow. The purpose of this experiment was to investigate the flotation of the porous carriers and its effect on the performance of denitrification. Figure 6 shows the time courses of the
BIOREACTORS FOR DENITRIFICATION
VOL. 84, 1997
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0
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50
60
[dl
FIG. 6. Time courses of nitrate and nitrite concentrations during continuous denitrification in the gas-lift reactor and expanded bed reactor with downflow. Reactor A: gas-lift reactor; Reactor B: expanded bed reactor with downflow; #: concentration of trace metal elements was Symbols: n , nitrate; a, nitrite. reduced to half of the original concentration.
N03-N and NOI-N concentrations in the early phase of continuous denitrification. The experiment was started in the gas-lift reactor with a carrier volume fraction of 10%. After batch cultivation for 5 d the continuous denitrification was initiated by pumping of the synthesized feed medium containing 20 mol-N03/m3 at an HRT of 20.8 h. Five days after the start of continuous feeding the inlet nitrate ions at 20 mol/m3 had been almost completely converted to nitrogen gas, and then the nitrate loading rate was increased to 5.6 kg-N/m3carrier/d by reduction the HRT to 12 h. The increase in nitrate loading rate caused carrier flotation, and homogeneous distribution of carrier particles could not be achieved even at higher gas circulation rates. Nitrate removal also became very unstable. Thirty-two d after the start of the experiment the carrier particles inside the gas-lift reactor were transferred into the expanded bed reactor with a working volume of 0.95 dm3. The circulation flow rate in this reactor was varied in a range of 4.0 to 7.5 dm3/min. In spite of a higher loading rate of 12.3 kg-N/m3-carrier/d, the distribution of carrier particles was markedly enhanced, and more than 98% nitrogen removal was achieved. After the maximum denitrification rate had been achieved, the concentration of trace metal elements in the influent was varied for investigation of its relationship to the denitrification rate. As shown by the data during the experiment without trace metal elements, the performance in denitrification dropped. For confirmation of this obvious effect, the trace metal element concentration was first returned to the original level and then reduced to half. The residual nitrate concentration change corresponded to the change in concentration of the trace metal elements. The results obtained in this experiment show the importance of homogeneous distribution of the
carrier particles and the presence of trace metal elements such as iron, manganese and molybdenum in order to achieve efficient denitrification. The denitrification was continued for 220 d for investigation of the stability of this reaction system. In the late phase of denitrification the carrier particles were gradually enlarged by the excess growth of cells on the carrier particle surfaces, which caused an increase in carrier particle density and instability of the system. Since the homogeneous distribution of carrier particles in this expanded bed reactor is based on the delicate balance between the buoyancy force and drag force, this reactor was sensitive to changes in the physical properties of the carrier particle and the liquid. Design of column reactor with jet pump and its performance in denitrification To establish a compact and
highly efficient system for denitrification using immobilized cells, it is necessary to develop new reactors which have the following characteristics: the ability to distribute the floating carrier particles at a high nitrate loading rate and a high carrier packing density; and a structure that allows generation of moderate shear stress on the carrier particle surface to detach the excess cells and to degas the carriers. The jet pump is a device for pumping fluids by means of a high velocity jet of the same or another kind of fluid. The principal phenomenon involved is the transfer of momentum from the high velocity fluid to the lower velocity fluid during the mixing process, with the result that all fluid leaving the mixing tube has about the same velocity. This pump is used to overcome to many pumping problems due to its low cost, simplicity in operation and ability to mix thoroughly the two fluids. In this work the jet pump was used as a distributor of floating carriers. The analysis and design of pump units to use the same liquid (self-entrainment)
148
J. FERMENT.BIOENG.,
MATSUMURA ET AL. 30
of low viscosity have been placed on a firm basis. The energy efficiency of jet pumps, p, is expressed by Eq. 1, as the ratio of the rate of mechanical energy addition to the suction liquid to the rate of net loss of mechanical energy by the drive liquid:
(1) where QS and Qj are the volumetric flow rates of the suction liquid and the drive liquid, respectively, hi, h2 and hd are the heads at the pump connection points for the drive line, suction line and discharge line, respectively, A4 is the capacity ratio given by [Qs/Qj], and H is the head ratio given by [(hd-&)/(hl -&)I. The performance characteristics of jet pumps depend strongly on the area ratio R of the cross-sectional area of the jet nozzle to the cross-sectional area of the mixing tube. The jet pump for our reactor should have a low discharge pressure but high suction flow rate QS. For such a jet pump, a low area ratio of about 0.12 was recommended to achieve a capacity ratio of M=2.2 and a head ratio of H=O.l at a pump efficiency of t7=0.22 (12). As shown in Fig. 4, the drive liquid was supplied into the mixing tube through the annulus of the jet pump, while the suction fluid was introduced through the central tube together with the carriers. This jet pump structure was effective in avoiding mechanical damage to the carriers. The diameter of the downflow tube set at the center of the reactor with a working volume of 12.5 dm3 was 32mm, and the tank diameter was 190mm. Considering the rising velocity of a single bubble, the linear velocity of liquid inside the downflow tube would be around 20 cm/s. This linear velocity corresponds to a suction liquid volumetric flow rate QS of 9.7 dm3/min; this value was calculated from the drive liquid volumetric flow rate Qj of 4.4 dm3/min and M=2.2. Under these operating conditions, homogeneous distribution of the carrier particle was possible for a carrier volume fraction of up to 30%. For the column reactor with the jet pump, the maximum nitrate loading rates which allowed complete denitrification were measured during a continuous experiment with the AQUACELTM carrier (particle size 3 mm cubic, pore size 500 pm, IEC 1.3 meq/g) at 30°C. The volume fraction of the carrier was increased to 20%, and the nitrate loading rate was gradually increased up to 28 kg-N/m3-carrier/d by reduction of the HRT from 12 to 3 h and increase of the N03-N influent concentration from 20 to 1,050 g/m3. In this reactor the distribution of the floating carriers was markedly enhanced, and a suitable shear stress to prevent excess cell growth was exerted on the carrier particle surfaces in the mixing tube of the jet pump. The stable denitrification lasted for more than five months, and complete denitrification was achieved even at a high nitrate loading rate of 15 kgN/m3-carrier/d (Fig. 7). Denitrikation of actual wastewater discharged from an electroplating factory The stirred tank reactor with a downcomer shown in Fig. 5 is practical for largescale treatment because of its simple structure. The rotation speed required for homogeneous distribution of floating carriers can be estimated from the equations below: When the hollow cylindrical casing with a radius of R is rotating at an angular velocity of w, liquid pressure PC is generated at the outlet of the elbow tube:
I 0
5
10
15
20
25
30
Nitrate loading rate [kg-Nlmbzarrierid]
FIG. 7. Relationship between nitrate loading rate and nitrate reduction rate measured in the column reactor with a jet pump.
