ELSEVIER The Chemical Engineering Journal 61 ( 1996) 241-246

THE BIOCHEMICAL ENGINEERING

JOURNAL

The magnetically stabilized fluidized bed bioreactor: a tool for improved mass transfer in immobilized enzyme systems? ’

Colin Webb a, Hong-Ku Kang a, Gillian Moffat a, Richard A. Williams a, Angel-M. Est&ez b, Jorge Cutllar b, Eladio Jaraiz bV2, Miguel-Angel GalAn b

‘Department of Chemical Engineering, UMIST, Manchester, UK b Departamento de Ingenieria Quimica, Universidad de Salamanca, Salamanca, Spain

Received 1 August 1995; accepted 1 August 1995

Abstract

Studies were carried out using novel magnetic carrier particles in a magnetically stabilized fluidized bed (MSFB) to determine the degree of liquid phase mass transfer enhancement achievable over a range of magnetic field strengths. The carrier particles, beads of K-carrageenan incorporating magnetite, ranging in size from 0.25 to 1 mm are used for enzyme immobilization. Results using a 113 mm diameter column showed that a more than five-fold increase in liquid velocity could be achieved when a current of 40 A was applied to a single magnetic coil. The “escape” velocity of the particles from the bed was found to be proportional to the magnetic field intensity squared. The effect was further increased (almost doubled) by inserting a steel rod into the centre of the bed.

Keyworak: Magnetically stabilized fluidized bed (MSFB); Immobilized enzyme.; Mass transfer

1. Introduction

Fluidized beds have been used in chemical processing for many years and have more recently found application in bio- processing, when enzymes or cells are immobilized on or within particulate solids. The basis of fluidization is the bal- ance of forces which exists when a fluid flows through a bed of particles, creating a drag force opposing that of gravity. In most cases there is a positive density difference between the solid and fluid phases and fluidization is effected by passing the fluid upwards through the bed. If the particles are mag- netically susceptible and a magnetic field is applied to the bed then it becomes possible to take advantage of this additional force to give a further degree of freedom in the control of the fluidization. Such beds are referred to as being magnetically stabilized.

Magnetically stabilized fluidized beds (MSFBs) in which a weak, time-invariant external magnetic field is applied axi- ally, relative to the flow of the fluidizing medium, offer a number of potential advantages over conventional fluidized beds [ 11. These include the elimination of solids mixing, low pressure drop through the bed and ease of solids transporta-

’ Dedicated to the memory of E. Jaraiz. ’ Deceased.

0923~0467/96/$15.00 0 1996 Elsevier Science S.A. AU rights reserved SSDlO923-0467(95)03043-3

tion as well as the possibility of operation at increased fluid velocities and even countercurrent operation.

The development of MSFBs has recently been reviewed by Liu et al. [ 21. Much of the work carried out during the past 30 years has concentrated on systems in which the flui- dizing medium is a gas and often the process objective is separation. However, MSFBs also have application as reac- tors and have been proposed for use with immobilized enzyme bioreactions [ 31. Table 1 summarizes some of the reported applications of MSFBs in biotechnology.

That enzyme-based bioreactions can benefit from immo- bilization of the enzyme is well known. This is largely due to the fact that enzymes are water soluble and must therefore be separated from the reaction mixture if reuse or continuous operation is desired. Thus, immobilization on solid particles (acting as carriers) enables the enzymes to be retained in the bioreactor. In addition, contamination of the product stream with the enzyme is minimized.

Optimization of immobilized enzyme systems must be a compromise between obtaining a high volumetric reaction rate (for which particles should be as small as possible) and the ease of particle retention (for which large particles are better). Most immobilization techniques result in particles with densities only a little higher than that of water and oper- ation in packed beds must involve either very low liquid

Tab

le 1

So

me

biot

echn

olog

ical

app

licat

ions

of

mag

netic

ally

sta

biliz

ed f

luid

ized

bed

Flui

d C

olum

n di

men

sion

s (m

m)

Dia

met

er

Len

gth

D,

k

Supp

ort m

ater

ial

Supp

ort

Supp

ort

Flui

d di

amet

er

dens

ity

velo

city

(m

m)

(kg

m-s

) (X

103m

s-‘)

Mag

netic

fi

eld

inte

nsity

(mT

)

Pow

er

supp

ly

App

licat

ion

Ref

.

