Biodegradation kinetics of ferrous(II) cyanide complex ions by immobilized Pseudomonas fluorescens in a packed bed column reactor

Process Biochemistry 35 (2000) 615 – 622 www.elsevier.com/locate/procbio

Biodegradation kinetics of ferrous(II) cyanide complex ions by immobilized Pseudomonas fluorescens in a packed bed column reactor Arzu Y. Dursun, Zu¨mriye Aksu * Department of Chemical Engineering, Hacettepe Uni6ersity, Beytepe 06532, Ankara, Turkey Received 22 April 1999; received in revised form 27 April 1999; accepted 14 August 1999

Abstract Combined external mass transfer and intrinsic biodegradation effects on the observed biodegradation rate were investigated for ferrous (II) cyanide complex (ferrocyanide) ions removal with calcium – alginate gel immobilized Pseudomonas fluorescens in a packed bed column reactor. Assuming first-order biodegradation kinetics, observed first-order biodegradation rate constants (kp) were calculated at different flow rates. To investigate the effect of external film diffusion on the observed biodegradation rate, (1 − n) several external mass transfer correlation models of the type JD = KN − were tested and the mass transfer coefficients (kl) Re were calculated as a function of the mass flux (G) and the Reynolds number (NRe) at different K and n values. The intrinsic first-order biodegradation rate constants (k) and the surface areas per unit weight of dried cells for mass transfer (am) were determined from 1/kp versus 1/G n plots at the same n values. Combining the k and klam values, the observed first-order biodegradation rate constants (kp) were calculated again at the same n values and compared with the kp values determined from 0.507 experimental data. The mass transfer correlation JD =1.625 N − predicted accurately the experimental data. © 2000 Elsevier Re Science Ltd. All rights reserved. Keywords: Ferrous (II) cyanide complex (ferrocyanide) ions; Ca – alginate gel immobilized Pseudomonas fluorescens; Packed bed; External mass transfer coefficient; First-order biodegradation

1. Introduction Industries dealing with metal plating and finishing, the mining and extracting of metals such as gold and silver, production of synthetic fibres and the processing of coal generate large quantities of cyanide-containing wastes. In the presence of metal ions, such as nickel, copper, zinc and iron, cyanide forms complex compounds of varying toxicity and stability [1 – 5]. Ferrocyanides which are the well-known hexacyano complexes of iron are also very recalcitrant cyano– metal complexes [2,6,7]. Cyanide and metal cyanide complexes are potent inhibitors of cellular metabolism. Hence, cyanide and metal cyanide complexes in industrial waste waters must be treated or reduced to the * Corresponding author. Tel.: +90-312-2977434; fax: + 90-3122992124. E-mail address: [email protected] (Z. Aksu)

lowest levels possible before they can be discharged [8,9]. Several methods (alkaline chlorination, hidrogen peroxide, ozon oxidation, biological oxidation, biosorption) are available for cyanide and metal cyanide complex ions removal and/or detoxification but most of these chemical techniques suffer from major drawbacks. They are not always well adapted and efficient with regard to technological, cost and disposal considerations [2–5,10–12]. The technical and economical concerns, related to these methods makes the biological processes viable in some cases. Despite cyanide’s toxicity to living organisms biological treatments are feasible alternatives to chemical methods without creating or adding new toxic and biologically persistent chemicals. The biological degradation of metal cyanide complex anions is based upon functions carried out by living cells and involves the metabolism and/or transformation of cyanide complex anions to products less toxic to the environment. Dif-

0032-9592/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 9 9 ) 0 0 1 1 0 - 7

