Journal of the Taiwan Institute of Chemical Engineers 44 (2013) 780–785

Kinetic characteristics of biodegradation of methyl orange by Pseudomonas putidamt2 in suspended and immobilized cell systems

Tran Phuong Thao, Hsiang-Chien Kao, Ruey-Shin Juang *, John Chi-Wei Lan **

Biorefinery and Bioprocess Engineering Laboratory, Department of Chemical Engineering and Materials Science, Yuan Ze University, Chung-Li 32003, Taiwan

A R T I C L E I N F O

Article history:

Received 2 April 2012

Received in revised form 6 January 2013

Accepted 13 January 2013

Available online 17 March 2013

Keywords:

Azo dye

Biodegradation

Immobilization

Ca-alginate

Mass transfer

Diffusion

A B S T R A C T

Azo dyes, which are considered the most recalcitrant and persistent among all groups of dye, were

biodegraded by various types of bacteria. In this study, the decolorization of methyl orange was

determined under various conditions by non-immobilized and immobilized Pseudomonas putida mt2. In

both systems, the decolorizations were more favorable under anoxic condition. Optimal conditions for

decolorization were investigated. The optimal temperature for suspended cell operation is in the range

between 33 and 35 8C and the decolorization seems not to be suitable in acidic condition. However, the

optimal range of pH and temperature in the immobilized cell system were obtained at 7–9 and 35–37 8C,

respectively. Kinetics parameters of nmax and km were predicted up to 7.5 mg/(g h) and 283 g L�1,

respectively, for non-immobilized system; and 6.3 mg/(g h) and 257 g L�1, respectively, for immobilized

Ca-alginate system. Mass transfer coefficients were calculated as 0.75 � 10�5 and 6.20 � 10�5 for each

system.

� 2013 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

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jou r nal h o mep age: w ww.els evier . co m/lo c ate / j t i c e

1. Introduction

Azo dyes are the most important and diffuse group of syntheticdyes, and have azo bond as the color-producing group, which areclassified as the most recalcitrant and persistent group. Over7 � 105 metric tons of synthesis dyes are produced worldwideevery year for dyeing and printing [1]. It has been estimated thatapproximately 10% of the dye stuff used during dyeing processesdoes not bind to the fibers and is discharged with wastewater [2].Several studies indicated that most azo dyes are toxic, carcino-genic, and mutagenic, which have substantial effects on environ-ment and results in a threat to public health [3–5]. It waschallenging to decolorize wastewater with recalcitrant syntheticdye compounds released from industrial effluents [6].

Several physical and chemical methods were used andindicated as efficient approaches for degradation of azo dyes,such as precipitation, adsorption, filtration, reverse osmosis, andadvanced oxidation processes (reduction, complexometric meth-ods, ion exchange and neutralization) [7–10]. However, suchcomplex procedures may have relatively high cost and lead tosludge formation or secondary pollution. Biological treatments

* Corresponding author.

** Corresponding author at: Bio-refinery and Bioprocess Engineering Laboratory,

Department of Chemical, Engineering and Materials Science, Yuan Ze University,

135 Yuan-Tung Road, Chung-Li 32003, Taoyuan, Taiwan.Tel.: +886 3 4638800x3550;

fax: +886 3 4559373.

E-mail address: lanchiwei@saturn.yzu.edu.tw (J.-W. Lan).

1876-1070/$ – see front matter � 2013 Taiwan Institute of Chemical Engineers. Publis

http://dx.doi.org/10.1016/j.jtice.2013.01.015

were discovered as the satisfaction of spontaneous demand to offerthe complete clean-up of pollutants in a natural and economicallyfeasible manner [11]. For aerobic decolorization, bacteria must beacclimatized for a long period under aerobic chemostatic growth inthe presence of a simple azo compound. Subsequently, the bacteriasynthesize an azoreductase that specifically corresponds to theazo compound added in the presence of oxygen. In contrast,decolorization under anaerobic activity is relatively nonspecificwith respect to the included azo compounds. This indicates that ananaerobic process is more useful for the removal of mixed azo dyein wastewater [12].

