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Biochemical Engineering Journal 97 (2015) 40–49

Contents lists available at ScienceDirect

Biochemical Engineering Journal

jo ur nal home p age: www.elsev ier .com/ locate / bej

haracteristics of a novel low density cell-immobilized magneticupports in liquid magnetically stabilized beds

akaria Al-Qodah a,∗,1, Mohammad Al-Shannag b, Eman Assirey c, Wasim Orfali d,halid Bani-Melhem e, Kholoud Alananbeh f, Nahla Bouqellah f

Chemical Engineering Department, Taibah University, Saudi ArabiaChemical Engineering Department, Faculty of Engineering and Technology, The University of Jordan, 11942 Amman, JordanChemistry Department, Taibah University, Saudi ArabiaCivil Engineering Department, Taibah University, Saudi ArabiaHashemite University, Faculty of Natural Resources and Environment, Department of Water Management and Environment, Al-Zarqa, JordanDepartment of Biology, Taibah University, Saudi Arabia

r t i c l e i n f o

rticle history:eceived 16 September 2014eceived in revised form 13 January 2015ccepted 21 January 2015vailable online 2 February 2015

eywords:

a b s t r a c t

The adsorptive and hydrodynamic characteristics of low density non-porous magnetic supports used forbiocatalyst immobilization have been investigated. The magnetic particles consist of a sand core havebeen covered with a magnetite layer followed by a layer of nano size activated carbon or fly ash withthe aid of epoxy resin. The resulted particles showed good adsorbing behavior toward resting cells ofEscherichia coli from batch culture of different cell concentrations. The maximum adsorption capacity forboth particles were calculated using Langmuir isotherm model as 18 (1.22 million cells) and 14 (0.95

agnetic supportsell immobilizationpoxy resinagnetic stabilized beds

million cells) mg/g, respectively. The hydrodynamic study in a liquid magnetically stabilized beds con-firms that these particles have lower fluidizing velocity and attain a higher expanded stabilized bed thanparticles of magnetic dense core. These results confirm the applicability of these particles to immobilizemicroorganisms for different applications. It was found that the conversion using these particles was 30%higher than that using traditional dense magnetic particles due to the longer residence times in the bed.

© 2015 Elsevier B.V. All rights reserved.

. Introduction

Cell Immobilization has generated considerable interest in theeld of bioreactor design and lead to the design of new contac-

ors to accommodate the resulting relatively large and sphericalmmobilized biocatalyst particles. Among these new contactors,uidized beds and packed beds are being considered as promisingeactors suitable for carrying out bioprocesses with immobilizediocatalysts [1,2]. Subsequently, magnetically stabilized fluidizededs (MSFBs) have been applied to conduct bioprocesses with

mmobilized biocatalyst on magnetic particles as a solid phase,

hereby presenting further options in continuous reactor systems3–5]. This technology is expected to show potential applications in

∗ Corresponding author. Present address: Chemical Engineering Department,aibah University, P. O. Box 344, Madinah, Saudi Arabia. Tel.: +966 48475837.

E-mail addresses: z alqodah@hotmail.com, zqudah@taibahu.edu.saZ. Al-Qodah).

1 Permanent address: Chemical Engineering Department, Al-Balqa Appliedniversity, P. O. Box 340558, Marka, Amman 11134, Jordan.

ttp://dx.doi.org/10.1016/j.bej.2015.01.004369-703X/© 2015 Elsevier B.V. All rights reserved.

various areas such as bioseparations and immobilized biocatalystsystems [6–12].

Accordingly, many types magnetic carriers or supports forimmobilized biocatalysts have been previously prepared and tested[13,14]. Non-porous magnetic supports as carrier for immobiliza-tion have many advantages as they seem to be more resistant todiffusional limitations, attrition, and fouling than porous supports[15,16]. In addition, these carriers usually have reasonable chemicalinertness.

A variety of non-porous magnetic materials have been used,including iron, cobalt and their oxides methods of their preparationhave been classified into four groups: direct use of a coupling agent,adsorption, encapsulation, and formation of a thin film of polymer.Goetz et al. [17,18] developed a versatile magnetic silica supportwhich can be derivatized readily for both adsorption chromatogra-phy and enzyme immobilization by well-known techniques. Thissupport was prepared by electrostatically depositing alternating

layers of colloidal silica and cationic polymer onto macroscopicnickel core particles. The polymer is then burned out and the silicapartially sintered to yield a porous shell with 5–80 m2/g of surfacearea. Horak et al. [19] presented a comprehensive review for all

Z. Al-Qodah et al. / Biochemical Engine

Nomenclature

ACMC Activated carbon magnetic coreACSC Activated carbon sand coreFAMC Fly ash magnetic coreBs Saturation flux density (mT)C Aqueous cell concentration in equilibrium (mg/l)CC Cell countdp Particle diameter (mm)Hb Bed height after applying the magnetic field (cm)Hbo The initial bed (cm)MSC Sand particles covered with magnetiteMSFBs Magnetically stabilized fluidized bedsOD Optical density (–)Qe Adsorbed cell density (mg/g)Qm Adsorption capacity (mg/g)Kd Dissociation constant (mg/l)Umf Minimum fluidization velocity (m/s)Ue Expansion velocity (m/s)SC Sand core�b Bed porosity (–)�so The initial porosity of the magnetic particles (–)

3

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2

ba

� Bed density(kg/m )�s Particle density (kg/m3)

ublished methods used in the preparation of magnetic nanoparti-les and microspheres used as separation media in different fieldsf chemistry, biochemistry, biology, and environment protection.owever, most of the previous methods led to the production of

elatively high density magnetic particles since the core of the parti-les is usually made of dense magnetic material such as iron, cobalt,r magnetite. The values of the minimum fluidization velocity inSFBs could vary from 3 to 6 times that in the absence of magnetic

eld depending on the particles properties and on the magneticeld intensity [20]. However, these values of high medium veloc-

ties are not applicable in bioreactors for two main reasons. First,io reaction rates are generally low and in continuous processeshe substrate should have enough residence time in the reactoro attain sufficient conversion. Second, liquid recirculation is notossible in processes accompanied by product inhibition. Further-ore, the use of high density magnetic support at high magnetic

elds could lead to a state of aggregated frozen bed rather than sta-ilized fluid-like bed. For these reasons, a possible way to overtakehe above limitations of using MSFBs as a bioreactor is to reduce the

agnetic particles densities. Several reports indicated that MSFBsf low density magnetic particles can be operated at acceptable

iquid flow rates with the aid of the stabilization action of the mag-etic field [21,22]. However, most of these low density carriers are

ragile and unable to be repeatedly cleaned with harsh reagents oro withstand sterilization conditions for repeated use [23]. For thiseason, the main objective of the present work was to conduct aew, simple method for the preparation of non-porous low densityagnetic supports for biocatalyst immobilization. The applicabil-

ty of the prepared magnetic particles with different types and sizesas tested as a support for cell immobilization using resting cells

f Escherichia coli. Indeed, the study assessed the hydrodynamicsnd catalytic properties of these particles in liquid magneticallytabilized bed.