Gas

-liq

uid

Liq

uid

Liiu

id

Liq

uid

Gas

-liq

uid

Liq

uid

28

12

28

13

13

92

20

230

NS

100

102

270 30-7

0

NS

0.22

Acr

ylam

ide-

FqO

, 0.

12

Ca

algi

nate

-F%

04

NS

NS

NS

Poly

acry

lam

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gel,

cubi

c sh

ape,

50

% M

s-Z

n fe

rrite

3.

6

Ni s

pher

es

0.1-

0.1s

NS

0.5-

3.65

N

S N

S

1200

0.

5-4

24-9

4 N

S

NS

1.2

4 1.

3A.8

V

NS

NS

4 10

w

1450

N

S W

6.2

10-5

A

NS

5.7

5.6

17w

t41

[31

r51

[61

[71

[81

Liq

uid

Liq

uid

Liq

uid

14

50

20

250

520 30

K-C

arra

gecr

W-F

e&,

Alg

inat

~F~O

,

Ni s

pher

es

0.25

-l

34

0.1-

0.15

1100

NS

NS

0.8-

4.7

3.3-

12.5

5.7

NS

20 5.6

2A,

1OV

2A,3

5V

17w

bnm

obiii

zed

gluc

ose

ox&

se

Imm

obili

zed

lura

se

Aff

inity

ch

rom

atog

raph

y

Bio

sepa

ratio

ns

Imm

obili

zed

C&

S

Cel

l deb

ris

filtr

atio

n

Mal

tode

xtri

n hy

drol

ysis

plan

t ce

ll cu

ltme

Cel

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spen

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pr

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r91

r101

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NS,

info

rmat

ion

not

supp

lied.

C. Webb et al. /The Chemical Engineering Journal 61(1596) 241-246 243

velocities or the use of retaining screens. In the former case, liquid phase mass transfer can be poor while, in the latter, unreasonably high pressure drops and plugging of the bed can result. Usually, fluidization provides a solution to the problems associated with packed bed operation; however, for small, near neutral buoyancy particles, minimum fluidizing velocities are very low and elutriation cannot be avoided. Thus the problem of poor liquid phase mass transfer remains.

Using magnetically susceptible particles as carriers, and stabilizing the bed magnetically, should permit higher liquid flow rates through the reactor, since the drag force exerted by tbe liquid can be compensated by magnetic forces. Better mixing and decreased mass transfer resistances in the liquid phase should therefore be possible while, at the same time, the risk of elutriation of particles from the bioreactor is reduced. The use of such particles offers a further advantage in that they can later be separated and even transported using magnetic fields.

In previous work using cylindrical magnetic particles it was shown that significant increases in liquid velocity could be achieved using a MSFB compared with conventional flui- dization [ 111. This work focusses on the behaviour of beds of smaller, spherical particles [ 121 with and without mag- netic fields applied, in order to determine the degree of mass transfer enhancement possible over a range of magnetic field strengths.

2. Experimental details

2. I. Magnetic carrier particles

A wide variety of magnetic carrier particles have been used for biotechnological applications, many of them involving enzyme immobilization. These have been reviewed recently by Pieters et al. [ 131. The particles used in the present work were spherical beads of K-carrageenan, produced by a simple and inexpensive emulsion polymerization technique incor- porating magnetite into the gel [ 141. K-carrageenan was melted and mixed with precipitated magnetite at a sufficiently high temperature to avoid solidification (65-70 “C). The mixture was then poured into a stirred vessel containing hot oil in order to produce small droplets. This mixture was then cooled to solidify the droplets as beads. The beads thus pro- duced were smooth spherical particles, uniformly bright black in colour with a density of 1059 kg rnm3. The size range produced can be predetermined by operating at the appropri- ate stirrer speed. The beads used in this study (diameter 0.37- 0.92 mm) were produced using stirrer speeds in the range 200-400 rev mm-‘.

