Non-porous magnetic supports for cell immobilization

JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 71, No. 2, 114-117. 1991

Non-Porous Magnetic Supports for Cell Immobilization ZAKARIA AL-HASSAN, 1. VIARA IVANOVA, 2. ELENA DOBREVA, 2 IVAN PENCHEV, I JORDAN HRISTOV, 1 ROSEN RACHEV, 2 AND RUMEN PETROV l Higher Institute of Chemical Technology, Department of Chemical Engineering, Bul. Kl. Ohridski 8, Sofia, 1 and Institute o f Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev BI. 26, Sofia 1113,2 Bulgaria Received 17 August 1990/Accepted 9 November 1990 A new method for covering magnetic particles with a stable non-porous layer of a material like zeolite or activated carbon was used for the preparation of support materials with good properties for the immobilization of yeast Saccharomyces cerevisiae cells. The immobilized cells can be used in batch and continuous alcoholic fermentation. A productivity of 35.6 g ethanol/l, h was reached. The adsorption isotherms of the immobilized yeast cells were determined. Yeast cell immobilization on non-porous magnetic supports obeyed the Langmuir isotherm equation. Satisfactory results were obtained also from repeated batch fermentations with fixed cells on supports additionally treated with glutaraldehyde or by simple adsorption.

Magnetic supports for biocatalyst immobilization offer several advantages when compared to non-magnetic systems. Besides allowing separation of the support from the suspended solids in the process medium, the ease and power of magnetic collection permits the use of very small particles. In addition, this allows the use of non-porous particles while still retaining a reasonable specific surface area for biocatalyst immobilization (1). Magnetic supports for immobilized biocatalists were first used by Robinson et al. (2) to separate small-size immobilized enzyme particles from other insoluble materials using magnetic devices. Subsequently, workers elsewhere reported immobilization of enzymes to magnetic supports (3-5). The parallel application of magnetic particles in bioaffinity adsorbents was investigated by Dunnill and Lilly (6). More recently, Robinson et aL (Robinson, P. J. and Dunnill, P., UK Patent, 1,403,359) have reported the possible uses and fields of application of enzyme magnetic supports. These include hydrolysis of carbohydrates, processing of waste materials, alcoholic and other kinds of fermentation, and specific redox reactions of organic materials. Another advantage of such magnetic supports is their use in a magnetically-stabilized fluidized bed, thereby presenting further options in continuous reactor systems. Sada et al. (7-9) have found that the mass-transfer resistance at the gas--liquid interface can be reduced by the spinning of magnetic beads under revolving magnetic field, and that the values of the enhancement factor due to catalytic oxidation of glucose are larger than these calculated on the basis of film theory. Also, in fluidized bed reactors with immobilized enzyme magnetic particles the dilution rates can be increased without appreciable effusion of the particles (10, 11). Magnetic supports which have been used are both porous and non-porous. Halling and Dunnill (12) have concluded that non-porous magnetic particles seem to be more resistant to diffusional limitations, attrition and fouling than porous supports. A variety of non-porous magnetic materials have been used, including iron, cobalt and their oxides, and the methods of their preparation have been classified into four groups: direct use of a coupling agent, adsorption, encapsulation, and formation of a thin film

of polymer. It must be noted, however, that the magnetic supports have been limited to enzyme immobilization. A few reports have appeared on the immobilization of cells on magnetic particles (13-15). The first objective of the present work was to devise a new, simple and flexible method for the preparation of non-porous magnetic supports for cell immobilization. The second objective was to test different methods for cell immobilization using the yeast Saccharomyces cerevisiae on different types and sizes of the prepared magnetic particles. MATERIALS AND METHODS Preparation o f magnetic supports Magnetite (Fe304) was covered with a stable layer of material with good adhesion properties, such as zeolite (Petrov, R., PhD Thesis, Higher Institute of Chemical Technology, Sofia, 1989) or activated carbon, with the aid of epoxy resins. The resins were obtained by condensation of epichlorohydrin with biphenol. These resins can be solidified using aliphatic chains which polymerize with the resin and modify the chain structure to a cyclic solid one having a high melting point (16). The magnetic particles were prepared by mixing 100 g of the epoxy resin with 9.0g 1.6=hexanediamine and with 1 kg of magnetite. After l0 min of continuous mixing every particle was coated with a thin film of the resin. After that, zeolite or activated carbon powder was added in excess and vigorously mixed for 15 rain. During this time the powder attached to the magnetite particle surface was forming a layer of zeolite or activated carbon. The resulting covered particles were left for 48 h to complete the solidification process at room temperature. They were then screened and washed to remove any excess powder and left to dry again. The resulting particles were larger in size and had lower density than magnetite. These steps can be repeated to increase the particle size and reduce their density if necessary. Microorganism The yeast S. cerevisiae, a production strain, was used in this investigation. Cell immobilization Two types of yeast cell immobilization were performed--with and without a bifunctional reagent such as glutaraldehyde, i.e. the first method was adsorption and the second was adsorption and the