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ferent mechanisms contribute to cyanide complex ions degradation in an aerobic biological treatment such as biological metabolism, adsorption onto the biomass, chemical reaction with organic compounds. Some microorganisms can adapt to grow in the presence of metal cyanide complex ions by inducing the synthesis of enzymes for the degradation of cyanide complex ions or by synthesising cyanide-resistant enzymes. The most commonly isolated microorganisms which degrade cyanide complex ions are the Pseudomonas species. Metal cyanide complex ions could be used for the growth of the microorganism as a source of nitrogen. For instance, strains of bacterium P. fluorescens were shown to utilize cyanide as a source of nitrogen for growth. They can convert cyanide to carbon dioxide and ammonia via cyanide oxgenase system. Although the degradation of simple cyanides have been widely reported, much less is known about the degradation of metal cyanide complexes [2 – 5,13 – 18]. Immobilized microorganism technology is used increasingly for removing the pollutants from contaminated ecosystems. Immobilization provides high cell concentration. Immobilized cells in a continuous fixed bed reactor avoid washout of cells, even under conditions of negligible cell growth, while the volumetric reaction rates and overall productivity compare favourably with those of suspension cultures. In addition, the flexibility of reactor design and the improved thermal and operational stability are further advantages of using immobilized cells. The separation of treated water from biomass is easy. A large volume of wastewater can be treated continuously using a defined quantity of immobilized cells in the column. Reuse of microorganism is also possible [19 – 24]. A good support material should be rigid and chemically inert, should bind cells firmly, and should have high loading capacity. Ca – alginate provides these specifications. The entrapment of cells in Ca – alginate is a promising method for microbial degradation of toxic substances and have been used since 1975. Ca – alginate is not toxic to cells and the immobilization method is practical [21,23,24]. Although immobilized cells exhibit a slower substrate (toxic substance) utilization rate than suspension culture organism, especially at lower inlet substrate concentrations and lower feed flow rates high yields can be obtained in the column. The removal in the packed column system is affected strongly by solute diffusion into the pores of Ca – alginate gels. The diffusion phenomena explain the higher removal yields obtained at lower flow rates. However, higher flow rates lower removal rates because of inefficient residence times between living cells and solute. Moreover, the immobilized cells are exposed to higher substrate concentrations without loss of cell viability and protection of organisms against changes in temperature and pH by immobilization in Ca – alginate is provided [5,21,23,26].

Ferrous(II) cyanide complex ion biodegradation was investigated in a packed bed column reactor operated in continuous mode using Ca–alginate immobilized P. fluorescens in this study and combined external mass transfer and intrinsic biodegradation effects on the observed biodegradation rate were carried out.

2. Model description Numerous papers have been devoted to modelling simultaneous diffusion and conversion of substrate by immobilized biocatalysts. In such systems the substrate is transferred from a liquid phase to a solid phase in which the reaction occurs.

2.1. Biodegradation and obser6ed biodegradation rate constant The mass balance for ferrous(II) cyanide complex ions in the packed bed column reactor is given by Eq. (1). In the development of this equation, steady state, plug flow, no axial dispersion and spherical immobilized cell particles are assumed.

 

HQ dC × 6× 10 − 2 = − r W dz

(1)

where r is the biodegradation (ferrous(II) cyanide complex ion removal) rate (mg g − 1 h − 1), Q is the volumetric flow rate (ml min − 1), H is the height of the column (cm), W is the total amount of dried cells in the immobilized particles (g) and dC/dz is the concentration gradient along the column length (mg l − 1 per cm). Assuming a first-order biodegradation (this is a correct assumption especially at lower substrate concentrations), the relationship between the biodegradation rate and the substrate (ferrous(II) cyanide complex ion) concentration in the column is given as: r= kpC

(2)

where kp is the observed first-order biodegradation rate constant (l g − 1 per h). Substituting Eq. (2) into Eq. (1) Eq. (3) is obtained.

 

HQ dC × 6× 10 − 2 = − kpC W dz

(3)

Eq. (4) is found by integrating Eq. (3) using boundary conditions of at z= 0; C=Co and at z= H; C=C.

 

ln

Co W = kp × (103/60) C Q

(4)

where Co is the inlet substrate concentration (mg l − 1) and C is the outlet substrate concentration (mg l − 1). The observed first-order biodegradation rate constants (kp) can be calculated from Eq. (4) at different flow rates at a constant dried cell quantity in the immobilized particles [19,20,22,23,26].