2.2. The magnetically stabilizedjuidized bed

Preliminary experiments using particles of different sizes were carried out in a small-scale MSFB (column diameter 14 mm, column length 250 mm) encompassed by two coils.

Fig. 1. Experimental set-up for the 113 mm diameter MSFB: 1, MSFB column; 2, recirculation tank; 3, pump; 4, rotameters; 5, distributor; 6, magnetic particle bed; 7, power supply; 8, magnetic coil.

A small d.c. power supply was used to provide up to 2.2 A of current to the coils. Experiments were performed to deter- mine the maximum stable operating flow rate for beds of particles with mean diameters ranging between 0.37 and 0.92 mm. The bed was considered to have become unstable when significant particle movement was observed. The superficial liquid velocity through the bed at this transition point was recorded as U,. The effect of the magnetic field on bed expan- sion characteristics was also measured.

The experimental set-up for the remainder of the experi- ments is shown schematically in Fig. 1. The magnetic carrier particles were introduced into the Perspex column (1) to form a randomly packed bed (6). The main characteristics of the bed and the coil (8) for producing the magnetic field are summarized in Table 2.

The distributor (5) supporting the magnetic carrier parti- cles comprised a perforated plate, made of Perspex, with 28 1 orifices of 2 mm diameter (arranged in a hexagonal config- uration) plus a wire mesh screen. The total free area was around 8%.

Power was supplied to the coil by a d.c. power supply (7)) producing currents of up to 45 A. Water from the tank (2) was pumped (3) upwards through a parallel group of three rotameters (4) connected to the column. The distributor (5) ensured a uniform flow of water through the bed of particles (6). Having passed through the bed, the water was recircu- lated to the tank (2).

During operation, the height and behaviour of the bed were observed as the flow rate through the column was gradually increased. Runs were first performed with no magnetic field applied, in order to observe the behaviour of the bed under conventional fluidization conditions. An “escape velocity” U, was defined for the purpose of determining the stable operating limit for the bed. This was chosen as the liquid superficial velocity at which a small but constant flow of particles from the bed was observed at the top of the coil. Although a little subjective, and dependent on the initial bed height, this criterion provided a reproducible basis for com-

244

2

C. et al. Chemical Engineering (19%) 241-246

of the stabilized fluidixed

Bed Coil

(mm)

Diameter Ma.%

(g)

Number of Layers Inner

(mm)

Outer

(mm)

Height

62 Copper 9 140 133

Fig. Effect of current I transition velocity for a of particle in a MSFB.

parison of data obtained under different magnetic field intensities.

3. Results and discussion

The results of preliminary experiments using a range of different particle sizes in the small-scale MSFR are summa- rized in Fig. 2. The graph shows the effects both of bead size and of magnetic field strength, as current to the coil. In line with normal fluidization behaviour, the liquid velocity U, required to render the bed unstable increased with increasing bead size. In addition, increasing the current to the coil, and therefore the magnetic field strength, also resulted in a con- siderable increase in transition velocity. Although only pre- liminary, and based on the rather subjective “transition” velocity, the results show clearly that enhanced stability of beds of magnetically susceptible beads can be achieved through the application of modest magnetic fields.

More detailed study of the bed expansion behaviour of the particles in the small-scale MSFR showed that for a given liquid velocity through the bed the bed height was less when a magnetic field was applied. A typical set of results is shown in Fig. 3. Such compaction of the bed leads to lower bed

d 0 0.5 1 1.5 2 2.5 3 :

u [x 1 OS (m/s)

5

Fig. 3. Effect of a magnetic field on the expansion characteristics of a bed of magnetically susceptible particles. The figure shows data for bed height against superficial liquid velocity (I when no current was applied (0 A) and with a current of 2.192 A applied.

C. Webb et al. /The Chemical Engineering Joumal61(19%) 241-246 245

0 5 IO 15 20 25 30 35 40

I (A)

Fig. 4. Effect of applied current Ion escape velocity U, for a 113 mm diameter MSFl3 using particles in the sire range 0.5-0.7 mm.