. Materials and methods

Sea sand of different sizes was obtained from the red sea, Yanboaeach. Magnetite (Fe3O4) of particles diameter (dp) less 5 �m andctivated carbon of nano size were obtained from Sigma–Aldrich,

ering Journal 97 (2015) 40–49 41

Germany. Acetophenone was obtained from Fulka, Chemie GmbH(Buchs, Switzerland), (S)-1-phenylethanol from Sigma–Aldrich,(Taufkirchen Germany) and 2-propanol from Carl Roth GmbH (Karl-sruhe, Germany). �-NADH was obtained by Jülich Fine Chemicals(Jülich, Germany) and used as delivered. All other chemicals andreagents with high purity: buffer, salts and solvents were obtainedfrom commercial sources. HPLC deionized water was used to pre-pare the required solutions.

Fly ash of nano-size was obtained from the activated sludgecombustion pilot plant of Prof. Dr.-Ing. J. Werther (Institute of SolidsProcess Engineering & Particle Technology, Hamburg University ofTechnology, Hamburg–Harburg, Germany). Hot curing epoxy sys-tem, based on Araldite LY 564 which is a low-viscosity epoxy resinand the hardener Aradur 2954 which is a cycloaliphatic polyamineneeded to prepare the epoxy resin were provided by Prof. Dr.-Ing.K. Schulte (Institute of Polymer Composites, Hamburg University ofTechnology, Hamburg–Harburg, Germany). Lyophilized E. coli tuner(DE3) cells containing alcohol dehdrogenase ADH-‘A’ were used forimmobilization on the magnetic support. These cells were kindlyprovided by Prof. Wolfgang Kroutil, Institute of Chemistry, Uni-versity of Graz (Graz, Austria) [24]. The cells were resting and notable to grow which facilitates an accurate measurement of enzymeactivity during the adsorption process.

2.1. Preparation of magnetic supports

Sea sand was cleaned from impurities by rinsing with 0.1 M HClsolution followed by washing by distilled water and then by 0.1 MNaOH and finally washed with abundant amount of distilled water.The clean sand particles were left to dry at 105 ◦C for overnightthen cooled, sieved into different fractions and kept in closed plasticbags. Araldite LY 564 (5 ml) and the hardener Aradur 2954 (2.1 ml)were mixed in a suitable glass dish under the fume hood for 5 minto ensure homogeneity. The mixture started to polymerize causinga modification of the chain structure to a cyclic solid of a high melt-ing point. After this period, the viscosity of the mixture started toincrease and the homogenous mixture of the epoxy resin which wasformed by condensation was mixed with 10 g of 500 �m diametersand particles. After 5 min of intensive manual mixing with suit-able glass rod, the epoxy resin was equally distributed as a thinfilm which was coating the particles surface and starts to formstrong bonds with it. At that point, 20 g of magnetite powder wasadded and thoroughly mixed until the magnetite powder coveredthe sand particles which disintegrated into individuals and left for16 h under the hood to complete the solidification process. In thenext day, the particles were sieved to remove the excess magnetite,washed several times with distilled water and dried at 105 ◦C for3 h and cooled. These particles were then mixed again, for 5 min inthe same procedure as before, with a mixture of 5 ml of Araldite LY564 and 2.1 ml of the hardener Aradur 2954. 20 g of activated car-bon powder or fly ash were added and thoroughly mixed until thecarbon covered the whole magnetite layer. The resulting coveredparticles were left for 48 h to complete the solidification process atroom temperature. Then, the cleaning procedure was repeated toget rid of the excess and unbounded activated carbon.

2.2. Characterization of magnetic supports

The magnetic particles were then sieved and subjected to sev-eral characterization experiments in order to measure the bulkdensity (�b), molded density (�s), bed porosity (�b), minimum flu-idization velocity (Umf) in the absence of magnetic field, and the

saturation flux density (Bs). After that, the magnetic particles werekept in glass bottles for further immobilization experiments. Scal-ing up of this production method will be the subject of furtherexperiments.

4 Engineering Journal 97 (2015) 40–49

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2 Z. Al-Qodah et al. / Biochemical

The morphology and surface structure of magnetic parti-les considered were observed qualitatively using the scanninglectron microscope (SEM) of the model XL-30W/TMP, manufac-ured by FEI Company. Briefly, sand particles were attached to anluminum stub using adhesive copper tapes. Each sand sample wasxed on a separate stub. Samples then were sputter coated withold, using a sputter coater of the model of SCD 005 manufacturedy BAL-TEC. The SEM Imaging was performed by inserting eachample into the scanning electron microscope. The high tensionsed was 15 kV, with a beam current of 56 micro amperes. Theseest conditions can be obtained from the data bar on each image. Aecondary electron detector used to produces five micrographs forach one of the samples, of the following order: 25×, 100×, 500×,000×, 5000×.

.3. Cell immobilization

Lyophilized E. coli cells (75 mg) were rehydrated in 243.145 mlhosphate buffer (50 mM, pH 7.5, 1 mM NADH) and 5 g of theagnetic particles were added at 30 ◦C in a screw cap bottlesith 130 rpm for 30 minutes. After this period, the residual free

r un-adsorbed cells in the suspension were separated from themmobilized cells particles by decantation which was subjected tohe activity assay. In addition, two experiments were conducted tonsure that the immobilized cells on the magnetic supports are stillctive. These experiments were conducted by replacing 5 g of themmobilized cells particles instead of the lyophilized E. coli cells inhe reaction medium. The decanted suspension (50 ml) was usedo rinse the solid particles in order to remove any loosely boundells to the magnetic particles. Then, this suspension was joinedo the first cell suspension to be used in the activity experiments.he solid particles were then washed twice with 50 ml of distilledater and the washed particles were tested for enzyme activity.

n adsorption isotherm experiments, different cell concentrationsere mixed with certain amount of the magnetic particles in 100 ml

ell solution in order to study the effect of cell concentration on theells adsorption capacity.