A.Y. Dursun, Z. Aksu / Process Biochemistry 35 (2000) 615–622

2.2. Bulk mass transfer (external film diffusion) and mass transfer coefficient

Several correlations for mass transfer rates use the above formula but vary in the dependence of JD on NRe, i.e.

When fluid flows through a bed of particles, there exist regions (boundary layer) near the surface of the particles where the fluid velocity is very low. In such regions around the exterior of particles, a near-stagnant film of fluid is present through which the substrate needs to be transported. This transport takes place primarily by molecular diffusion; since this rate may be quite slow, the observed reaction rate can be decreased significantly by this external film diffusion. The local rate of film diffusion of the substrate from the fluid bulk to the surface of the immobilized cells may be considered to be proportional to the area for mass transfer and the driving force for mass transfer, i.e. the concentration difference between the bulk and the external surface of the immobilized cell: rm = klam(C− Cs)×10 − 3

(5)

where rm is mass transfer rate (mg g − 1 h − 1), kl is external mass transfer coefficient (cm h − 1), am is surface area per unit weight of microorganism cells for mass transfer (cm2 g − 1), Cs is substrate concentration at the surface of the cell (mg l − 1) [19 – 23,25 – 27]. Average transport coefficients between the bulk stream and particle surface in the packed bed column reactor can be correlated in terms of dimensionless groups which characterize the flow conditions. Chilton and Colburn in 1934, as a basis for the correlation of mass transfer data, suggested the following relationship [27]:

 

klr m G rD

=f(NRe)

60Qr G= Ao

(6)

(7)

where Q is the volumetric flow rate of ferrous(II) cyanide complex ion solution (ml min − 1), A is the cross sectional area of column (cm2), o is the void fraction of the column. The Reynolds number can be defined as dp G m

(8)

where dp is the diameter of the particle (cm). The term in parentheses in Eq. (6) is the Schmidt number and the group on the left-hand side is a dimensionless group which is symbolized by JD as a function of NRe JD =

(1 − n) JD = KN − Re

(10)

Different mass transfer coefficients have different values of K and n. The value of n varies from 0.1 to 1.0; this range encompasses all the values of the exponent in Eq. (10) that have presented in the chemical engineering literature [23,26,27]. Wilson and Geankoplis [27] proposed the following correlation for the external mass transfer from the liquids in the packed bed column reactor JD =

1.09 − 2/3 N Re o

(11)

McCune and Wilhelm presented the below correlation for the estimation of external mass transfer and Rowito and Kittrell showed the applicability of this correlation to glucose oxidase enzyme immobilized on porous glass beads system [26]. 0.507 JD = 1.625N − Re

(12)

This correlation has been used successfully also to determine the bulk mass transfer coefficients for phenol removal using immobilized P. putida in a packed bed column reactor by Aksu and Bu¨lbu¨l [23]. An alternative correlation is that of Chu et al. [26]. This correlation has been employed successfully to estimate bulk mass transfer coefficients for a packed bed of collagen enzyme chips: 0.78 JD = 5.7N − Re

(13)

2/3

where m, r and D are the fluid viscosity, density and diffusivity, respectively, and G is the mass flux (g cm − 2 per h). G can be calculated from Eq. (7).

NRe =

617

klr 2/3 N G Sc

(9)

Finally, the last work was done by Nath and Chand [26] to estimate the external mass transfer coefficients proposing the following correlation; 0.59 JD = 5.7N − Re

(14)

They showed the applicability of the above correlation for the continuous bioconversion of sugars to ethanol in the immobilized yeast cells on activated bagasse chips.