* Corresponding author. 114

VOL 71, 1991

MAGNETIC SUPPORT FOR ALL IMMOBILIZATION

creation o f b o n d s between the yeast cells by glutaraldehyde. F o r each i m m o b i l i z a t i o n procedure, 5 g o f the supp o r t was used. S u p p o r t s were treated with 1% N a O H for 1 h at 45°C with continuous mixing. After that, they were sequentially washed with distilled water, 1% HCI, and distilled water again to neutralize the p H . The magnetic particles were mixed for 3 h at 25°C with 5 ml o f cell suspension with different concentrations o f yeast cells. The a d d e d glutaraldehyde concentration was 0.4 ml o f 2.5%o solution per 5 ml o f cell biomass. A f t e r the fixation process the particles were washed with an a b u n d a n t quantity o f distilled water to remove the excess cells and glutaraldehyde. The quantity o f the fixed biomass was measured by counting the cells in the washing solution. The zeolite particles were 1.6-2.0 m m in diameter, and the activated carbon ones 1.2-1.6 m m or 1.0-1.2 mm. Fermentation F e r m e n t a t i o n m e d i u m (g//): 140-150 g glucose; 2 . 5 g yeast autolysate, 5 g peptone; 3 g KH2PO4; 25 mg CaC12; 3 g (NH4)2SO4; 25 mg M g S O 4 (heptahydrate), tapwater, p H 4.5. F e r m e n t a t i o n unit: a glass column with total volume o f 7.5 c m 3, packed with 10 g o f immobilized material. The m e d i u m volume was 3.0 cm 3 and the dilution rate 0.5 h ~ (based on the m e d i u m volume). Six repeated batch anaerobic fermentations were carried out in flasks containing 50 ml o f fermentation m e d i u m with the immobilized magnetic supports. Each fermentation was stopped after 42 h, the particles were washed with distilled water and then used again in fresh medium. F e r m e n t a t i o n control: ethanol (by g a s - c h r o m a t o g r a p h y on a PV 17 fused silica capillary c o l u m n - - 2 5 m, E r b a Science 4300 g a s - c h r o m a t o g r a p h ; temperature 35°C; carrier gas N 2 - 2 m l / m i n ; detector FID; calculation by the m e t h o d o f internal s t a n d a r d - - n - b u t a n o l ) , glucose (by the m e t h o d o f Somogyi (17)), released cell biomass (cells per gram support or per ml medium) and productivity were monitored. RESULTS A N D DISCUSSION Adsorption isotherms for S. cerevisiae The purpose o f constructing sorption isotherms was to determine the m a x i m u m possible cell loading and to model the sorption for potential scale-up in future work. The sorption experiments were done at 25°C in a buffer over a wide range o f

115

TABLE 1. Isotherm coefficients Support

A

Zeolite Activated carbon d 1.2- 1.6mm Activated carbon d = 1.0 = 1.2 m m

B

5.9001 ~

11.7242 a

5.0751 a

28.1111 a

12.9763 a

83.3755 a

a x I O 6.

cell concentrations. Figure 1 shows the sorption isotherms of S. cerevisiae on activated carbon and zeolite. Each point represents a separate experiment. The best d a t a fit was given by Eq. 1 using the m a r q u a r d nonlinear regression technique: A.X Y= B+X'