It was reported that free cells degradation is inhibited at a highconcentration of dye [13]. The application of immobilization canprotect microbial cells from possible toxic effects caused bypollutant metabolites or changes in the environment conditions.The rapid development of this technology can be explained by itsinherent advantages over the use of free cells, such as improvedbiocatalysts stability. It allows continuous operation withoutwashout, separation of suspended biomass from the effluent, cellreuse for extended period of time, and protection of cells frominhibitory-products or contamination [14–16]. In a large numberof studies using immobilized cells on azo dye decolorization,entrapment was the most widely used technique for immobiliza-tion, and alginate was chosen as the suitable matrix material. Sucha mild method is characterized by preservation viability of cells toachieve all of the advantages of heterogeneous catalysis because ofits nontoxic characteristic [17,18]. However, the diffusion limita-tion can reduce the degradation efficiency because substrates must

hed by Elsevier B.V. All rights reserved.

T.P. Thao et al. / Journal of the Taiwan Institute of Chemical Engineers 44 (2013) 780–785 781

be transported across external boundary layers (external masstransfer) and within the matrix (internal mass transfer) [19–21]

In this study, Pseudomonas putida mt2 was applied as thebiological carrier to degrade methyl orange at both non-immobilized and immobilized Ca-alginate bead systems toinvestigate the decolorization properties and degradation limita-tion of this resistant dye. The optimality of each system wasstudied. Kinetics parameters and mass transfer coefficient werealso evaluated and discussed.

2. Materials and methods

2.1. Materials and cell cultivation

The dye, methyl orange (C14H14N3NaO3S), was obtained fromMerck Chemicals (Taiwan). Solutions were made by dissolvingrequisite quantity of the dye without further purification indistilled water. All other chemicals used in this study werepurchased from Sigma (USA).

One colony of P. putida mt2 on agar medium was cultivated in50 mL of medium 1 ((NH4)2HPO4 3 g/L, KH2PO4 1.2 g/L, NaCl 5 g/L,MgSO4.7H2O 0.2 g/L, yeast extract 0.5 g/L, glucose 3 g/L andmethyl orange 0.05 g/L) for cell growth, in which the carbonsource consisted of 3 g/L glucose concentration and 50 mg/Lmethyl orange concentration. Biomass was harvested at late-exponential phase and subsequently transferred to 100 mL ofmedium 2 for biodegradation study. The medium 2 consisted theidentical composition to medium 1, except that the dyeconcentration was doubled and the glucose concentration wasconsiderably decreased to 100 mg/L. The fresh medium 2 in the500 mL flask were flushed with filtered O2 or N2 before cells added.The suspended cell culture was sealed with parafilm on the top offlask to avoid penetration of air. Subsequently, the culture wasperformed under several of experimental conditions in anincubator at 200 rpm or without agitation (static condition).

2.2. Decolorization in non-immobilized system

The decolorization was operated under static and shakeconditions for comparison of aerobic and anaerobic biodegrada-tion. The static condition was chosen for the following experimentswith various conditions of pH, temperature, cell mass, and initialdye concentration. Samples were collected at regular intervals andanalyzed. Residual dye was determined through the absorbance offiltered supernatant at optical density of 465 nm, and biomass wasmeasured at 600 nm by Spectrophotometer [22].

2.3. Preparation of immobilized Ca-alginate cell beads and

decolorization by immobilized cell beads

The immobilization was performed according to the methoddescribed by Keweloh and Heipieper [23]. Biomass was harvestedfrom medium 1 at stationary phase and subsequently re-suspendedin 20 mL of 2% (w/v) sodium alginate. The final mixture was droppedinto 200 mL CaCl2 2% (w/v) solution by gravity through syringe andneedle. The drops of alginate solution were gelled to form 3 mmdiameter spheres upon contact with CaCl2 solution. The immobi-lized P. putida mt2 beads were stored in 2% CaCl2 solution at 30 8C for1 h to 2 h complete gel formation.