P, = ,oo2R2/2
(2)
This pressure induces liquid flow inside the downcomer against the gravity head at the outlet of the downcomer. The linear velocity of liquid inside the downcomer U is calculated using Bernoulli’s equation as U= (w2R2- 2Zg)“*
(3)
where 2 is the liquid depth at the center of tube, and g is the acceleration due to gravity. ing the rising velocity of a single bubble, the rotation speed may be estimated from Eq. 3. requirement, L,, may also be estimated, as
the elbow Considermaximum The power
L, = Gw2R2/2
(4)
where G is the mass flow rate. The pilot-scale reactor with a working volume of 500dm3 was used for denitrification of actual wastewater discharged from an electroplating factory. Based on Eq. 3 and the dimensions of the reactor shown in Fig. 5, the rotation speed necessary to produce a linear velocity of 20cm/s inside the downcomer was estimated to be 300rpm. Since the buoyancy force of the carrier particles is lower than that of the gas bubbles, this rotation speed could be considered to be the maximum rotation speed. Under these operating conditions homogeneous TABLE 2.
Metal Mg Fe MO Mn Na K cu co Zn Ni Pb Al Cd Cr
Concentrations of metals in electroplating wastewater and synthetic wastewater Electro;t;yOg
_ , 3.2-4.3 5.5-5.9 0.36-0.39 0.44-0.56 3200 1830 0.2-0.51 so.41 0.24-0.27 0.11-0.16 SO.076 so.031 SO.0028 0.26-0.43
waste
Synthetic waste (ma/0 . - , 0.81 0.20 0.08 0.20 690 420 -
BIOREACTORS FOR DENITRIFICATION
VOL. 84. 1997
0
.E .Z s
600
z m 0 S!
4oo
.% =
0
20
40
60
60
100
0
20
40
60
60
100
149
120
200 0
Time
120
[d]
FIG. 8. Time courses of nitrate and nitrite concentration during continuous denitrification of electroplating wastewater in the stirred tank reactor with a downcomer. Symbols: q , influent Nor-N; n , effluent Nor-N; 0, influent NO*-N; 0, effluent NO*-N; A, temperature; %, trouble in methanol feeding.
annual concentrations of N03-N, N02-N and BOD after these treatments are 460, 18 and 30g/m3, respectively. The wastewater upper limit in this region is 20g/m3 for total nitrogen; hence the existing anaerobic biofilm process is inadequate for meeting this limit. The concentrations of metals and phosphate in the wastewater were measured. As shown in Table 2, the concentrations of important heavy metals for denitrification such as Fe, MO, and Mn were higher than those in the synthesized wastewater. The total phosphate concentration in the wastewater, however, was only 4.2g/m3,
distribution of the carrier particles was possible for a carrier volume fraction of up to 40%. This factory discharges wastewater containing cyanide, nitrite and nitrate at high concentrations with a displacement of 50 m3/d, and has a conventional wastewater treatment system. The cyanide is converted to ammonia by thermal hydrolysis followed by stripping. The other wastewater containing nitrate and nitrite is discharged into a storage tank together with domestic wastewater. This mixed wastewater is treated by an activated-sludge process followed by an anaerobic biofilm process. The average
HRT[h]
20’10/
8
I
0
20
3
15:
I
I
40
; I
2
60 Time
80
100
120 --
Id]
FIG. 9. Time courses of nitrogen removal rate and ratio during continuous denitrification of electroplating wastewater in stirred tank reactor with a downcomer. Symbols: 0, nitrogen removal ratio; 0, nitrogen removal rate; %, trouble in methanol feeding.
150
J. FERMENT.BIOENG.,
MATSUMURA ET AL.