(1)

where X is the free cell concentration in 107 cells/g support and Y is the adsorbed cell concentration in 106 cells/g support, A and B are coefficients describing the m a x i m u m possible cell loading and the sorptive immobilization constant o f yeast cells on these magnetic particles. A free cell concentration greater than 8 x 107 cells/g support "saturates" the available sorption sites on the zeolite and on activated carbon with a particle diameter of 1.2-1.6 mm. This concentration was 12 x 107 cells/g for the smaller activated carb o n supports. The isotherm model can be used to estimate the free cell concentration needed to make biocatalysts with a given a m o u n t o f a d s o r b e d cells. The constants can provide a basis for cell binding comparisons among different possible supports and different sorption conditions and sizes for the same support (Table 1). Batch fermentations with the immobilized S. cerevisiae The results from batch fermentations using glutaraldehyde-treated zeolite and activated carbon supports are shown in Fig. 2. It can be seen that the ethanol concentration in the 4th batch was found to be only about 35.0 m g / m l medium and the yield reached only 55-60% o f the theoretical yield, but there existed a tendency toward augmentation o f the ethanol concentration in the effluent. The cell concentration in the medium (daughter cells could not be retained on the supports) in the first two batch

4 0 0 [ ~

t g 80

A

9 ~o

500

~6,0 -

~

¢- B

-

c

× 4.0 %

2oo I

~ 2 o. ]

!~ o (/3

uJ 100

1.0

50

i

2'o 4'o do s:o ,oo

L

;4o' ,6o

Free cell concentration, 10 7 X cells/g support FIG. 1. Adsorption isotherms of S. cerevisiae on activated carbon and zeolite. A, Carbon, d=l.0-1.2mm; B, zeolite, d=1.62.0 ram; C, carbon, d = 1.2-1.6 mm.

0.~

1

2

5 4 5 6 Botch number FIG. 2. Results from batch fermentation with glutaraldehydetreated zeolite and activated carbon magnetic supports. Symbols: O, ethanol from zeolite particles; O, ethanol from activated carbon particles with diameter 1.0-1.2mm; v, released cell biomass in cells/ml x 108 from the three supports.

116

AL-HASSAN ET AL.

J. FERMENX. BXOEN6.,

runs was low, but continued to increase until the 4th batch run. Then, it nearly held a constant value of 1.0x 108 cells/ml in the following three batch runs. It can be concluded that in the first three fermentations the viable cell content on the magnetic particles was low and the substrate was utilized for cell growth and breeding. Then, in the 4th run, the biofilm thickness reached a steady state value. This hypothesis is in agreement with the ethanol concentration profile, which indicates low but increasing values in the first three batch runs and relatively high and constant values after the 4th batch run. It must be noted that the immobilized cells p r o d u c e most of the ethanol. In batch ethanol fermentation with free cells o f this strain the biomass concentration is about 1.0 × 109 cells/ml, which is I0 times higher than that o f the free cells in the present case (Ivanova, V., P h D Thesis, Higher Institute of Chemical Technology, Prague, Czechoslovakia, 1986). Figure 3 shows results from the fermentation using adsorbed yeast cells on zeolite and activated carbon magnetic supports without treatment with glutaraldehyde. It is evident that this i m m o b i l i z a t i o n is also effective. With these magnetic supports, an ethanol concentration o f about 65.0-70.0 m g / m l fermentation m e d i u m and a yield o f 8590%0 of the theoretical one were reached in the sixth batch run. A t the beginning o f the fermentation process in this case, the ethanol concentration was low and the yield was from 30% to 45% o f the theoretical yield. A t the same time, the released biomass concentration reached 1.2 x 108 cells per ml fermentation liquid, i.e. the substrate was also utilized for cell biomass p r o d u c t i o n in the first two batch runs. A f t e r that, the released biomass decreased slowly and a higher ethanol yield was obtained from the 3rd to the 6th batch runs. It can be noted that with this type o f immobilization, the results obtained are better than those obtained with glutaraldehyde treatment. The ethanol concentration is 1.5-2.0 times higher and the immobilized cell supports can be used for longer periods o f time. This fact can be attributed to the negative effect o f the glutaraldehyde on the yeast cells. Continuous fermentation with immobilized S. cerevisiae C o n t i n u o u s fermentations were p e r f o r m e d with the immobilized S. cerevisiae cells on the magnetic supports with and without glutaraldehyde treatment. The immobilized cells used in the previous batch fermen-