The beads with immobilized cells after 2 h storage in CaCl2

were washed by deionized water and adapted to 50 mL freshmedium 1 at 30 8C and shaken at 100 rpm for 3 h. Subsequently,beads were rewashed by deionized water and transferred tomedium 2 with limited concentration of glucose to obtain dyeremoval. Experiments were operated under various conditions,including dissolved oxygen, pH, temperature, cell mass and initial

dye concentration. The determination of residual dye is describedin Section 2.2.

2.4. Model description

2.4.1. Diffusion effects on nonporous support material

It was assumed that all cells are equally active, substratediffuses through a thin liquid film surrounding the support surfaceto reach the reactive surface, and the intrinsic kinetic parametersn0max and km are unaltered.

At steady state, the reaction rate is equal to the masstransfer rate (Js; based on Fick’s law) and can be defined usingEq. (1):

Js ¼ kLð½Sb� � ½Ss�Þ ¼ v0max½Ss�

km þ ½Ss�(1)

where kL is the liquid mass transfer coefficient (cm/s), Sb is thesubstrate concentration in bulk liquid, Ss is the substrateconcentration at surface, n

0max is the maximal reaction rate per

unit of external surface area (g/(cm2 s)), and km is the MichaelisMenten constant. Because the value of [Ss] is not amenable to directexperimental observation, (1) can be solved graphically accordingto the method reported by Shuler et al. [24].

2.4.2. Diffusion effects in a porous matrix

It was assumed that the porous structure is isothermal andspherical in shape, and microorganisms are uniformly distributedin the particle. In addition, the substrate between the exterior andinterior of the support was not partitioned. Therefore, Eq. (2) can bewritten in the basic of Fick’s law stating that diffusion rate is equalto reaction rate at steady state.

d2C

dr2þ 2

r

dC

dr¼

r p

Den (2)

where C is substrate concentration within the immobilizedparticles (mg/dm3); r is the radical position within the bead; rp

is the density of dried microorganism (g/dm3); De is effectivediffusion coefficient of dye within the bead (cm2/s) and n is theactual biodegradation rate.

The biodegradation kinetics can be expressed by the first orderkinetics if low concentration of dye substrate was introduced, andbiodegradation rate (n) can be defined in Eq. (3)

v ¼ k � C (3)

where k is the first order biodegradation rate constant (dm�3

solution g�1 dried cell weight h�1) and C is substrate concentrationwithin the immobilized particles (mg/dm3). To solve Eq. (2) withboundary conditions which C = Cs at r = R and dC/dr = 0 at r = 0.Consequently, Eq. (4) can be yielded:

C

CS¼ sinhð3’ � r=RÞðr=RÞsinh3’

(4)

where R is the radius of the bead (cm), Cs is the substrateconcentration at the bead surface and w is the Thiele modulus for aspherical particle which can be obtained by the following Eq. (5).

’ ¼ R

3

ffiffiffiffiffiffik0

De

s(5)

where k’ is equaled to kr � 3:6, and r is density of bead.

The effectiveness factor, h, was defined as the ratio of the actualreaction rate to the rate evaluated at outer surface conditions (ns)(see Eq. (6)).

n ¼ h � ns (6)

pH

4 5 6 7 8 9 10

spec

ific

deco

loriz

atio

n ra

te(m

g g

cell-1

h-1

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4C

onversion (%)

0

20

40

60

80

100

120

specific degrada tion ra teconver sio n

Fig. 2. Effect of pH on the specific decolorization rate and conversion of methyl

orange by non-immobilized Pseudomonas putida mt2.