2.3. Combined bulk mass transfer and biodegradation reaction The first-order biodegradation rate (Eq. (2)) can also be written as r= kCs

(15)

where k is the intrinsic first-order biodegradation rate constant (l g − 1 per h). Since the rate of mass transfer equals to biodegradation rate at steady state, so equating Eqs. (5) and (15) solving for the unknown surface concentration and Eq. (16) is found:

A.Y. Dursun, Z. Aksu / Process Biochemistry 35 (2000) 615–622

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Cs =

klamC k+klam

(16)

The effects of mass transfer and biodegradation rates on the observed biodegradation rate are shown in Eq. (17). kp =

kklam k+klam

(17)

or 1 1 = kp 1 1 + k klam

(18)

At a constant temperature, the contributions of biodegradation and mass transfer resistance on the kp were represented by (1/k) and (1/klam) terms. kp is now related to the parameters of the external film diffusion model by substituting Eq. (10) into Eq. (9) and solving for the mass transfer coefficient, giving kl = AG n where A=

3.2. Preparation of Ca–alginate gel immobilized P. fluorescens beads At the end of exponential growth phase, P. fluorescens cells were centrifuged and resuspended in 2% Na–alginate. The ratio of Na–alginate to biomass was chosen as 3. This mixture was then dropped into 20% calcium chloride solution using a peristaltic pump. The drops of Na–alginate solution were gelled into 0.25 cm diameter spheres upon contact with chloride solution. Ca–alginate gel for immobilizing P. fluorescens particles has been stored in calcium chloride solution at 4°C for at least 2 h to complete gel formation. In this way insoluble and stable immobilized beads were obtained.

(19)

  

K m r rD

sterilized by autoclaving at a pressure of 1.1 atm. and a temperature of 121°C. After the culture was inoculated into 100 ml enrichment medium (in 1:10 ratio) in a 250-ml conical flask, it was incubated at 25°C in an agitated shaker (100 rpm) for 24 h.

− 2/3

dp m

3.3. Continuous packed bed column reactor studies − (1 − n)

×10 − 3

(20)

Substituting Eq. (19) into Eq. (17) finally leads to: 1 1 1 1 = + n kp Aam G k

(21)

Using the plots of the experimentally measured values of 1/kp versus 1/G n for different K and n values takes place in JD correlation, am and k values can be obtained from the slopes and intercepts. Since the am value is known, klam values can be calculated at different flow rates and combined with the k values to obtain the kp values as shown in Eq. (17). The JD correlation predicts accurately our experimental data can be chosen for this system when comparing these kp constants with the kp constants found from Eq. (4). Then, comparing the k and klam constants, it is also possible to decide which step has limited the biodegradation rate. 3. Materials and methods

3.1. Microorganism and growth conditions P. fluorescens (P70) obtained from Universitat Hohenheim Institut Umwelt und Tierhygiene Sowie Tiermedizin Mit Tierklinik, Germany, was used in this study. The bacterium was grown in an enrichment medium containing the following ingredients (amounts given per l): glucose, 5 g; peptone, 1 g; yeast extract, 1 g; KH2PO4, 0.5 g; K2HPO4, 0.5 g; (NH4)2SO4, 0.5 g; MgSO4 · 7H2O, 0.05 g at pH 6.5. In addition 18 g l − 1 agar was used for solid growth media. All media were

Biodegradation studies were carried out in a packed bed column reactor with an inside diameter of 2 cm and a bed depth of 27.5 cm. The column contained a known weight of immobilized cells (56.99 g of immobilized cell particles contain 34.53 g of wet cells which is equal to 7.41 g of dried microorganism: average immobilized particle size 0.248 cm; immobilized particle density is 1.19 g ml − 1, determined with a picnometer). The temperature of column during the experiments was kept constant at 25°C. The previous batch stirred reactor studies showed that no growth was observed in media containing iron(II) cyanide complex ions as the sole source of carbon and nitrogen. The presence of organic nutrients such as glucose in the medium has a major influence on the degrading iron(II) cyanide complex ions. Addition of glucose led to bacterial growth and biodegradation of ferrocyanide ions so glucose was used as the carbon and energy source in the biodegradation medium. Initial experiments also showed that microorganism could degrade ferrocyanide only in the absence of another nitrogen source so ferrocyanide was used as the sole source of nitrogen [2]. Because the salts containing Mg2 + , K+, PO4− 3 ions caused the dissolution of Ca– alginate beads, the components in the feed solution were optimized also to improve the stability of beads. Feed solution was prepared by mixing the ferrocyanide solution autoclaved separately and the sterilized solution containing other optimized ingredients (as glucose, 0.465 g l − 1; KH2PO4, 0.0084 g l − 1; K2HPO4, 0.0108 g l − 1; and MgSO4, 0.05 g l − 1). After the sterilization, the pH of feed solution was adjusted to 5.0 with dilute sterilized H2SO4 and NaOH solutions. Feed solution