Langmuir adsorption isotherm expressed by Eq. (1) was used toodel the adsorption data shown in Fig. 7 [7]:

e = QmC

Kd + C(1)

here Qe (mg/g) and C (mg/l) are adsorbed cell density and aqueousell concentration at equilibrium, respectively. Qm is the adsorptionapacity (mg/g) and Kd is dissociation constant (mg/l).

To determine the values Qm and Kd for the particles a linear formf Langmuir model could be expressed as:

1Qe

= Kd

(QmC)+ 1Qm

(2)

y plotting 1/Qe versus 1/C, the values of Qm and Kd could be deter-ined. In this linear plot 1/Qm represents the slope and Kd/Qm

epresents the intercept.In addition, the effect of pH on the loading capacity of the mag-

etic particles was investigated by repeating similar steps at pHalues of 5, 9, and 11 at 30 ◦C and 100 mg/l initial cell concentra-ion. Ionic strength experiments were carried out using the same

edium at pH of 7.5 with NaCl concentrations ranging from 0.05o 0.5 M.

.4. Activity assay

The decanted or free cell suspension (48.629 ml) was added to0.8 ml of 2-propanol (18% v/v) and 571 �L of 80 mM acetophe-one. The mixture was shaken at 30 ◦C and 130 rpm. Samples wereaken periodically for preparation and analysis. The activity assay

Fig. 1. Reduction of acetophenone to (S)-1-phenylethanol.

for the immobilized cells was performed by adding 1 g of the immo-bilized cells particles to 48.629 ml phosphate buffer (10 mM, pH7.5, 0.2 mM NADH) at 30 ◦C in screw cap bottles with 130 rpm for30 min. After that, 10.8 ml of 2-propanol (18% v/v) and 571 �L of80 mM acetophenone were added and the mixture was shakenat 30 ◦C and 130 rpm, then the concentrations of acetophenoneand (S)-1-phenylethanol were monitored with time by gas chro-matography (GC) analysis. From these results the cell loading onthe particles can be obtained. The enzyme catalyzed reduction ofacetophenone is described in Fig. 1.

2.5. Sample preparation

Each sample was extracted twice with 100 �L of ethyl acetate(20 s vortex, centrifugation for 1 min at 13,000 rpm). 80 �L of eachethyl acetate phase were transferred to another Eppendorf tube.The aqueous phase was extracted again with 100 �L of ethyl acetate(20 s vortex, centrifugation for 1 min at 13,000 rpm) and 80 �L ofethyl acetate phase were combined to the first ethyl acetate phase.The combined phases were analyzed via gas chromatography. Theconversion of acetophenone to phenyl ethanol by alcohol dehdro-genase ADH-‘A’ produced by E. coli was estimated by measuringthe concentration of residual acetophenone and the produced (S)-1-phenylethanol at different times after mixing.

2.6. Analytics

The concentrations of the acetophenone/(S)-1-phenylethanolwere determined by gas chromatography: HP 6890 GC, FS-Cyclodexß-I/P column (50 m × 0.32 mm), carrier gas: hydrogen. Method: FIDtemperature 250 ◦C, hydrogen 40 ml/min, make-up (He) 45 ml/min,air 350 ml/min, split temperature 220 ◦C, split ratio 30.0/1, splitflow 90.0 ml/min, primary column pressure 1.118 bar. The temper-ature was kept at 140 ◦C for 14 min.

Libra S12 spectroimagemeter (Biochrom Ltd., Cambridge UK)was used to measure the optical density (OD) of the cell suspen-sion before and after immobilization with the magnetic particles.A microscope (Axioskop, Zeiss, Germany) with a Color Video Cam-era TK-C1381 (JVC, Japan) was used to measure the cell count (CC).The calibration curves of optical density and cell count against cellconcentration are shown in Fig. 2. As shown in the figure, the rela-tionships between both the cell count and the optical density withcell concentration are linear with squared correlation coefficient(R2) closed to unity.

2.7. Hydrodynamics of magnetically stabilized bed using lowdensity magnetic particles

The purpose of conducting the hydrodynamic experiments wasto test the performance of the prepared magnetic particles underthe effect of magnetic field and liquid velocity, and to compare their

Z. Al-Qodah et al. / Biochemical Engineering Journal 97 (2015) 40–49 43

C (mg/l)0 50 100 150 200 250

OD

(-)

0

2

4

6

8

10

12

BlankACSC magnetic particlesACMC magnetic particlesFASC magneti c particles

FE

pd4mocti[r

mmwfflmifi

2

nitmtsstv

Fig. 3. (a) Schematic of the experimental apparatus: (1) column; (2) supportinggrid; (3) magnetic particles; (4) power supply; (5) feed tank and stirrer; (6) efflu-

TS

ig. 2. Calibration curves between optical density and cell count for suspensions of. coli against cell concentration (g/l).

erformance with magnetic particles of similar sizes but of higherensities. A transparent plexiglas column with inner diameter of5 mm and height of 0.75 m was used to perform this task. Theagnetic system was made of a cast steel corewith painted sheets

f 9 mm thickness. A copper coil was consisted of 1500 turns of aopper wire of 9 mm diameter. A image and a schematic diagram ofhe reactor setup are shown in Fig. 3(a) and (b); more details aboutts operation can be found in the study of Al-Qodah and Al-Shannag8]. Bed porosity was determined by measuring the volume of waterequired to fill the voidage of the bed using a graduated cylinder.

In this part of the study, experiments were performed in theode ‘magnetizing first’ [4,20]. Magnetizing first means that theagnetic field was set at the desired value and the distilled wateras fed into the column in a suitable flow rate to transfer the bed

rom the packed to the expanded state. Subsequently, the waterow rate was gradually reduced until eventually was turned off. Theinimum expansion velocity (Ue), the minimum fluidization veloc-

ty (Umf) and bed porosity (�b) were determined for each magneticeld value.