T.P. Thao et al. / Journal of the Taiwan Institute of Chemical Engineers 44 (2013) 780–785782

If the actual rate is assumed to equal to the diffusion rate ofsubstrate into the immobilized particle at R, and can be written inEq. (7).

n ¼ 4pR2De �dC

dr

� �r¼R

(7)

After the necessary regulations, the effectiveness factor wasderived by Eq. (8) as the function of w

h ¼ 1

’1

tanh3’� 1

3’

� �(8)

3. Results and discussion

3.1. Suspension cell system

3.1.1. Oxygen effects

Aerobic and anaerobic decolorizations were compared incapability of free cells degradation with static and shakenincubation. The experiments were performed at 30 8C and naturalpH with the initial biomass of 1.3 g/L for 3 conditions. Although thebiomass was cultivated under aerobic condition, decolorizationwas more favorable under anaerobic condition. The results areshown in Fig. 1. No dye degradation was observed under shakencondition, whereas 100% dye was removed in static condition and80% in anaerobic shaking after 3 days incubation. In accordancewith these results, several previous studies demonstrated thatoxygen had an inhibitory effect on azo reductase reaction. Thisphenomenon may be attributed to the predominant competitionfor NADH utilization by aerobic respiration, because NADH acts asthe electron donor for the azo bond cleavage caused bybiodegradation [25].

However, Ben et al. [22] demonstrated that acid orange 52 wasbiologically degraded by P. putida mt2 at rich culture in both staticand 100 rpm shaking incubation after 10 and 52 h, respectively. Incontrast to our case, cell mass did not increase during degradation(data not shown), and in that rich medium, P. putida mt2 grewunder static condition to reach the 3.4 g/L from initial concentra-tion of 1.45 g/L. Growth of biomass may be one of reasons forimproved decolorization performance. Moreover, the higherconcentration of glucose in medium can be the catalytic agentfor dye biodegradation, as demonstrated in related literature. Itdepicted that the presence of glucose as carbon source is theinduced factor to boost enzyme reactions [11,14].

Time (h)

0 20 40 60 80

Res

idua

l dye

(mg

L-1)

0

20

40

60

80

100

120

staticanaerobic sh akingaerobic shaking

Fig. 1. Effect of static and shaken conditions at 200 rpm on decolorization of

100 mg/L methyl orange by free Pseudomonas putida mt2.

3.1.2. Optimal pH

Specific degradation rate and conversion curves were estab-lished to determine the biologically feasible and optimal pH fordegradation. In this experiment, the initial dye concentration wasfixed at 100 mg/L in the second state with various pH values in arange from 5 to 9. Fig. 2 shows that the favorable pH conditions formethyl orange decolorization are from 6 to 9, and the optimal pH isapproximately 7. The decolorization may not be suitable in acidicmedium (i.e., pH = 5) when the specific degradation rate andconversion decrease to less than 0.4 mg/g cell h�1 and 30%,respectively. This is consistent with Hsueh’s conclusion [25] thatmethyl orange was almost completely decolorized at pH 6–10, butthe decolorization efficiency was less than 10% at pH 5. It wasassumed that the alteration in chemical structure of methyl orangeto protonated azo dye formation (i.e., –N55N– ! [–NH–N55]+) atlow pH may lead to the inhibition in decolorization at acidiccondition [26].

3.1.3. Optimal temperature

The variation effects of temperatures on decolorization in Fig. 3demonstrate that an optimal temperature range is narrow becausethe high degradation rate and conversion were obtained at 33 8Cand 35 8C. The conversions reached approximately 100% in theoptimal range, but decreased to 60% at 37 8C and low temperaturerange of 25 8C–30 8C. A similar optimal range was achieved withanother type of bacteria, Lactobacillus casie TISTR 1500, in methylorange degradation [12], or with other types of Pseudomonas, suchas GM3 or luteola in other types of azo dye degradation [1,27].