and phosphate supplementation was necessary. The concentration of methanol as an electron donor was fixed at 1.5 times the theoretical requirement, and the weight ratio of phosphate to nitrogen (P/N) was fixed at 0.04 by addition of KH2P04. The seed sludge in this experiment was obtained from the activated sludge tank in this factory, and was immobilized in the cellulose carriers (particle size 5 mm cubic, pore size 1,000 pm, IEC 1.3 meq/g). The volume fraction of the carrier particles to the reactor working volume was 20%, and the carriers were homogeneously distributed at a rotation speed of 98 rpm during the initial stage of continuous denitrification. Figures 8 and 9 show the time courses of N03-N and N02-N concentrations and the nitrogen removal rate and ratio during the 120d continuous denitrification, respectively. Continuous denitrification was initiated at an HRT of 20 h after four batch treatments for proliferation of denitrifiers. Tend after the start of continuous feeding, the inlet N03-N of 650g/m3 and NOz-N of 80 g/m3 had been almost completely converted to nitrogen gas at an HRT of 10 h, and then the HRT was shortened to 2 h. In spite of the marked fluctuation of the influent concentrations of N03-N and NOz-N, the system was able to keep the effluent N03-N concentration lower than 5 g/m3 and completely remove N02-N. Trouble in the methanol feeding occurred at 30 and 80 h after the start of the experiment, which caused rapid increases in the N03-N concentration. The system, however, was quickly restored to its original state after repair of the feed pump. This means that the immobilized system is tolerant to troubles in operation. When the HRT was shortened to 3 h, the flotation of the carrier particles was obvious and the rotation speed was increased to 150rpm. As seen from Fig. 9, the nitrogen removal rate reached was 20.4 kg-N/m3-carrier/d. This excellent performance might be due to homogeneous distribution of carriers and moderate shear stress on the surfaces of the carriers. The moderate shear stress was applied to the carrier particle surfaces at the outlet of the elbow tube, which would be effective for preventing excess accumulation of cells on the carrier particle surfaces and for enhancement of transport of nutrients into the carriers. As shown in Fig. 8, the fluctuation of the concentrations of N03-N and N02-N in the influent was so marked that the methanol concentration could not be maintained at a given level. In this experiment oversupply of methanol occurred, which induced excess accumulation of cells and residual BOD. An on-line monitoring method for NH4-N, N03-N and N02-N concentrations should be developed, and an automatic feed control system for the electron donor should be developed to complete this AQUACELTM system. In conclusion, cells immobilized in macro-porous cellulose carriers were used for denitrification. Based on the results of long-term continuous denitrification experiments with different types of reactors, the following conclusions were reached. (i) Even when macro-porous carriers were employed, flotation of the carrier particles was caused by vigorous evolution of nitrogen gas at high nitrogen loading rates. (ii) Homogeneous distribution of floating carrier particles was essential to achieve highly efficient denitrification. (iii) New reactors in which hydrodynamic jet flow and centrifugal force were used were effective for homogeneous distribution of carriers, and have the ability to expose the surface of carriers to moderate shear stress.
NOMENCLATURE : acceleration due to gravity, m/s* : mass flow rate, kg/s : head at pump connection point for the drive line, m : head at pump connection point for suction line, m : head at pump connection point for discharge line, m : head ratio defined by [(ha -h2)/(h1 -hd)], L, :power requirement, W M : capacity ratio defined by Qs/Qj, PC :liquid pressure generated by rotation, Pa QS : volumetric flow rate of suction liquid, m3/s Qj : volumetric flow rate of drive liquid, m3/s R :radius of hollow cylindrical casing, m U : linear velocity of liquid inside the downcomer, m/s Z :liquid depth at the center of the elbow tube, m ‘j; : energy efficiency of jet pump, p : density, kg/m3 w : angular velocity, rad/s g G hl hz hd H
ACKNOWLEDGMENT The authors wish to acknowledge the Tsukuba Advanced Research Alliance (TARA) for partly financing this work. REFERENCES 1. Sbuval, IL I. and Gruaer, N.: Infant methemoglobinemia and other health effects of nitrate in drinking water. Prog. Water Technol., 8, 183-188 (1977). of 2. Tramper, J. and de Man, A. W. A.: Characterization Nitrobucter agilis immobilized in calcium alginate. Enzyme Microb. Technol., 8, 472-476 (1986). 3. SomIno. T.. Nakamura. H.. Mori. N.. Kawaauchi. Y.. and Tada, M.: ‘Immobilization of nitrifylng bact&ia in porous pellets of urethane gel for removal of ammonium nitrogen from wastewater. Appl. Microbial. Biotechnol., 36, 556560 (1992). 4. Nilsson, I., Ohlson, S., Haggstrom, L., Moiin, N., and Mosbach, K.: Denitrification of water using immobilized Pseudomonas denitrificans cells. Eur. J. Appl. Microbial. Biotechnol., 10, 261-274 (1980). 5. Nilsson, I. and Ohlson, S.: Columnar denitrification of water by immobilized Pseudomonas denitrtficans cells. Eur. J. Appl. Microbial. Biotechnol., 14, 8690 (1982). 6. Kokufnta, E., Shimohashi, M., and Nakamura, I.: Immobilization of Paracoccus denitrificans cells with polyelectrolyte comolex and denitrifving activitv of the immobilized cells. J. Ferment. Technol., 64,533-538-(1986). 7. Taya, M., Miora, H., Ucbiyama, IL., Iijima, S., and Kobayashi, T.: Denitrification with immobilized Paracoccus denitr@kns and preservation characteristics of immobilized cells. Hakkokogaku, 66, 235-240 (1988). 8. Lin, Y. F. and Chen, K. C.: Denitrification and methanogenesis in a co-immobilized mixed culture system. Wat. Res., 29, 3543 (1995). 9. Matsnmura, M., Yamamoto, T., Shinabe, K., and Yasuda, K.: Rapid nitrification with immobilized cells using macro-porous cellulose carrier. Wat. Res., 31, 1027-1034 (1997). _ 10. Matsumura, M., Takehara, S., and Kataoka, II.: Continuous butanoVisopropano1 fermentation in down-flow column reactor coupled with pervaporation using supported liquid membrane. Biotechnol. Bioeng., 39, 148-156 (1992). 11. Sumino, T., Nakamura, H., Mori, N., and Kawaguchi, Y.: Immobilization of nitrifylng bacteria by polyethylene glycol prepolymer. J. Ferment. Bioeng., 73, 37-42 (1992). 12. Folsom, R. G.: Jet pumps with liquid drive. Chem. Eng. Prog., 44, 765-770 (1948).