.8. Acetophenone conversion in magnetically stabilized bed

In this part, experiments were also performed in the mode ‘mag-etizing first. The bed was filled by the magnetic particles after

mmobilization. The initial bed height in all runs was 5 cm. Thenhe magnetic field intensity was set at the desired value, and the

edium containing acetophenone was fed into the column in ordero transfer the bed from the packed to the expanded state. Sub-

equently, the medium flow rate was gradually reduced to theuitable flow rate to achieve the optimum conversion. The run con-inued until the exit substrate concentration reached a steady statealue.

ent stream, (7) pump; (8) effluent receiver; (9) magnetic system; (b) image of theexperimental apparatus: (1) DC power supply; (2) water influent pump; (3) feedtank; (4) effluent tank; (5) magnetic system; (6) copper solenoid; (7) column; (8)magnetic particles; (9) column support; (10) level control tank.

able 1ome characteristics of different magnetic particles.

Type of magnetic particles Class �b (kg/m3) �s (kg/m3) Shape factor (−) dp(mm) Umf (cm/s) �b (−) Bs (mT) Reference

ACMC Nonporous 1500 2692 0.8 0.45 0.91 0.443 590 [4]ACSC Nonporous 1260 2290 0.95 0.45 0.75 0.45 390 This studyFASC Nonporous 1190 2165 0.95 0.45 0.68 0.45 380 This studySteel particles Nonporous 4321 7450 Sphere – – 0.42 – [25]Covered magnetite Porous 1655 – – 0.9 – – [26]Uncovered magnetite Porous 2800 – – 1 – – [26]Covered with zeolite Nonporous 2100 3500 0.8 – 0.4 – [27]�-carrageanan + magnetite Porous 1059 – – 0.37–0.92 – – – [28]Polyacrylamide + 50% magnetite + 50% ferrite Porous 1450 1450 – 3.6 – – – [29]

44 Z. Al-Qodah et al. / Biochemical Engineering Journal 97 (2015) 40–49

Fig. 4. SEM images SC, MSC, ACSC and FASC particles: (a) 25× (b) 5000×.

Engineering Journal 97 (2015) 40–49 45

3

3

fiApcotoTTbsnansoTFvvt0iftaitnsttTntrcAFhaf

bpip1

3

dniu43jic

Time (min)0 20 40 60 80 100 120

%C

onve

rsio

n (-

)

0

5

10

15

20

25

30

ACMC magnetic particlesACSC magnetic particlesFASC magnetic particlesBlank

Fig. 5. Percent conversion of acetophenone by alcohol dehdrogenase ADH-‘A’ versustime for E. coli in blank suspension and that after mixing with ACMC, ACSC and FASCmagnetic particles. Cell concentration in all experiments was 0.3084 mg/ml.

C (mg/l)0 50 10 0 15 0 200 25 0

OD

(-)

0

2

4

6

8

10

12

BlankACSC magnetic particlesACMC magneti c particlesFASC magnetic particles

Fig. 6. Variation of optical density (with its error bar) with cell concentration for

Z. Al-Qodah et al. / Biochemical

. Results and discussion

.1. Characteristics of the magnetic particles

Three types of magnetic particles were used in this study. Therst one is activated carbon magnetic core particles (ACMC) used byl-Qodah [4]. The other two types of magnetic particles were pre-ared in this study. The first type named as activated carbon sandore magnetic particles (ACSC) consists of sand core, inner layerf magnetite and an outer layer of activated carbon. The secondype named as fly ash sand core magnetic particles (FASC) consistsf sand core, inner layer of magnetite and an outer layer of fly ash.hese particles are nonporous and were prepared in different sizes.hese nonporous low density magnetic particles are suitable foriofilm formation to be used as a catalyst in fluidized magneticallytabilized beds. Some physical properties of the new prepared mag-etic particles and other magnetic particles found in the literaturere given in Table 1. It could be seen from Table 1 that ACSC and FASConporous magnetic particles are of lower densities. The bulk den-ities of ACSC and FASC are about 85 and 80%, respectively, of thatf ACMC particles. All particles were rather spherical. As shown inable 1 the values of the minimum fluidization velocities of ACSC,ASC and ACMC are 0.75, 0.68 and 0.91 cm/s, respectively. Thesealues indicate a substantial decrease in the minimum fluidizationelocities in the non magnetic core particles compare to ACMC par-icles. The bed porosity of ACSC, FASC and ACMC were 0.45, 0.45 and.44, respectively. On the other hand, the saturation magnetic flux

ntensity, Bs, is different for the three particles: 590, 390 and 380 mTor ACMC particles, ACSC and FASC, respectively. This is attributedo the magnetite core in ACMC particles leading to higher densitynd magnetic saturation flux intensity and consequently the min-mum fluidization velocity. The saturation magnetization is equalo the product of the net magnetic moment for each atom and theumber of atoms present per unit volume. The reduction in den-ity and consequently the minimum fluidization velocity suggesthe operation with relatively low superficial velocity to transferhe bed into the stabilized flow regime as will be illustrated later.he morphology of the particles surface was studied from the scan-ing electron micrographs (SEM) as shown in Fig. 4. Fig. 1a showshe 100× magnified images for SC, MSC, ACSC and FASC particles,espectively. It could be seen that the added layers to the sandore are quite then and the surface roughness increases in MSC,CSC and FASC compared with MSC due to the attached layers. Inig. 4b the images are 5000× magnification. All particles seems toave corrugations in the surface but the most corrugated are ACSCnd FASC. This rough surface is advantageous in the case of biofilmormation.

The prepared particles are both nonporous and low densityecause they have a nonporous sand core. Nonporous magneticarticles with an outer biofilm are characterized by the absence of

nternal mass transfer resistance [28,30]. In addition, these non-orous magnetic are thermo-stable and withstand sterilization at20 ◦C.