3.2. Immobilized cells system

3.2.1. Oxygen effects

As shown in non-immobilized system, the anaerobic decolori-zation of dye was more efficient than the aerobic condition. Thisdid not confirm that an identical result would be obtained withimmobilized system because the entrapment of immobilized cellinside the beads may limit the interaction between cells andoxygen, leading to a reduction of the aerobic respiration. Thisproposal was tested with static and 100 rpm shaking condition forimmobilized cells.

The results demonstrated that dye was completely degradedafter 50 h in static batch, but only 13% of dye was removedcontemporaneously in shaking batch operation (refer to Fig. 4). Incomparison with non-immobilized cell, the slops of dye concen-tration removal did not change considerably at the initial and finalstage of degradation process. This may have occurred because the

Tempe rature (oC)25 30 33 35 37

Spe

cific

deg

rada

tion

rate

(m

g g

cell-1

h-1

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Conversion (%

)

0

20

40

60

80

100

120

specific deg rada tion rateequilibriu m con versio n

Fig. 3. Effect of temperature on the specific decolorization rate of methyl orange by

non-immobilized Pseudomonas putida mt2. Time (h)

0 20 40 60 80

Res

idua

l dye

(mg

L-1)

0

20

40

60

80

100

120

pH 5pH 6pH 7pH 8pH 9

Fig. 5. Time courses of residual concentration of methyl orange using immobilized

ca-alginate Pseudomonas putida mt2 in different pH conditions.

T.P. Thao et al. / Journal of the Taiwan Institute of Chemical Engineers 44 (2013) 780–785 783

immobilization protected cells from the disadvantages of medium,such as high level of dye or toxic products as well as from theoxygen impact upon enzyme activity [28]. It has been indicatedthat the transformed intermediates of azo dyes are highly toxic[28]. Based on these results, the anaerobic condition was applied infurther studies.

3.2.2. Optimal pH condition

The decolorization processes of immobilized P. putida mt2 wereessentially controlled by various pH values of medium. Theresidual dye was studied after certain degradation time at variouspH conditions ranging from 5.0 to 9.0 (see Fig. 5). The optimal pHfor bead-immobilized cells slightly shifted towards more basicthan for non-immobilized cells (pH 7) when the maximaldecolorization for methyl orange was observed at pH of 7.0, 8.0,and 9.0.

3.2.3. Optimal temperature

The evaluation of optimal temperature for decolorization ofmethyl orange in the immobilized cell system was depicted inFig. 6. Over the range of 27–37 8C, the degradation time decreasedwith increasing of temperature. The optimal range for immobilizedcells was 35–37 8C when the dye was completely removed after40 h. The completion of degradation was obtained several hourslater than with the optimal range at lower temperatures.

Time (h )

0 20 40 60 80

Res

idua

l dye

(mg

L-1)

0

20

40

60

80

100

120

staticshake

Fig. 4. Effect of static and shaken conditions at 200 rpm on decolorization of

100 mg/L methyl orange by immobilized Pseudomonas putida mt2.

3.3. Kinetics study of dye decolorization

Degradations with various initial dye concentrations wereperformed at optimal pH and temperature for both systems toestablish the kinetics studies. Lineweaver–Burk plots (Eq. (9)) wereapplied to establish the kinetics parameters for decolorization.

1

n¼ km

nmax½S�þ 1

nmax(9)

The predicted values of km and nmax were calculated from linearequations of reciprocal plots (Fig. 7) and summarized in Table 1.The entrapment P. putida mt2 in Ca-alginate exhibited the lowermaximal specific degradation rate (6.3 mg/(g h)) when comparedwith non-immobilized cells (7.5 mg/(g h)). The km values were 283and 257 g/L, and these figures can be considered approximateresults for both systems. From other research, Ca-alginate mayreduce the contact of dye and cells by the exclusion effect and theobstruction effect, which leads to smaller specific degradation ratethan that of free cells [20,27]. The smaller result in maximalspecific decolorization of immobilized cells can be related andexplained by diffusion limitation in a different operated system. Inthis case, the small distinction was attributed to the decompositionof immobilized beads when ion exchange occurred between Ca-alginate and monovalent ions of medium. However, Radha et al.