Development
of Bioreactors for Denitrification
MASATOSHI
MATSUMURA,‘*
Institute of Applied Biochemistry,
with Immobilized Cells
HIROSHI TSUBOTA,’ OSAFUMI ITO,’ PI-CHAO WANG,’ AND KIMIAKI YASUDAZ
University of Tsukuba, Tsukuba, Ibaraki 305’ and Biomaterial Co. Ltd., 1-21-I Junka, Fukui, Fukui 910,= Japan
Received 29 January 19971Accepted15 May 1997 A macro-porous cellulose carrier (AQUACELTM) was used for immobilization of denitrifying bacteria to develop a practical, high-performance nitrogen removal system. When the immobilized cells were used for denitriiication at a high nitrogen loading rate, flotation of carriers caused by the evolution of nitrogen gas occurred. For countering the problem of carrier flotation, new reactors in which hydrodynamic jet flow and centrifugal force are used. In these new reactors the floating carriers were distributed homogeneously and complete denitriiication was achieved even at high loading rate of 20 kg-N/m3-carrier/d. This AQUACEL system was used for efficient denitrification of wastewater discharged from an electroplating factory. [Key words: denitrification, porous carrier, immobilization, floating carrier, jet pump] rate in denitrification processes in which cells immobilized in alginate (5) or polyvinyl alcohol (8) are used. In our previous work (9), we used a positively charged macro-porous cellulose carrier for immobilization of nitrifying bacteria by an adsorption method. This carrier (AQUACELTM) promotes stable adhesion of bacteria onto the carrier walls, and allows transfer of nutrients into the carrier. This results in the formation of a high concentration of active bacteria necessary to achieve a high nitrification rate of 12 kg-N/m3-carrier/d. In this study, this macro-porous cellulose carrier was used for denitrification. During rapid denitrification, much nitrogen gas is produced from nitrate, and release of nitrogen gas from the carrier can be a serious problem, as any nitrogen gas retained in the carrier tends to buoy the carrier with a corresponding loss of denitrification efficiency (10). We developed reactors in which the floating carriers are distributed in the reactor homogeneously without excessive mechanical stress, which could otherwise lead to reduced carrier life expectancies. The reactors were tested with synthetic wastewater and actual wastewater discharged from an electroplating factory for determination of their operational efficiencies.
Nitrogenous compounds are major pollutants of water and occur in domestic waste, in agricultural waste and aqueous waste from key industries such as fertilizer production, paper manufacturing, metal finishing and food processing. Nitrate contamination of groundwater supplies is a growing problem throughout the world. Nitrates are proven precursors to methemoglobinemia (blue-baby syndrome). This syndrome is caused by bacterial reduction of nitrates to nitrites in the infant’s upper gastrointestinal tract (1). Once absorbed into the circulatory system, nitrites then convert the blood pigment hemoglobin to methemoglobin which is incapable of transporting oxygen from the lungs to the body’s tissues. Since the recognition of the significance of nitrogenous compounds as pollutans, intensive efforts have been devoted to the development of processes for nitrogen control and the modification of conventional waste treatment processes so as to effect nitrogen removal. The biological process which consists of successive nitrification and denitrification is one of the most economical for the removal of nitrogen in the form of ammonia, nitrite and nitrate from aqueous waste. In this biological nitrogen removal system, the nitrification generally is the ratelimiting step because of the extremely low growth rate of obligate autotrophic nitrifying bacteria. Use of immobilized cells in nitrification overcomes this disadvantage and shortens greatly the hydraulic retention time in continuous nitrification (2, 3). In response to the need for the development of a more compact nitrogen removal system for wastewater and of an efficient system for purification of high nitrate groundwater, immobilized cells have also been used for denitrification. These immobilized cells were prepared by entrapment using alginate (4, 5), a polyelectrolyte complex (6), photocrosslinkable resins (7) and polyvinyl alcohol (8). This entrapment method requires use of specialized equipment and sufficient time to prepare an adequate number of gel beads for large-scale wastewater treatment. Traditionally, mass transfer behavior in the gel matrix has often been recognized as a rate-limiting factor in an immobilized cell system. Diffusional restriction causes a remarkable drop in the nitrate reduction
MATERIALS
AND METHODS
Macro-porous cellulose carrier The AQUACELTM carrier is produced from cellulose with a polyethyleneimine (PEI) coating to form a stable cross-linked structure. The result is a large, effective surface area of 3-7 m2/g (as measured by the BET absorption isotherm method), a porosity of 97%, an ion exchange capacity (IEC) of 0.8-1.3 meq/g and a density of about 1.05 kg/dm3 in water. The particle and mean pore sizes of the AQUACELTM carrier in cubic form are variable in the ranges of l-5 mm and lOO-1,260 pm, respectively. A scanning electron micrograph of the exterior of an AQUACELTM is shown in Fig. 1. Reactors used for continuous denitrification Four different reactors were used for continuous denitrification. They were a gas-lift reactor, an expanded bed reactor with downflow, a column reactor with a jet pump and a stirred tank reactor with a downcomer. The performance of these reactors was evaluated mainly from the
* Corresponding author. 144
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u
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FIG. 1.
SEM image of macro-porous cellulose carrier AQUACELr”.
viewpoint of the extent of homogeneous distribution of the carrier which strongly influenced the rate of performance for nitrate removal. Since gas-lift reactors are commonly used for biological processes in which the cells are immobilized in a gel matrix which is highly susceptible to damage by mechanical stress (1 l), such a reactor was also used in our first experiment. The working volume of this reactor was 1.3 dm3, and the head space gas was circulated at a volumetric flow rate of 1 dm3/ min for distribution of carrier particles. Flotation of lnorganicmediuml
IOrganicrnediurn
1.