.2. Adsorption isotherms

The purpose of conducting the isotherm experiments was toetermine the maximum possible cell loading capacity of the mag-etic particles and to fit the experimental data to the appropriate

sotherm models [31]. The sorption experiments were done at 30 ◦Csing ACSC, FASC and ACMC magnetic particles with diameters of50 �m. After mixing the cell suspensions with the particles for

0 min, the immobilized cells on the magnetic particles were sub-

ected to acetophenone solution in order to test the ability of themmobilized cells to convert acetophenone and the degree of thisonversion according to the cell loading on the particles. On the

three E. coli: blank or no contacting with magnetic particles and after contactingwith ACMC, ACSC and FASC magnetic particles.

other hand, the supernatant was used to wash the particles thenwas subjected to the assay test to measure the activity of the un-immobilized cells.

Fig. 5 shows the percent conversion of acetophenone by theimmobilized cells in the three magnetic particles ACSC, FASC andACMC in addition to a blank cell suspension. It is evident from Fig. 5that the conversion of acetophenone increases with time. In addi-tion, Fig. 5 shows that there is no significant difference betweenthe quantities of cells attached to ACSC and ACMC, whereas FASCparticles adsorbed less E. coli cells. These results indicated that allthe three magnetic particles could be considered as good carriers forcell immobilization. In addition and as previously mentioned, theseexperiments insure that the immobilized cells on the three mag-netic supports were still active after immobilization. The resultsof these experiments confirmed that the magnetic particles haveno adverse effects on the cells activity. In addition, the percentconversion using the immobilized cells on ACSC, ACMC and FASCparticles was about 50, 50 and 32% of that in the blank (free cells),

respectively.

Fig. 6 shows the variation of optical density of the suspensionsbefore and after mixing with the magnetic particles, as a function

46 Z. Al-Qodah et al. / Biochemical Engineering Journal 97 (2015) 40–49

C (mg/l)0 50 100 150 20 0 250

Qe

(mg/

g)

0

3

6

9

12

15

1/C (l/mg)0.00 0.02 0.04 0.06 0.08

1/Q

e (g

/mg)

0.1

0.2

0.3

0.4

ACSC magnetic particlesACMC magnetic particlesFASC magnetic particles

Ft

oct

p1ttAplEt

vuutriIchifid1wt1aatcTi

3

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pH (- )

4 6 8 10 12

Qe (

mg/

g)

4

6

8

10

12

14

ACSC magnetic particlesACMC magneti c particlesFASC magneti c particles

ig. 7. Adsorption isotherm of E. coli cells on ACMC, ACSC and FASC magnetic par-icles at pH 7.5 and T = 30 ◦C.

f initial cell concentration. It is clear from Fig. 6 that the residualells in the supernatant solution were higher for FASC particles fromhose of ACSC and ACMC.

It is evident from Fig. 6 that the conversion in the blank sus-ension, ACSC, ACMC and FASC solutions reached 26, 12, 12 and7% within 100 min, respectively. This reveals that the cell concen-ration in the ACSC and FASC supernatants are 46, 46 and 65% ofhat in the blank suspension containing 0.3084 mg E. coli cells/ml.ssuming that the rest of cells are immobilized on the magneticarticles, accordingly, 54, 54 and 35% of the cells were immobi-

ized on the ACSC, ACMC and FASC, respectively. This implies that. coli cells are easier to be adsorbed on ACSC and ACMC particleshan on FASC particles.

Fig. 7 shows the variation of the adsorbed cells loading, Qe,ersus the cell concentration in the solution. It is evident in the fig-re that the cell loading increases as the cell concentration increasentil the cell concentration reaches 50 mg/l. In addition, the adsorp-ion capacity of the three carriers are comparable within this lowange of cell concentrations. Beyond this value, the cell loadingncreases in a slower rate until it reaches a relatively constant value.t could be seen from Fig. 7 that at equilibrium, where the parti-le become saturated, the ACSC and ACMC particles seem to haveigher adsorption capacity than that of FASC particles. These results

ndicated that the cells form a monolayer on magnetic particles sur-ace rather than multi layers. For this reason, Langmuir adsorptionsotherm expressed by Eq. (1) was used to model the adsorptionata shown in Fig. 7. In addition, Fig. 7 contains an insert by plotting/Qe versus 1/C to determine the values Qm and Kd for the particles,here in this plot 1/Qm represents the slope and Kd/Qm represents

he intercept. The values of Qm for ACSC, ACMC and FASC were5.2, 15.1 and 11.8 mg/g, where the values of Kd were 20.2, 20.3nd 16 mg/l, respectively. It could be seen in Fig. 7 that Q reachesbout 80% of Qm in the first 30 min when the cell initial concen-ration was 50 mg/l, then its increase becomes smaller as the celloncentration increases until it reaches a relatively constant value.his is a typical trend which characterizes the adsorption processes

n which Langmuir monolayer adsorption model is dominant [32].

.3. Effect of pH on the cell adsorption

To investigate the effect of pH on the adsorption of the cells on

he magnetic particles, the sorption experiments were done at 30 ◦Csing ACMC, ACSC and FASC particles of 250–300 �m diameters.he cell suspensions of 100 mg/l concentration were mixed withhe particles for 30 min at different pH values; the supernatant was

Fig. 8. Effect of pH on the adsorption capacity using the magnetic particles.

used to wash the particles and then it was subjected to the assaytest to measure the activity of the un-immobilized cells. The resultsare shown in Fig. 8. As shown in the figure, the adsorption capac-ity on three types (ACMC, ACSC and FASC) of magnetic supportswas maximum at pH of around 7.5. Below and above this pH value,the adsorption capacity decreases. Using ACMC and ACSC parti-cles, the adsorption capacity decreased from 12 to 8 and for FASC itdecreased from 10 to 7 as pH increases 7.5–11, respectively. Theseresults could be explained by the fact that pH affects the electro-static charges on the magnetic particle surfaces and consequentlythe cell loading. It seems that the iso-electric point of the supportsoccurs at pH of around 7.5 and the cell adsorption favors this pHvalue where the net charge on the surface is zero. These resultsagree with those of Wang et al. [33] who investigate the specificadhesion of E. coli with surface-exposed cellulose-binding domainto cellulosic materials.

The effect of ionic strength was also investigated using NaClsolutions of different concentrations at pH 7.5, temperature of30 ◦C, initial cell concentration of 100 mg/l using both ACSC andFASC of 450 �m diameter. It was noted that a slight decrease of2% in cell loading occurs at the highest 0.5 M NaCl. Other authors[34] reported an increase of cell loading with increasing ionicstrength and others [35] reported no net apparent effect of ionicstrength on the cell adsorption. However, in viable cell immobiliza-tion, it is expected that under the experimental conditions, a highionic strength might destabilize immobilized cells and preventsthe interaction between cells and supports surfaces, and therebyreduces the cell adhesion [36].