0 300 600 900 1200 I500 1800

12 (AZ)

Fig. 5. Relationship between applied current squared I* and escape velocity U, for the conventional MSFR (0) and for the MSFR with a centrally placed steel rod ( n ) .

porosities which, in turn, give rise to higher local velocities for a given superhcial velocity, resulting in increased liquid phase mass transfer coefficients.

Following the preliminary tests with relatively small quan- tities of beads, larger-scale experiments were carried out in the 113 mm diameter column using particles in the size range OS-O.7 mm. The results for escape velocity over a range of magnetic field strengths (in terms of applied current) are shown in Fig. 4. Again, there was a clear enhancement in operational stability with a five-fold increase in liquid veloc- ity being possible for an applied current of 40 A compared with that with no magnetic field applied. The stable operating regime is represented by all points on or below the line in Fig. 4. The shape of the curve is also interesting since it suggests that the extent of stability enhancement itself increases with increasing field strength.

In earlier work, Jaraiz and Estevez [ 151 showed that the force acting on a magnetically susceptible particle in a mag- netic field Ho was proportional to the applied current I squared (i.e. HG a f ) . Since, for small particles settling under an applied force, Stokes’ law is valid and since Stokes’ law states that the terminal settling velocity UT is proportional to the force applied (i.e. in this case, UT a HG) then it follows that the terminal settling velocity and, therefore, the escape velocity U, should also be proportional to the applied current squared (i.e. U, a Z2). Fig. 5 shows the data for U, plotted against P. The straight line confirms the relationship.

Also shown in Fig. 5 are the results of a further experiment in which a steel rod was placed into the centre of the bed of particles. Jaraiz and Briz [ 161 have suggested that the pres- ence of a magnetizable central core concentrates the magnetic

55 I I I I I

- Kinetic rate

Magnetic field

Mass transfer rate

15 I I I I I I I IO

0.0001 o.obo2 0.0003 0.0004 0.0005 O.ObO6

Superficial fluid velocity (m/s)

Fig. 6. Model prediction of MSFR reaction mass transfer rates at different flow rates showing also the magnetic field strength required to enable operation at the necessary superficial fluid velocities.

246 C. Webb et al. /The Chemical Engineering Journal 61(19%) 241-246

field and can be used to increase particle retention in the bed at high liquid throughputs. In this case, a rod of magnetizable stainless steel (430) of 13 mm diameter and 165 mm length was placed centrally in the MSFB and the same series of escape velocity determinations was repeated. Fig. 5 shows clearly that a further enhancement in superficial liquid veloc- ity was obtained which, again, appeared to be proportional to the applied current squared.

In terms of bioreactor operation involving immobilized enzymes, the most important parameters are catalyst activity and mass transport efficiencies. Using an MSFB, enhanced mass transfer rates can be achieved since the flow rates through the reactor are higher. Fig. 6 shows a prediction of the mass transfer rates through an MSFB, based on a mass transfer correlation for conventional fluidized bed reactors [ 171. These mass transfer rates have been compared with the kinetic rate for starch hydrolysis using immobilized gluco- amylase [ 181, which is also shown in Fig. 6. It is clear that by increasing fluid velocity the overall mass transfer rates can be significantly improved. The plot also shows that the magnetic field required to retain the magnetic carrier particles in the MSFB increases with increasing fluid velocities.

4. Conclusions

The MSFB reactor enables an additional force to that of gravity to be used in the control of throughput for reactors involving beds of magnetically susceptible particles. Thus in the case of immobilized enzyme systems it becomes possible to operate processes continuously and with lower mass trans- fer resistances. In an operating system both internal andexter- nal mass transfer limitations can be reduced because it becomes possible to use smaller particles (potentially reduc- ing internal resistances) at higher superficial liquid velocities (improving external mass transfer). The starch hydrolysis example referred to above is just one candidate system cur- rently being explored for use with the MSFB in conjunction with immobilized enzymes [ 191. In cases where there is a clear mass transfer limitation the MSFB reactor makes it possible, in principle, to achieve the full kinetic potential of the system.

Acknowledgements

The authors would like to thank the British Council and the Ministerio de Education y Ciencia for jointly providing financial support through an Acci6n Integrada grant (refer- ence, ( 1992/93) /7440). Further financial support from EA

Technology Ltd. and SERC, in the form of a research dentship for Gillian Moffatt, is also acknowledged.