5ffluent
@I+----J/ Water recirculation
FIG. 2. downflow.
’
Schematic configuration
L I of expanded bed reactor with
i
FIG. 3. Schematic configuration of column reactor with jet pump. 1. Ejector; 2. liquid of circulation pump; 3. water jacket; 4. wire screen; 5. inlet of carbon source; 6. inlet inorganic component; 7. DO sensor; 8. pH sensor; 9. overflow tube; 10. drain tube.
gel beads entrapping active cells is caused by vigorous evolution of gas and its accumulation inside the gel matrix. The homogeneous distribution of these floating gel beads could be realized by balance of the drag force caused by downward liquid flow with the buoyancy force of the gel beads in the reactor shown in Fig. 2 (10). Figure 3 shows a novel column bioreactor for denitrification with immobilized cells and a macroporous cellulose carrier. The column reactor with a jet pump has the ability to draw the carrier particles which float to the top of the reactor downward to be reintroduced tangentially at the bottom of the reactor. The downflow in a pipe set at the center of the reactor is induced by a jet pump with an inlet on the clear water side of the reactor so that the carrier particles do not pass through the circulation pump. The top of the downflow tube is funnel-shaped, and the diameter of the column reactor is smaller around the top of the downflow tube. This structure is useful for enhancing the suction of the floating carrier particles into the downflow tube. The carrier particles pass through the jet pump as shown in Fig. 4. Since the diameter of the suction tube is much larger than that of the carrier particles, they could pass through the jet pump without sustaining mechanical damage, but a moderate shear stress was exerted on the carrier particle surfaces in the mixing part where the driving liquid and the entrained liquid met. The tangential flow causes a turbulence in the liquid in the reactor, which promotes homogeneous distribution of the carrier particles. This column reactor with a working volume of 12.5 dm3 was mainly used for basic research to determine the optimum operating conditions
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.I. FERMENT.BIOENG.,
MATSUMURA ET AL. TABLE 1. Qs
Suction
Qs+Qj ed liquid
liquid
Qi drive liquid
FIG. 4. Schematic configuration of ejector and carrier movement inside the ejector.
for denitrification. The other reactor, with a working volume of 500 dm3, used for denitrification of actual wastewater discharged from an electroplating factory was a stirred tank reactor with a downcomer. A tubular centrifugal force generator shaped like an elbow tube was installed at the end of the downcomer (Fig. 5). The centrifugal force generator was enclosed in a hollow cylindrical casing to relieve fluid resistance, which results in reducing the mechanical damage to carriers and the power requirement for rotation. The hollow cylindrical casing has the same radius as the maximum radius of rotation of the centrifugal force generator. It also has an opening in its circumferential wall, which communicates with the outlet of the centrifugal force generator. The floating carrier particles in this reactor flow into the downcomer together with the downward liquid flow induced by the centrifugal force,
E
______
3
=I+-1
---
b 4 J
rr --
2
P $!
E
8
2
II
KNOP Na2HP0, KH2P04 MgSOd-7HsO CaC12.2H20 FeS04-7Hz0 MnS09-HZ0 Na2Mo0,,Hz0 Methanol Tap water
Composition of synthetic wastewater 2.02 or 4.64 g 5.31 g 1.36g 0.1 g 1.99 mg l.Omg 0.35 mg 0.5 mg 0.80or 1.6Og II
and a moderate shear stress is exerted on the carrier particle surfaces at the outlet of the downcomer. Experimental procedures for continuous denitrification The start-up procedure was simple. The denitrifying seed inoculum was prepared from activated sludge from an existing wastewater treatment plant. A desired amount of dried porous cellulose carrier was soaked in the sludge suspension for 3 h inside the reactors. During this time the carrier particles swelled, and the bacterial cells were trapped in the particles. The sludge suspension was replaced with fresh synthesized medium containing nitrate ion, and then batch cultivation was carried out for proliferation of denitrifying bacteria. When the residual concentration of nitrate became lower than 5 mol/m3, the continuous denitrification experiments were initiated by pumping of synthetic feed medium or actual wastewater containing nitrate ion into the respective reactors. The nitrogen loading rate was gradually increased by increase of the nitrogen feed concentration or shortening of the hydraulic retention time (HRT). The basic composition of the synthetic wastewater feed medium for denitrification is shown in Table 1. Methanol was used as an electron donor for denitrification. Its concentration was fixed at 1.5 times the theoretical requirement calculated from the stoichiometric relationship between reduction of nitrate and the hydrolysis of methanol (0.83 mol-methanol/l .O mol-nitrate). For avoiding contamination of the feed medium inside the storage tank, the carbon source and the salt solution were separately supplied. These solutions were fed into the reactor at the same flow rates. Analytical procedures Water analyses were performed according to the methods described in the Japanese Industrial Standards (JIS) K0102. Nitrate and nitrite concentrations were determined calorimetrically using the brucine and the sulfanilamide-naphthylethylenediamine methods, respectively. Metals were identified using a Jarrel ash inductively coupled argon plasma analyzer (Model 975, USA). Dissolved organic carbon was determined by Total Organic Carbon (TOC) Auto Analyzer (Shimadzu SOOOA) after filtration with a nitrocellulose membrane of 0.2 pm pore size. RESULTS AND DISCUSSION
FIG. 5. Schematic configuration of stirred tank reactor with downcomer. 1. Influent tube; 2. wire screen; 3. motor; 4. overflow tube; 5. hollow cylindrical casing; 6. drain tube; 7. downcomer; 8. suction port of carriers; 9. exhaust port of carriers.