Time (h)

0 10 20 30 40 50 60

Res

idua

l dye

(mg

L-1)

0

20

40

60

80

100

120

27 oC30 oC33 oC35 oC37 oC

Fig. 6. Time courses of residual concentration of methyl orange using immobilized

ca-alginate Pseudomonas putida mt2 in different temperature conditions.

1/V

(mg-1

g h

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Rsqr = 0.9899

a

1/Ss (mg-1L)

0.00 0.01 0.02 0.03 0.04 0.05

1/V

(mg-1

g h

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Rsqr = 0.9606

b

Fig. 7. Typical reciprocal plot of specific degradation rate vs. dye concentration for

non-immobilized cells (a) and immobilized cells (b).

Ss (m ol L-1)

0.000 0.00 1 0.0 02 0.00 3

J or

V' x

1010

(mol

s-1

cm

-2)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

B1

B2B3

A

Fig. 8. Graphical solution for diffusion coefficient of liquid film: curve A obtained

from v0max½Ss�=km þ ½Ss� and B curves derived from calculation of kL ([Sb] – [Ss]).

T.P. Thao et al. / Journal of the Taiwan Institute of Chemical Engineers 44 (2013) 780–785784

[11] also revealed the various values of km and nmax for 7 types ofdye, including acid orange, vat magenta, and methylene blue oracid red 114. The maximal degradation rates for immobilized Ca-alginate beads were slightly lower than suspended cells, and notconsiderably lower, such as in reactive red 22 [27].

3.4. Investigation of mass transfer behavior in decolorization

operation

3.4.1. Mass transfer and liquid diffusion coefficient in suspension cell

system

In contrast to chemical degradation of phenol at aerobiccondition, dye decolorization with static condition includesdiffusion limitation, even in non-immobilized cell systems. Ifaerobic-shaking can provide substantial interaction between celland substrate, it can be considered without diffusion limitation.The static operation in this study involved liquid film bounding celllayer when all cells settled at the bottom of the flask. It wasassumed that the cell layer covered the square bottom of the flask,cells were equally active, and intrinsic kinetic parameters nmax andkm were unaltered.

Table 1Comparison of kinetic characteristics for degradation of methyl orange.

System Maximum specific

degradation rate

(nm ; mg g�1 h�1)

Michaelis–Menten

constant (g/L)

Suspension cell system 7.5 283

Immobilized cell system 6.3 257

The results were illustrated in Fig. 8. Curve A resulted from theright side of Eq. (1), and 3 lines, B, are the mass transfer equation atdifferent dye concentration (derived from left side of Eq. (1)). Theslope of line B is the diffusion coefficient across the liquid filmtabulated in Table 2. The diffusion coefficient (kL average = 7.53 �10�6 cm/s) is the average value of the 3 slopes.

3.4.2. Mass transfer and effective diffusivity of methyl orange within

the porous matrix

This study assumed that the external mass transfer for thebeads equaled the external mass transfer for non-immobilized cell.It was due to both systems were in static condition, which thisassumption is considerable. The effectiveness factor was calculat-ed by Eq. (6), in which ns is the degradation rate (after first 24 h)with internal diffusion limitation (rate in Ca-alginate beadssystem), and n is the degradation rate (after first 24 h) undernon-immobilized system. Table 3 presents effectiveness factorwith 100 mg/L of initial dye concentration, pH at 7.0, 35 8C, and1.8 g/L biomass. From Eq. (8), Thiele modulus w with a value of 0.44was found by using Matlab software.

It was assumed that the biodegradation of methyl orange dyeby P. putida mt2 follows first order reaction kinetics at lowconcentration of dye. The first order rate constant k (dm3 solutiong�1 biomass h�1) was subsequently obtained from the slope oflinear equation of biodegradation rate, and substrate concentra-tion. The k’ was transferred from k base on the density of Ca-alginate bead (r = 0.9235).