Catalyst regeneration is very important in process economyespecially if the catalyst is expensive and not easy to be prepared.Ease regeneration usually reduces the catalyst cost and extend itsperiod of operation. Regeneration by washing and sterilization ofthe magnetic supports was tested to examine their stability andability for repeated use [7]. The exhausted particles were regener-ated by a treatment with 1.0 M NaOH solution for 4 h then washedby distilled water. After that, the particles were rinsed by 0.1 M HClsolution followed by frequent distilled water washes. The value ofQm of the ACSC regenerated particles was 14.8 mg/g, which is verysimilar to the original particles. Moreover, the particles were sub-jected for sterilization procedures at 121 ◦C and 5 bar, for 30 min.Visual observations indicated that the structure of the particles wasnot affected by the sterilization conditions. These results insure

the potentiality of the prepared magnetic particles as supports forgrowing cells and the use in repeated batches experiments, whichwill be the subject for further investigation. One can assume that

Z. Al-Qodah et al. / Biochemical Engineering Journal 97 (2015) 40–49 47

Magnetic field intensity (mT)

0 20 40 60 80 100 120

Lui

quid

vel

ocit

y (c

m/s

)

0

1

2

3

4

5

ACSC magneti c particlesACMC magnetic particlesFASC magnetic particles

"Initial packed bed regime"

"Stablized expanded bed regime"

"Fluidised bed regime"

Flv

ga

3

imuiiwmpotti

itb

�

wtmo

ssribalvottF

m

ig. 9. Phase diagram of MSFBs using three different magnetic particles. Dashedines represent expansion velocities; solid lines represent minimum fluidizationelocity.

rowing cells will easily generate biofilms of considerable stabilityround the activated carbon or fly ash surface.

.4. Hydrodynamic behavior of the magnetic particles in MSFBs

Phase diagrams are usually constructed based on the exper-mental results in order to understand the performance of the

agnetic particles under the mutual effect of the magnetic fieldsed for stabilization or separation purposes and the liquid flow

n the bed [36]. These experiments were conducted in “magnetiz-ng first” mode in which the magnetic particles were poured in the

ater filled column to form a packed bed of certain height. Then,agnetic intensity was set at the desired value and applied to the

acked bed. After that, distilled water was fed to the column inrder to transfer the bed in different flow regimes by increasinghe liquid velocity (Ul). The phase diagrams of MSFBs using thehree types of magnetic particles ACMC, ACSC and FASC are shownn Fig. 9.

One more important property in fluidized system is bed poros-ty, since it affects the size of the fluid-bed equipment and residenceime of the liquid phase in the contractor. The bed porosity, �b cane estimated using:

b =(Hbo

Hb

)�s o (3)

here Hbo and Hb are the initial bed height and that after applyinghe magnetic field, respectively, and �so is the initial porosity of the

agnetic particles which was measured by measuring the amountf water required to fill the voidage between the particles.

Fig. 9 shows that each phase diagram usually consists of threeubsequent flow regimes. These are: the initial packed bed, thetable expanded bed and the fluidized bed, respectively. Theseegimes correspond to the case where the applied magnetic fieldntensity is more than 10 mT to induce sufficient cohesion forcesetween the magnetic particles. It can be seen from Fig. 9 that forll particles considered, the packed bed continued to exist until theiquid velocity exceeded what is called the minimum expansionelocity (Ue.). Beyond Ue, the bed began to stably expand but with-ut particle movement. This uniform expansion continued untilhe liquid velocity exceeded a second transition velocity named

he minimum fluidization velocity, Umf (above the upper curve ofig. 9).

With further increase of Ul the bed breaks down at the mini-um fluidization velocity, Umf. Above this transition velocity, the

Fig. 10. MSFBs porosity under the effect of constant magnetic field intensity of60 mT and various liquid velocities.

bed existed in the fluidized regime. It can be seen from Fig. 9 thatin contrast to Ue, Umf increased as the applied intensity increases.For instance, Ue for ACSC was independent of the magnetic fieldvalue with a value of 0.66 cm/s. On the other hand, Umf for the sameparticles increased from 0.66 to 3 cm/s as the magnetic field inten-sity increased from 0 to 113 mT. In addition, Fig. 9 revealed thatboth Ue and Umf were strongly dependent on the particles densi-ties. For instance, when the magnetic field intensity was 43 mT, Ue

and Umf for ACMC, ACSC and FASC were 0.92, 0.65 and 0.55 and 2,1.7 and1.45 cm/s, respectively. Furthermore, the effect of increasingthe intensity on Umf was more significant with ACMC rather thanACSC and FASC particles. This indicated that the magnetic particleswith the sand core that were prepared in this investigation are eas-ily stabilized and/or fluidized. Moreover, it was observed that bedsof such particles did not suffer from aggregation problem as thosehaving magnetic core. Since this behavior is favorable in processeswhere mass transfer is of primary concern, the prepared particlesare considered as good solid phase in MSFBs.

It can be seen from Fig. 10 that the bed expansion started asUl exceeded Ue. This regular expansion continued until the bedbreak down at Umf, at which the porosity is named as the maximumporosity (�max) where the stabilized bed can attain. Fig. 10 revealesthat for the same magnetic field intensity and liquid velocity, bedporosity, � and �max increased as the particles density decreased.However, � and �max at any value of liquid velocity were higher forlower density particles. This implies that it is possible to expand thebed of low density magnetic particles or increase its porosity whileoperating with relatively low superficial velocity. As mentionedabove, this condition suits bioprocesses. In addition, the operationwith low liquid velocity will minimize the attrition and slough-ing of the biofilm surrounding the magnetic carrier. In other word,this will facilitate the biofilm stability and the predictability of anymathematical model for the immobilized system [2]. As a result ofthis new arrangement of particles, �max exceeds a value of 0.7 asFig. 10 indicates. This means that the bed height is almost doubledand the particles are just touching each others. Consequently, theeffective outer surface area of the particles is improved and morecell deposition is expected in this regime.