Continuous denitrification with gas-lift reactor and expanded bed reactor with downflow A 220-d continuous denitrification experiment with the AQUACELTM carrier (particle size 3 mm cubic, pore size 300 pm, IEC 1.3 meq/g) was conducted using the gas-lift reactor and the expanded bed reactor with downflow. The purpose of this experiment was to investigate the flotation of the porous carriers and its effect on the performance of denitrification. Figure 6 shows the time courses of the
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FIG. 6. Time courses of nitrate and nitrite concentrations during continuous denitrification in the gas-lift reactor and expanded bed reactor with downflow. Reactor A: gas-lift reactor; Reactor B: expanded bed reactor with downflow; #: concentration of trace metal elements was Symbols: n , nitrate; a, nitrite. reduced to half of the original concentration.
N03-N and NOI-N concentrations in the early phase of continuous denitrification. The experiment was started in the gas-lift reactor with a carrier volume fraction of 10%. After batch cultivation for 5 d the continuous denitrification was initiated by pumping of the synthesized feed medium containing 20 mol-N03/m3 at an HRT of 20.8 h. Five days after the start of continuous feeding the inlet nitrate ions at 20 mol/m3 had been almost completely converted to nitrogen gas, and then the nitrate loading rate was increased to 5.6 kg-N/m3carrier/d by reduction the HRT to 12 h. The increase in nitrate loading rate caused carrier flotation, and homogeneous distribution of carrier particles could not be achieved even at higher gas circulation rates. Nitrate removal also became very unstable. Thirty-two d after the start of the experiment the carrier particles inside the gas-lift reactor were transferred into the expanded bed reactor with a working volume of 0.95 dm3. The circulation flow rate in this reactor was varied in a range of 4.0 to 7.5 dm3/min. In spite of a higher loading rate of 12.3 kg-N/m3-carrier/d, the distribution of carrier particles was markedly enhanced, and more than 98% nitrogen removal was achieved. After the maximum denitrification rate had been achieved, the concentration of trace metal elements in the influent was varied for investigation of its relationship to the denitrification rate. As shown by the data during the experiment without trace metal elements, the performance in denitrification dropped. For confirmation of this obvious effect, the trace metal element concentration was first returned to the original level and then reduced to half. The residual nitrate concentration change corresponded to the change in concentration of the trace metal elements. The results obtained in this experiment show the importance of homogeneous distribution of the
carrier particles and the presence of trace metal elements such as iron, manganese and molybdenum in order to achieve efficient denitrification. The denitrification was continued for 220 d for investigation of the stability of this reaction system. In the late phase of denitrification the carrier particles were gradually enlarged by the excess growth of cells on the carrier particle surfaces, which caused an increase in carrier particle density and instability of the system. Since the homogeneous distribution of carrier particles in this expanded bed reactor is based on the delicate balance between the buoyancy force and drag force, this reactor was sensitive to changes in the physical properties of the carrier particle and the liquid. Design of column reactor with jet pump and its performance in denitrification To establish a compact and
highly efficient system for denitrification using immobilized cells, it is necessary to develop new reactors which have the following characteristics: the ability to distribute the floating carrier particles at a high nitrate loading rate and a high carrier packing density; and a structure that allows generation of moderate shear stress on the carrier particle surface to detach the excess cells and to degas the carriers. The jet pump is a device for pumping fluids by means of a high velocity jet of the same or another kind of fluid. The principal phenomenon involved is the transfer of momentum from the high velocity fluid to the lower velocity fluid during the mixing process, with the result that all fluid leaving the mixing tube has about the same velocity. This pump is used to overcome to many pumping problems due to its low cost, simplicity in operation and ability to mix thoroughly the two fluids. In this work the jet pump was used as a distributor of floating carriers. The analysis and design of pump units to use the same liquid (self-entrainment)
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of low viscosity have been placed on a firm basis. The energy efficiency of jet pumps, p, is expressed by Eq. 1, as the ratio of the rate of mechanical energy addition to the suction liquid to the rate of net loss of mechanical energy by the drive liquid:
(1) where QS and Qj are the volumetric flow rates of the suction liquid and the drive liquid, respectively, hi, h2 and hd are the heads at the pump connection points for the drive line, suction line and discharge line, respectively, A4 is the capacity ratio given by [Qs/Qj], and H is the head ratio given by [(hd-&)/(hl -&)I. The performance characteristics of jet pumps depend strongly on the area ratio R of the cross-sectional area of the jet nozzle to the cross-sectional area of the mixing tube. The jet pump for our reactor should have a low discharge pressure but high suction flow rate QS. For such a jet pump, a low area ratio of about 0.12 was recommended to achieve a capacity ratio of M=2.2 and a head ratio of H=O.l at a pump efficiency of t7=0.22 (12). As shown in Fig. 4, the drive liquid was supplied into the mixing tube through the annulus of the jet pump, while the suction fluid was introduced through the central tube together with the carriers. This jet pump structure was effective in avoiding mechanical damage to the carriers. The diameter of the downflow tube set at the center of the reactor with a working volume of 12.5 dm3 was 32mm, and the tank diameter was 190mm. Considering the rising velocity of a single bubble, the linear velocity of liquid inside the downflow tube would be around 20 cm/s. This linear velocity corresponds to a suction liquid volumetric flow rate QS of 9.7 dm3/min; this value was calculated from the drive liquid volumetric flow rate Qj of 4.4 dm3/min and M=2.2. Under these operating conditions, homogeneous distribution of the carrier particle was possible for a carrier volume fraction of up to 30%. For the column reactor with the jet pump, the maximum nitrate loading rates which allowed complete denitrification were measured during a continuous experiment with the AQUACELTM carrier (particle size 3 mm cubic, pore size 500 pm, IEC 1.3 meq/g) at 30°C. The volume fraction of the carrier was increased to 20%, and the nitrate loading rate was gradually increased up to 28 kg-N/m3-carrier/d by reduction of the HRT from 12 to 3 h and increase of the N03-N influent concentration from 20 to 1,050 g/m3. In this reactor the distribution of the floating carriers was markedly enhanced, and a suitable shear stress to prevent excess cell growth was exerted on the carrier particle surfaces in the mixing tube of the jet pump. The stable denitrification lasted for more than five months, and complete denitrification was achieved even at a high nitrate loading rate of 15 kgN/m3-carrier/d (Fig. 7). Denitrikation of actual wastewater discharged from an electroplating factory The stirred tank reactor with a downcomer shown in Fig. 5 is practical for largescale treatment because of its simple structure. The rotation speed required for homogeneous distribution of floating carriers can be estimated from the equations below: When the hollow cylindrical casing with a radius of R is rotating at an angular velocity of w, liquid pressure PC is generated at the outlet of the elbow tube:
I 0
5
10
15
20
25
30
Nitrate loading rate [kg-Nlmbzarrierid]
FIG. 7. Relationship between nitrate loading rate and nitrate reduction rate measured in the column reactor with a jet pump.