The internal diffusion coefficient inside the bead is listed inTable 4. The pores in the porous particles are not straight andcylindrical, but are a series of tortuous, interconnecting paths ofvarying cross-sectional areas. The effective diffusion coefficientwas determined by the molecular diffusivity and the pore structureof the immobilized particle, and considered less dependent fromthe particle size than from matrix structure [29–31]. In this study,the integrity of immobilized bead was not controlled (because ofsuch high monovalent ion in nutrient medium) [14], which

Table 2Summary of mass transfer in suspension cell system.

Line n’ (mol/(s cm)) So (mol/cm3) Slope = kL (cm/s)

B1 0.1368 � 10�10 2.0960 � 10�6 0.653 � 10�5

B2 0.0364 � 10�10 4.6030 � 10�7 0.79 � 10�5

B3 0.0238 � 10�10 2.9210 � 10�7 0.815 � 10�5

Table 3The effectiveness factor with particle size of 0.3 cm.

dp (cm) n (mg/(g h)) h

Non-immobilized cell 1.343 1

0.3 1.204 0.897

Table 4Calculated values of Thiele modulus, first order rate constant and diffusion

coefficient.

w k’ (cm3/(cm3 s)) De (cm2/s)

0.44 0.0048 6.2 � 10�5

T.P. Thao et al. / Journal of the Taiwan Institute of Chemical Engineers 44 (2013) 780–785 785

resulted in the decrement of matrix linking, indicating that thediffusion coefficient was higher than that in indecomposable case.From the observation, a small number of beads were cracked after10 h of degradation. The phenol diffusion coefficient in Ca-alginatebeads (9.67 � 10�6 cm2/s) [19] with similar properties of Ca-alginate particles for dye diffusion was higher (6.2 � 10�5 cm2/s) inthis study. This result is limited because of the instability of thebeads occurred in the present study, but the process can bevisualized to some extent. In addition, the difference in molecularweights of dye and phenol might be one of reason. The methylorange diffusion coefficient in Ca-alginate was larger than that inwater (diffusion coefficient of methyl orange in water was1.4 � 10�5 cm2/s) [32]. This may occur because the biopolymerreduces the available volume for dye to move in; and the pathlength for movement of dye increases because of the impenetrablepart of polymer [20].

4. Conclusions

The degradation of methyl orange by P. putida mt2 under staticand shake conditions was studied in both suspension andimmobilized cell systems. The trends of dye degradation in bothconditions were similar when dye disappeared under anaerobicand static conditions, and no dye removal occurred under aerobiccondition. Anaerobic condition was used for further studies. Theoptimality of decolorization of methyl orange by P. putida MT2occurred at pH 7 and temperatures of 33–35 8C for anoxicsuspended biodegradation. However, the optimal temperaturefor Ca-alginate immobilized cells was 35–37 8C. Optimal pH valuesslightly shifted towards more basic for immobilized Ca-alginatesystem. The trend of declination slops differed for immobilized andnon-immobilized systems. Dye removal decreased considerablyafter the first period of suspended degradation, and more slowly athalf end of decolorization process, whereas steady decrement wasachieved with immobilized system. From the degradation ratekinetics of P. putida mt2, the maximal decolorization ratespredicted for non-immobilized and Ca-alginate immobilized beadswere up to 7.5 and 6.3 mg g cell�1 h�1 with saturated constantvalues of 283 ppm and 257 ppm, respectively. Diffusion coeffi-cients in these two systems were 0.75 � 10�5 cm2/s and6.2 � 10�5 cm2/s, respectively. The stability of beads is a crucialfactor; therefore, Ca-alginate was an unfavorable carrier in thiscase. A suggestion is to reduce the monovalent ions in the mediumor to replace it with another material for superior performance.

Acknowledgements

This work is supported by National Science Council (NSC99-2221-E-155-018-MY2) and special appreciation to Dr. Yuan fromVeolia water cooperation (Taiwan).

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