References

stu-

[ I] B.E. Terrauova and M.A. Bums, Continuous cell suspension processing using magnetically stabilised fluidised beds, Riotechnol. bioeng., 37 (1991) 110-120.

[2] Y.A. Liu, R.K. Hamby and R.D. Colberg, Fundamental and practical developments of magnetofluidised beds: a review, Powder Technol.,

64 (1991) 3-41. [3] E. Sada, S. Katoh, M. Shiozawa and T. Fukui, Performanceof fluidised

bed reactors utilising magnetic fields. Biotechnol. Bioeng., 23 (1981) 2561-2567.

[4] E. Sada, S. Katoh and M. Terashima, Enhancement of oxygen absorption by magnetite containing beads of immobilised glucose oxidase, Biotechnol. Bioeng., 23 (1981) 1037-1044.

[5] M.A. Bums and D.J. Graves, Continuous affinity chromatography using a magnetically stabilised fluidised bed, Biotechnol. Prog., l(2) (1985) 95-103.

[6] M.A. Bums and D.J. Graves, Application of magnetically stabilised fluidised beds to bioseparations, Reactive Poly., 6 (1987) 45-50.

[7] T.T. Hu and J.Y. Wu, Study on the characteristics of a biological fluidised bed in a magnetic field, Chem. Eng. Res. Des., 65 (1987) 238-242.

[ 81 B.E. Terranova and M.A. Bums, Continuous cell debris filtration using a magnetically stabilised fluidised bed, Biotechnol. Prog., .5( 3) ( 1989) 98-104.

[9] B.R. Pieters, Magnetic separations in biotechnology, MSc. Dissertation, University of Manchester Institute of Science and technology, Manchester, 1989.

[11

[12

[lo] J.L. Bramble, D.J. Graves and P. Brodelius, Plant cell culture using a novel biomactor: the magnetically stabilised fluidised bed, Biotechnol. Prog.. 6 (1990) 452-457. A.M. Estevez, J. Cuellar, E. Jaraiz and J.M. Rodriguez, Behaviour of magnetisable composites in a liquid-solid magneto-fluid&d bed: potential use as a bioreactor, Chem. Biochem. Eng. Q.. 9(2) ( 1995). R.A. Williams, B.R. Pieters, C. Webb and M.A. Longo-Gonzalez.

t

Formation and surface engineering of magnetic polymer beads for large scale bioprocesses, in R.A. Williams and N.C. de Jaeger (eds.), Advnaces in Measurement and Control of Colloidal Processes,

Butterworth-Heinemaun, Oxford, 1991, pp. 172-186. 131 B.R. Pieters,R.A. WilliamsandC. Webb,Magneticcarriertechuology,

in R.A. Williams (ed.), Colloid and Sulface Engineering:

Applications in the Process Industries, Butterworth-Heinemann, Oxford, 1991, pp. 248-286.

141 B.R. Pieters, R.A. Williams and C. Webb, Microparticles for immobilising macromolecules, UK Patent Appl. 8,908,662, 1989.

[ 151 E. Jaraiz and A.M. Estevez, Design concepts for a collar-type magnetic valve for small-size solids (MVS): theory and experiments, Powder

Technol., 53 (1987) l-9. [ 161 E. Jaraiz and J.A. Briz, Aparato para filtrar particulas por medio de

campos magneticos, Spanish Patent 550,363, 1987. [ 171 W.K. Beck, Mass transfer in fluidised beds, in J.F. Davidson and D.

Harrison (eds.), Fluidisation, Academic Press, New York, 1976. [ 181 G. Moffat, R.A. Williams, C. Webb and R. Stirling, Enzyme hydrolysis

in a magnetically stabilised fluidised bed reactor (MSFBR), Proc. IChemE Research Event, 1995, IChemE, Rugby, 1995, pp. 989-991.

[ 191 G. Moffat, R.A. Williams, C. Webb and R. Stirling, Slective separations in environmental and industrial processes using magnetic carrier technology, Miner. Eng., 7 (1994) 1039-1056.