P, = ,oo2R2/2
(2)
This pressure induces liquid flow inside the downcomer against the gravity head at the outlet of the downcomer. The linear velocity of liquid inside the downcomer U is calculated using Bernoulli’s equation as U= (w2R2- 2Zg)“*
(3)
where 2 is the liquid depth at the center of tube, and g is the acceleration due to gravity. ing the rising velocity of a single bubble, the rotation speed may be estimated from Eq. 3. requirement, L,, may also be estimated, as
the elbow Considermaximum The power
L, = Gw2R2/2
(4)
where G is the mass flow rate. The pilot-scale reactor with a working volume of 500dm3 was used for denitrification of actual wastewater discharged from an electroplating factory. Based on Eq. 3 and the dimensions of the reactor shown in Fig. 5, the rotation speed necessary to produce a linear velocity of 20cm/s inside the downcomer was estimated to be 300rpm. Since the buoyancy force of the carrier particles is lower than that of the gas bubbles, this rotation speed could be considered to be the maximum rotation speed. Under these operating conditions homogeneous TABLE 2.
Metal Mg Fe MO Mn Na K cu co Zn Ni Pb Al Cd Cr
Concentrations of metals in electroplating wastewater and synthetic wastewater Electro;t;yOg
_ , 3.2-4.3 5.5-5.9 0.36-0.39 0.44-0.56 3200 1830 0.2-0.51 so.41 0.24-0.27 0.11-0.16 SO.076 so.031 SO.0028 0.26-0.43
waste
Synthetic waste (ma/0 . - , 0.81 0.20 0.08 0.20 690 420 -
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.E .Z s
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z m 0 S!
4oo
.% =
0
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FIG. 8. Time courses of nitrate and nitrite concentration during continuous denitrification of electroplating wastewater in the stirred tank reactor with a downcomer. Symbols: q , influent Nor-N; n , effluent Nor-N; 0, influent NO*-N; 0, effluent NO*-N; A, temperature; %, trouble in methanol feeding.
annual concentrations of N03-N, N02-N and BOD after these treatments are 460, 18 and 30g/m3, respectively. The wastewater upper limit in this region is 20g/m3 for total nitrogen; hence the existing anaerobic biofilm process is inadequate for meeting this limit. The concentrations of metals and phosphate in the wastewater were measured. As shown in Table 2, the concentrations of important heavy metals for denitrification such as Fe, MO, and Mn were higher than those in the synthesized wastewater. The total phosphate concentration in the wastewater, however, was only 4.2g/m3,
distribution of the carrier particles was possible for a carrier volume fraction of up to 40%. This factory discharges wastewater containing cyanide, nitrite and nitrate at high concentrations with a displacement of 50 m3/d, and has a conventional wastewater treatment system. The cyanide is converted to ammonia by thermal hydrolysis followed by stripping. The other wastewater containing nitrate and nitrite is discharged into a storage tank together with domestic wastewater. This mixed wastewater is treated by an activated-sludge process followed by an anaerobic biofilm process. The average
HRT[h]
20’10/
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I
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; I
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Id]
FIG. 9. Time courses of nitrogen removal rate and ratio during continuous denitrification of electroplating wastewater in stirred tank reactor with a downcomer. Symbols: 0, nitrogen removal ratio; 0, nitrogen removal rate; %, trouble in methanol feeding.