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Table 1 The experimental values of Q, C/Co and kp calculated from Eq. (4) at various flow rates at 100 mg l−1 of inlet ferrocyanide concentration (T, 25°C; pH of inlet ferrocyanide solution, 5; W= 7.41 g) Q (ml min−1)

C/Co

kp×103 (l g−1 per h)

0.09 0.20 0.31 0.41 0.62

0.15 0.32 0.38 0.46 0.63

1.43 1.83 2.38 2.56 2.33

aerated before sending to column throughout the course of the experiments. Feed ferrocyanide solution at the known concentration was passed continuously through the stationary bed of immobilized P. fluorescens particles. The flow rate was regulated with a variable speed pump. Samples were taken out from the effluent at different time intervals and analyzed for ferrocyanide ions. The experiment was continued for 48 h. All the experiments were carried out at least twice. The values used in calculations were mostly the arithmetic average of the experimental data.

3.4. Analysis of ferrocyanide Ferrocyanide ion concentration in the exit stream of column was determined titrimetrically in 1:1 HCl using fenontralin indicator and cerium (IV) sulphate [28].

4. Results and discussion The previous studies on biodegradation of ferrocyanide ions by Ca– alginate gel immobilized P. fluorescens in a batch stirred reactor showed that the ferrocyanide removal rate was affected slightly by the temperature and pH of medium, because of the protecting effect of Ca–alginate on the microorganism against to the changes of outlet conditions. A wide optimum range for temperature and pH were determined as 25 – 35°C and 4.0–7.0 [24]. For column studies, the optimum pH and temperature values were chosen as 5.0 and 25°C, respectively.

Fig. 1. Plots of 1/kp vs. 1/G n (R shows the linear regression coefficient).

Flow rate, ferrocyanide ion concentration, particle and column sizes and bacterial biomass quantity are the main operating characteristics of a packed bed column and they affect greatly the external mass transfer rate and observed biodegradation rate at steady state. The effect of the flow rate, one of the parameters affecting the external mass transfer resistance or indirectly observed biodegradation rate was investigated in this part of the study. For this purpose flow rates were varied from 0.09 to 0.62 ml min − 1 at 100 mg l − 1 inlet ferrocyanide ion concentration. The experimental values of Q, C/Co and kp evaluated from Eq. (4) at 100 mg l − 1 of inlet ferrocyanide ion concentration are given in Table 1. It is reported that the observed first-order biodegradation rate constants increased with increasing flow rates up to 0.41 ml min − 1. For investigating the external film diffusion effects on the observed biodegradation rate, Reynolds numbers and mass fluxes at studied flow rates were also calculated from Eqs. (7) and (8) using dp = 0.25 cm, m= 33.948 g cm − 1 per h, r= 0.997 g ml − 1, o= 0.45 and A= 3.14 cm2 values which were determined from experimental data. Calculated values of mass fluxes, Reynolds numbers, 1/kp and 1/G n for n=0.22, n=1/3 (=0.33), n= 0.41 and n= 0.49 are listed in Table 2. Plots of 1/kp versus 1/G n for all values of n are depicted in Fig. 1 and k and am values obtained from such plots

Table 2 Calculated values of G, NRe, 1/kp and 1/G n values found at various flow rates at 100 mg l−1 of inlet ferrocyanide concentration (T, 25°C; pH of inlet ferrocyanide solution, 5; W= 7.41 g) G (g cm−2 per h)

NRe

1/kp (g h l−1)