3.5. Performance of the immobilized magnetic particles in MSFBs

As mentioned above, immobilized cell particles were filledin the bioreactor to a height of 5 cm. Then the bed was mag-netized followed by medium pumping until the bed expandedand reached its maximum value. Then the flow was reduced

48 Z. Al-Qodah et al. / Biochemical Engine

Time (min)

0 20 40 60 80 100

%C

onve

rsio

n (-

)

0

3

6

9

12

15

ACSC magentic particlesACMC magnetic particlesFASC magnetic particles

Fig. 11. Variation percent conversion of acetophenone with time in liquid magnet-i1

bossabfiurFt6wffl6iii

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aopflmprtw

[

[

[

[

[

[15] M. Koneracka, P. Kopcansky, M. Timko, Ch. Rmchand, Z. Saiyed, M. Trevan,Immobilization of enzymes on magnetic particles, Methods Biotechnol. 22

cally stabilized bed at magnetic field intensity of 43 mT and medium flow rate of.6 ml/min.

ut the bed height was maintained at a higher value of thatf packed beds by the aid of magnetic forces. The bioconver-ion of acetophenone started and its concentration in the exittream was measured as a function of time until it reached

steady state value. The stable expanded bed height differsetween the three particles. The chosen value of the magneticeld intensity was 43 mT for all runs. At these conditions, the val-es of Umf for ACMC, ACSC and FASC were 2, 1.6 and 1.45 cm/s,espectively. Furthermore, the bed porosity for ACMC, ACSC andASC at the operating conditions were 0.50, 0.6 and 0.62, respec-ively. Consequently, the operating bed height for was 55, 62 and9 mm for ACMC, ACSC and FASC, respectively. Accordingly, theorking volume of the stabilized bed was 87.91, 98.6 and 109.8 cm3

or ACMC, ACSC and FASC, respectively. For the same mediumow rate of 1.6 ml/min, the residence time will be 61.6, 67.7 and8.7 min for the beds of ACMC, ACSC and FASC, respectively. Dur-

ng these runs the residual outlet of acetophenone was measuredn order to estimate the percent conversion. The results are shownn Fig. 11.

It is evident from Fig. 11 that the percent conversion increasedith time until it reached the steady state condition in three runs.

he steady state concentration or conversion was 12.1, 13.3 and3.5% for ACMC, ACSC and FASC, respectively. The main reason forhis difference could be attributed to the difference in the resi-ence time of the substrate in the three beds. Another factor of

mportance is the loading capacity of the particles from the E. Coliells during the immobilization process. However, it was shownn the isotherm study that the loading capacities of the three sup-orts were comparable. Accordingly, the low density of the sandore seems to have the major effect. The more bed expansion andonsequently the more residence time of the substrate produce

relatively higher substrate conversion in beds with low densityarticles.

The novel low density magnetic particles proved that they had dual advantage. The first important advantage was the reductionf the particles fluidization velocity. This permits the use of thesearticles in fluidized beds bioreactors characterized by low mediumow rates. The second crucial advantage for the intensification ofass transfer in magnetically stabilized beds is the relatively high

orosity of the stabilized bed. This will cause an increase in the

esidence time of the substrate in the bed. In addition, the massransfer area in the bed between the liquid and the solid phasesill increase as a result of the increased porosity.

[

ering Journal 97 (2015) 40–49

4. Conclusions

Spherical, low density and nonporous magnetic support for cellimmobilization has been fabricated by coating sea sand with twolayers of magnetite and activated carbon or fly ash. The structureof these particles makes them good supports for immobilized cellused in multi-phase bioreactors. The stability of the magnetic parti-cles under sterilization and cleaning conditions suggest their use inrepeated batch processes. Since the cells retain their activity afterimmobilization, this suggests that cells could be easily adsorbedon these particles. This predicts the applicability of these magneticparticles as supports for growing cells immobilization. The particleshave relatively low minimum fluidization velocities and their bedcould be easily expanded with low liquid velocities. Bed expansioncould facilitate mass transfer processes due to the increase of interparticles surface area. This indicate that the operational conditionsof these particles of low feed velocity and higher bed expansionare more attractive in fluidized bed bioreactor compared to otherdense particles since in the conversion bioreactor is low comparedto traditional ones. In addition, the low density particles produce ahigher bed and consequently, more residence time and a relativelyhigher conversion.

Acknowledgments

The author would like to thank Al-Balqa Applied University, Jor-dan for funding this research during the sabbatical leave. GreatAppreciation for Taibah University, Al-Madinah, KSA for hostingme during the sabbatical year.

References

[1] M. Shuler, F. Kargi, Bioprocess Eng Basic Concepts, second ed., Printice HallPTR, 2002.

[2] Z. Al-Qodah, W. Lafi, Modeling of antibiotics production in magnetothree-phase airlift fermenter, Biochem. Eng. J. 7 (2001) 7–16.

[3] E. Sada, S. Katon, M. Shiozawa, I. Matsui, Rates of glucose oxidation with acolumn reactor utilizing a magnetic field, Biotechnol. Bioeng. 25 (1983)2285–2292.

[4] Z. Al-Qodah, Hydrodynamic behavior of magneto air-lift column in atransverse magnetic field, Can. J. Chem. Eng. vol. 78 (3) (2000).

[5] Z. Zhang, D.A. O’Sullivan, A. Lyddiatt, Magnetically stabilized fluidized bedadsorption: practical benefit of uncoupling bed expansion from fluidvelocities in the purification of a recombinant protein from Escherichia coli, J.Chem. Technol. Biotechnol. 74 (1999) 270–274.

[6] R.E. Rosensweig, Fluidization: hydrodynamic stabilization with a magneticfield, Science 204 (1979) 57–60.

[7] D. Yong, S. Yan, Small-sized dense magnetic pellicular support formagnetically stabilized fluidized bed adsorption of protein, Chem. Eng. Sci. 60(2005) 917–924.

[8] Z. Al-Qodah, M. Al-Shannag, Application of magnetically stabilized fluidizedbeds for cell suspension filtration from aqueous solutions, Sep. Sci. Technol.42 (2007) 1–18.

[9] Y. Ren, J. Rivera, L. He, H. Kulkarni, D. Lee, Ph. Messersmith, Facile highefficiency immobilization of lipase enzyme on magnetic iron oxidenanoparticles via a biomimetic coating, BMC Biotechnol. 11 (2011) 63–67.