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and phosphate supplementation was necessary. The concentration of methanol as an electron donor was fixed at 1.5 times the theoretical requirement, and the weight ratio of phosphate to nitrogen (P/N) was fixed at 0.04 by addition of KH2P04. The seed sludge in this experiment was obtained from the activated sludge tank in this factory, and was immobilized in the cellulose carriers (particle size 5 mm cubic, pore size 1,000 pm, IEC 1.3 meq/g). The volume fraction of the carrier particles to the reactor working volume was 20%, and the carriers were homogeneously distributed at a rotation speed of 98 rpm during the initial stage of continuous denitrification. Figures 8 and 9 show the time courses of N03-N and N02-N concentrations and the nitrogen removal rate and ratio during the 120d continuous denitrification, respectively. Continuous denitrification was initiated at an HRT of 20 h after four batch treatments for proliferation of denitrifiers. Tend after the start of continuous feeding, the inlet N03-N of 650g/m3 and NOz-N of 80 g/m3 had been almost completely converted to nitrogen gas at an HRT of 10 h, and then the HRT was shortened to 2 h. In spite of the marked fluctuation of the influent concentrations of N03-N and NOz-N, the system was able to keep the effluent N03-N concentration lower than 5 g/m3 and completely remove N02-N. Trouble in the methanol feeding occurred at 30 and 80 h after the start of the experiment, which caused rapid increases in the N03-N concentration. The system, however, was quickly restored to its original state after repair of the feed pump. This means that the immobilized system is tolerant to troubles in operation. When the HRT was shortened to 3 h, the flotation of the carrier particles was obvious and the rotation speed was increased to 150rpm. As seen from Fig. 9, the nitrogen removal rate reached was 20.4 kg-N/m3-carrier/d. This excellent performance might be due to homogeneous distribution of carriers and moderate shear stress on the surfaces of the carriers. The moderate shear stress was applied to the carrier particle surfaces at the outlet of the elbow tube, which would be effective for preventing excess accumulation of cells on the carrier particle surfaces and for enhancement of transport of nutrients into the carriers. As shown in Fig. 8, the fluctuation of the concentrations of N03-N and N02-N in the influent was so marked that the methanol concentration could not be maintained at a given level. In this experiment oversupply of methanol occurred, which induced excess accumulation of cells and residual BOD. An on-line monitoring method for NH4-N, N03-N and N02-N concentrations should be developed, and an automatic feed control system for the electron donor should be developed to complete this AQUACELTM system. In conclusion, cells immobilized in macro-porous cellulose carriers were used for denitrification. Based on the results of long-term continuous denitrification experiments with different types of reactors, the following conclusions were reached. (i) Even when macro-porous carriers were employed, flotation of the carrier particles was caused by vigorous evolution of nitrogen gas at high nitrogen loading rates. (ii) Homogeneous distribution of floating carrier particles was essential to achieve highly efficient denitrification. (iii) New reactors in which hydrodynamic jet flow and centrifugal force were used were effective for homogeneous distribution of carriers, and have the ability to expose the surface of carriers to moderate shear stress.
NOMENCLATURE : acceleration due to gravity, m/s* : mass flow rate, kg/s : head at pump connection point for the drive line, m : head at pump connection point for suction line, m : head at pump connection point for discharge line, m : head ratio defined by [(ha -h2)/(h1 -hd)], L, :power requirement, W M : capacity ratio defined by Qs/Qj, PC :liquid pressure generated by rotation, Pa QS : volumetric flow rate of suction liquid, m3/s Qj : volumetric flow rate of drive liquid, m3/s R :radius of hollow cylindrical casing, m U : linear velocity of liquid inside the downcomer, m/s Z :liquid depth at the center of the elbow tube, m ‘j; : energy efficiency of jet pump, p : density, kg/m3 w : angular velocity, rad/s g G hl hz hd H
ACKNOWLEDGMENT The authors wish to acknowledge the Tsukuba Advanced Research Alliance (TARA) for partly financing this work. REFERENCES 1. Sbuval, IL I. and Gruaer, N.: Infant methemoglobinemia and other health effects of nitrate in drinking water. Prog. Water Technol., 8, 183-188 (1977). of 2. Tramper, J. and de Man, A. W. A.: Characterization Nitrobucter agilis immobilized in calcium alginate. Enzyme Microb. Technol., 8, 472-476 (1986). 3. SomIno. T.. Nakamura. H.. Mori. N.. Kawaauchi. Y.. and Tada, M.: ‘Immobilization of nitrifylng bact&ia in porous pellets of urethane gel for removal of ammonium nitrogen from wastewater. Appl. Microbial. Biotechnol., 36, 556560 (1992). 4. Nilsson, I., Ohlson, S., Haggstrom, L., Moiin, N., and Mosbach, K.: Denitrification of water using immobilized Pseudomonas denitrificans cells. Eur. J. Appl. Microbial. Biotechnol., 10, 261-274 (1980). 5. Nilsson, I. and Ohlson, S.: Columnar denitrification of water by immobilized Pseudomonas denitrtficans cells. Eur. J. Appl. Microbial. Biotechnol., 14, 8690 (1982). 6. Kokufnta, E., Shimohashi, M., and Nakamura, I.: Immobilization of Paracoccus denitrificans cells with polyelectrolyte comolex and denitrifving activitv of the immobilized cells. J. Ferment. Technol., 64,533-538-(1986). 7. Taya, M., Miora, H., Ucbiyama, IL., Iijima, S., and Kobayashi, T.: Denitrification with immobilized Paracoccus denitr@kns and preservation characteristics of immobilized cells. Hakkokogaku, 66, 235-240 (1988). 8. Lin, Y. F. and Chen, K. C.: Denitrification and methanogenesis in a co-immobilized mixed culture system. Wat. Res., 29, 3543 (1995). 9. Matsnmura, M., Yamamoto, T., Shinabe, K., and Yasuda, K.: Rapid nitrification with immobilized cells using macro-porous cellulose carrier. Wat. Res., 31, 1027-1034 (1997). _ 10. Matsumura, M., Takehara, S., and Kataoka, II.: Continuous butanoVisopropano1 fermentation in down-flow column reactor coupled with pervaporation using supported liquid membrane. Biotechnol. Bioeng., 39, 148-156 (1992). 11. Sumino, T., Nakamura, H., Mori, N., and Kawaguchi, Y.: Immobilization of nitrifylng bacteria by polyethylene glycol prepolymer. J. Ferment. Bioeng., 73, 37-42 (1992). 12. Folsom, R. G.: Jet pumps with liquid drive. Chem. Eng. Prog., 44, 765-770 (1948).