1/G 0.22

1/G 0.33

1/G 0.41

1/G 0.49

3.94 8.51 12.96 17.36 26.26

0.029 0.063 0.095 0.127 0.194

699.3 546.4 420.0 390.6 429.2

0.74 0.62 0.57 0.53 0.48

0.64 0.49 0.43 0.39 0.34

0.57 0.42 0.35 0.31 0.29

0.51 0.35 0.28 0.25 0.20

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620

Table 3 The values of k and am calculated from the 1/kp versus 1/G n plots for various values of n K

n

k×103 (l g−1 per h)

am (cm2 g−1)

5.7 2.42 5.7 1.625

0.220 0.330 0.410 0.493

Negative intercept 52.6 8.9 5.7

1.26 0.90 4.32

are tabulated in Table 3. The plot of 1/kp versus 1/G 0.22 gave negative intercept and was therefore not analyzed further. The magnitude of the intercept increases with increasing values of n while the magnitude of the slope decreases with increasing n. Thus, proposed film diffusional models could give satisfactory straight lines. Using ferrocyanide ion diffusivity found from the literature at T=25°C [7], kl values were determined from Eqs. (9) and (10) for all values of n and klam values were also calculated using am values found at the same n values and compared in Table 4. As the k and klam values were known, kp values were calculated from Eq. (17) again and compared with the kp constants found from Eq. (4) in Table 5. All the kp constants found from Eq. (17) for n = 0.33, n = 0.41 and n=0.49 are quite similar to the kp constants found from Eq. (4). In order to compare the validity of the mass transfer correlation more definitely, a normalized deviation, D(%), was calculated as follows.

N

% D(%) =

i=1

)

)

kp (Eq. (4)) − kp (Eq. (17)) kp (Eq. (4)) ×100 N

(22)

where the subscripts ‘p(Eq. (4))’ and ‘p(Eq. (17))’ show the kp values calculated from Eqs. (4) and (17) at various n values and N: the number of measurements. D(%) values obtained for n= 0.33 and n= 0.41 are slightly higher (D(%): 7.4 and 9.0, respectively) than that of n= 0.493 (D(%): 4.8). Thus, it was decided that 0.507 correlation provides a satisfacthe JD = 1.625 N − Re tory prediction of the kl values can be chosen for this system. The combined effects of intrinsic first-order biodegradation rate constant and external mass transfer coefficients on the observed first-order biodegradation rate constants for n=0.493 are compared also in Table 6. It is clear that the biodegradation is limited by both the diffusion of the substrate on the cell surface and the biodegradation of ferrocyanide ions by the microorganism. However, the effect of external diffusion on the observed biodegradation rate decreased with increasing flow rate. 5. Conclusion Biodegradation of ferrocyanide ions by Ca–alginate gel immobilized P. fluorescens was carried out in a packed bed column reactor, the packing material was assumed to be porous, spherical particles. A kinetic model was developed assuming plug flow, no axial dispersion, steady state and first-order biodegradation

Table 4 The comparison of kl and klam values found at all the studied flow rates for various values of n Q (ml min−1)

0.09 0.20 0.31 0.41 0.62

n = 0.33

n= 0.41

n = 0.493

kl (cm h−1)

klam×103 (l g−1 per h) kl (cm h−1)

klam×103 (l g−1 per h)

kl (cm h−1)

klam×103 (l g−1 per h)

1.19 1.54 1.77 1.95 2.24

1.50 1.94 2.23 2.46 2.82

1.73 2.37 2.82 3.18 3.76

0.45 0.66 0.81 0.93 1.14

1.94 2.83 3.48 4.02 4.93

2.11 2.89 3.44 3.88 4.59

Table 5 The comparison of the observed first-order biodegradation rate constants calculated from Eq. (4) with the ones calculated from Eq. (17) found at all flow rates for various values of n Q (ml min−1)

0.09 0.20 0.31 0.41 0.62

kp(Eq. 4)×103 (l g−1 per h)