10] S.D. Santana, A.S. Pina, A.C. Roque, Immobilization of enterokinase onmagnetic supports for the cleavage of fusion proteins, J. Biotechnol. 161(2012) 378–382.

11] P. Prikryl, J. Lenfel, D. Horak, M. Ticha, Z. Kucerova, Magnetic bead cellulose asa suitable support for immobilization of �-chymotrypsin, Appl. Biochem.Biotechnol. 168 (2012) 295–305.

12] C. Yi-Yu, T. Ming-Gen, C. Meng-Chun, W. Tzu-Fan, L. Long-Liu, Covalentimmobilization of Bacillus licheniformis (-glutamyl transpeptidase onaldehyde-functionalized magnetic nanoparticles, Int. J. Mol. Sci 14 (2013)4613–4628.

13] I. Safarik, M. Safarikova, Magnetic techniques for the isolation and purificationof proteins and peptides, BioMagn. Res. Technol. 2 (2004) 1–17.

14] W. Wei, D. Le, P. Hui, X. Xiao, Study of the epoxydized magnetic hydroxylparticles as a carrier for immobilizing penicillin G acylase, Enzyme Microb.Technol. 40 (2007) 255–261.

(2006) 217–228.16] P.J. Hailing, P. Dunnill, Magnetic supports for immobilized enzymes and

bioaffinity adsorbents, Enzyme Microb. Technol. 2 (1980) 1–10.

Engine

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

A

Z. Al-Qodah et al. / Biochemical

17] Z. Al-Qodah, V. Ivanona, E. Dobervo, I. Penchev, J. Hristov, R. Petrov,Non-porous magnetic support for cell immobilization, J. Ferment. Bioeng. 71(1991) 114–117.

18] V. Goetz, R. Magali, D.J. Graves, A novel magnetic silica support for use inchromatographic and enzymatic bioprocessing, Biotechnol. Bioeng. 37 (1991)614–626.

19] D. Horak, M. Babic, H. Mackov, M. Benes, Preparation and properties ofmagnetic nano- and microsized particles for biological and environmentalseparations, J. Sep. Sci. 30 (2007) 1751–1772.

20] J.H. Siegell, Liquid fluidized magnetically stabilized beds, Powder Technol. 52(1987) 139–147.

21] C.J. Fee, Stability of the liquid fluidized magnetically stabilized fluidized bed,AIChE J. 43 (1996) 1213–1219.

22] T.M. Cocker, C.H. Fee, R.E. Evans, Preparation of magnetically susceptablepolyacrylimide/magnetite beads for use in magnetically stabilized fluidizedbed chromatography, Biotechnol. Bioeng. 53 (1997) 78–87.

23] X.D. Tong, Y. Sun, Application of magnetic agarose support in liquidmagnetically stabilized fluidized bed for protein adsorption, Biotechnol.Progr. 19 (2003) 1721–1727.

24] K. Edegger, C.C. Gruber, T.M. Poessl, S.R. Wallner, I. Lavandera, K. Faber, F.Niehaus, J. Eck, R. Oehrlein, A. Hafner, W. Kroutil, Biocatalytic deuterium- andhydrogen-transfer using over-expressed ADH-‘A’: enhanced stereoselectivityand 2H-labeled chiral alcohols, Chem. Commun. 22 (2006)2402–2404.

25] A.-V. Ursu, I.D. Nistor, F. Gros, A.V. Arus , G. Isopencu, A.M. Mares ,

Hydrodynamic aspects of fkluidized bed stabilized in magnetic field, U. P. B.Sci. Bull. Series B 72 (2010).

26] V. Ivanova, J. Hristov, E. Dobreva, Z. Al-Qodah, I. Penchev, Performance of amagnetically stabilized bed reactor with immobilized yeast cells, Appl.Biochem. Biotechnol. 59 (1996) 188–198.

[

ll in-text references underlined in blue are linked to publications on Researc

ering Journal 97 (2015) 40–49 49

27] Z. Al-Qodah, M. Al-Busoul, M. Al-Hassan, Hydro-thermal behavior ofmagnetically stabilized fluidized beds, Powder Technol. 115 (2001)58–67.

28] C. Webb, H.K. Kang, G. Moffat, R. Williams, A.M. Estevez, J. Cuelar, E. Jaraiz,The Magnetically stabilized fluidized bed bioreactor: a tool for improved masstransfer in immobilized enzyme systems, Chem. Eng. J. 61 (2015)241–246.

29] T.-T. Hu, J.-Y. Wu, Study of the characteristics of biological fluidized bed in amagnetic filed, Chem. Eng. Res. Des. 65 (1987) 238–243.

30] Z. Al-Qodah, M. Al-Hassan, Phase holdup and gas-to-liquid mass transfercoefficient in magneto stabilized GLS airlift fermenter, Chem. Eng. J. 79 (2000)41–52.

31] Z. Al-Qodah, Adsorption of methylene blue with diatomite, J. Eng. Technol. 17(1998) 128–138.

32] H.M. Zalloum, Z. Al-Qodah, M.S. Mubarak, Copper adsorption onchitosan-derived Schiff bases, J. Macromol. Sci. Pure Appl. Chem 46 (2008)46–57.

33] A. Wang, A. Mulchandani, W. Chen, Whole-cell immobilization using cellsurface-exposed cellulose-binding domain, Biotechnol. Progr. 17 (2011)407–411.

34] J.L. Van Haecht, M. Bolipombo, P.G. Rouxhet, Immobilization of Saccharomycescerevisiae by adhesion: treatment of the cells by Al ions, Biotechnol. Bioeng.27 (1985) 217–223.

35] N. Yee, J.B. Fein, C.J. Daughney, Experimental study of the pH, ionic strength,and reversibility behavior of bac-teria-mineral adsorption, Geochim.

Cosmochim. Acta 64 (2000) 609–617.

36] P. Kilonzo, M. Bergougnou, Surface modifications for controlled andoptimized cell immobilization by adsorption: applications in fibrous bedBioreactors containing recombinant cells, J. Microbial. Biochem. Technol.(2012) http://dx.doi.org/10.4172/1948-5948.S8-001

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