1.43 1.83 2.38 2.56 2.33

n=0.33

n =0.41

n = 0.493

kp(17)×103 (l g−1 per h)

kp(Eq. 17)×103 (l g−1 per h)

kp(Eq. 17)×103 (l g−1 per h)

1.46 1.87 2.14 2.35 2.68

1.56 2.01 2.29 2.50 2.82

1.44 1.85 2.26 2.45 2.64

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Table 6 The comparison of the combined effects of intrinsic first-order biodegradation rate constant and mass transfer coefficients on the observed first-order biodegradation rate constants for n=0.493 kp×103 (l g−1 per h) 1.44 1.85 2.26 2.45 2.64

k×103 (l g−1 per h)

5.7

1/k (g h per l)

% Contribution of k

klam×103 (l g per h)

1/klam (g h−1 per l)

% Contribution of klam

0.18

26 34 38 42 47

1.94 2.83 3.48 4.02 4.93

0.52 0.35 0.29 0.25 0.20

74 66 62 58 53

(or low inlet ferrocyanide ion concentrations). The biodegradation kinetics of the system was investigated as a function of flow rate. Theoretically, the external (1 − n) film diffusion model of the type JD =KN − evaluRe ated. It is decided that the mass transfer correlation 0.507 JD =1.625N − can be used successfully to quantify Re the external film diffusion effects for the continuous biodegradation of ferrocyanide ions in a calcium –alginate immobilized P. putida packed bed reactor. The external mass transfer rate constants and the observed first-order biodegradation rate constants predicted from the model give an idea about the magnitudes of kinetic parameters and the controlling step of biodegradation. In comparison of the obtained mass transfer rate and intrinsic biodegradation rate constants, the effect of external film diffusion on observed biodegradation rate decreases with increasing flow rate, as expected. However, for this process both the biodegradation and mass transfer steps limited the observed biodegradation rate. We believe that application of ferrocyanide ions biodegradation by Ca – alginate immobilized P. putida in the purification of waste water can be suitable for large-scale column exploitation by using these kinetic parameters. This study will be use of in making realistic engineering estimates of the effect of external mass transfer on the observed reaction rates in immobilized cell bioreactors. Further studies will be attempted to quantify the effects of pore diffusion in immobilized cell particles.

am

Acknowledgements

Greek symbols o Void fraction in packed bed m Feed fluid viscosity (g cm−1 per h) r Feed fluid density (g ml−1)

The authors wish to thank to the Scientific and Technical Research Council of Turkey (TUBITAK), project no. YDABC ¸ AG 528, for the partial financial support of this study.

C Co Cs

dp D G H JD k kl kp NRe NSc Q r rm W z

surface area per unit weight of dried cells for mass transfer (cm2 g−1) outlet substrate (ferrous (II) cyanide complex ion) concentration (mg l−1) inlet substrate (ferrous (II) cyanide complex ion) concentration (mg l−1) substrate (ferrous (II) cyanide complex ion) concentration at the cell surface (mg l−1) particle diameter (cm) substrate (ferrous (II) cyanide complex ion) diffusivity (cm2 per h) mass flux of ferrous (II) cyanide complex ion solution (g cm−2 per h) height of the column (cm) dimensionless group given by Eq. (9) intrinsic first-order biodegradation rate constant (l g−1 per h) mass transfer coefficient (cm h−1) observed first-order biodegradation rate constant (l g−1 per h) the Reynolds number in the packed bed the Schmidt number in the packed bed volumetric flow rate (ml min−1) biodegradation (ferrous (II) cyanide complex ion) rate (mg g−1 per h) mass transfer rate (mg g−1 per h) dried microorganism quantity in packed bed (g) the change of the height of the column (cm)

References Appendix A. Nomenclature A

column superficial cross section area (cm2)

[1] Patterson JF. Industrial Wastewater Treatment Technology, 2nd ed. London: Butterworths, 1985:115 – 34. [2] Dursun AY, Calik A, Aksu Z. Degradation of ferrous (II) cyanide complex ions by Pseudomonas fluorescens. Process Biochem (in press).

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