tations were packed in a small column and washed for 8 h with sterile distilled water with a flow rate of 10.0 m l / h . The fermentation process was then carried out under the following conditions: The fermentation liquid was p u m p e d into the b o t t o m o f the column. The flow rate was 1.5 m l / h and the dilution rate was 0.5 h -~ (based on the medium volume in the column). The fermentations were run continuously for six days. The ethanol concentration, reducing sugars and released cell biomass concentration were continuously monitored. Figure 4 shows the results from continuous fermentation with yeast cells immobilized on zeolite magnetic supports with glutaraldehyde treatment. It can be seen that the average concentration o f ethanol was 55.0 g/l. The maxi m u m concentration was 62.0 g/l. Productivity of ethanol (based on medium volume in the fermentor) was equal to 30.0 g e t h a n o l / l . h after 144 h o f continuous running. The released cell biomass reached a maximal value of 0.78 x 108 cells/ml medium at 48 h and after that slowly decreased to 0.45 x 108 cells/ml. The maximal ethanol yield was 84% of the theoretical yield. Table 2 shows the results obtained from continuous fermentations with yeast cells immobilized on the three types o f magnetic supports by simple adsorption. It can be seen that in continuous fermentation this type o f immobilization gives better results than that using the glutaraldehyde treatment. In addition, the results obtained using different supports are c o m p a r a b l e (Table 2). As mentioned above, m a n y magnetic supports and methods o f preparing them are available, but it is highly desirable to have a magnetic support whose only difference from a conventional one is in its magnetic properties, and which can be readily prepared by simple procedures. In our study we have f o u n d that the zeolite and activated carbon magnetic particles obtained by the p r o p o s e d method can be used as supports for the a d s o r p t i o n o f yeast cells. This new method for the p r e p a r a t i o n of magnetic particles with the aid o f epoxy resins gives support materials having several advantages over those prepared by other methods. The magnetic particles have no adverse effects on the yeast cells, which exhibit catalytic behaviour similar to that of their non-magnetic counterparts because the magnetic part is completely isolated and there is no direct contact between the immobilized cells and the metal. In addition, this method is flexible because the epoxy resin can act as a

7O0

%

u~

600

7O0 ol

"6 50,0

g

600

0

E

m

0

5 4o.o

o

50.0

10

E

8 400

o 30.0 t~

[K

200

09

o

30 0

08j 06

250

:£

2oo

¥

"-o

04

150

c I

1

2

3

I

I

I

4

5

6

Batch number

FIG. 3. Results from batch fermentation using zeolite and activated carbon magnetic biocatalysts obtained by simple adsorption. Symbols: - 0 - , ethanol from zeolite particles; • , ethanol from the carbon support fixed yeasts (d 1.2-1.6 ram); - - , ethanol from activated carbon particles with diameter 1.0-1.2 mm; - - 0 , released cell biomass in cells/ml x 108 from the zeolite support.

50.0 Ld

~

02

2~ 01

.

1

2

t

,

J

5

4

5

100 6

Days

FIG. 4. Continuous fermentation with S. cerevisiaeimmobilized yeast cells on glutaraldehyde-treated zeolite particles. Symbols: ~, ethanol concentration; ~ , productivity; - - ~ , released cell biomass, 108 x cells/ml.

MAGNETIC SUPPORT FOR ALL IMMOBILIZATION

VoL 71, 1991 TABLE 2. Material

Activated carbon 1.0-1.2 mm

Activated carbon 1.2-1.6 mm

Zeolite 1.6-2.0 mm

117

Results from continuous fermentation using yeast cells immobilized by simple adsorption

Parameters

Time, h 24

48

72

96

120

144

Ethanol (g//)

34.2

50.5

59.8

65.3

66.4

71.2

Productivity (g/I. h)

17.2

25.3

29.9

32.6

33.2

35.6

Released biomass cells/ml x 108

1.02

0.78

0.54

0.45

0.38

0.24

Ethanol (g//)

27.6

54.8

53.2

58.6

66.8

70.7

Productivity (g/l. h)

13.8

27.4

26.6

29.3

33.4

35.4

Released biomass cells/ml x 108

0.92

0.72

0.47

0.30

0.25

0.15

Ethanol (g//)

31.5

46.6

48.1

50.9

56.8

65.0

Productivity (g/l. h)

15.8

23.3

24.0

25.5

28.4

32.5

Released biomass cells/ml x 108

0.94

binding agent between any metal having magnetic properties w i t h a n y s u p p o r t h a v i n g g o o d a d h e s i v e p r o p e r t i e s n e e d e d f o r cell i m m o b i l i z a t i o n . It was f o u n d t h a t t h e fixed cell p a r t i c l e s c a n b e u s e d i n r e p e a t e d b a t c h a n d c o n t i n u o u s a l c o h o l i c f e r m e n t a t i o n . It m u s t b e n o t e d t h a t t h e s i m p l e a d s o r p t i o n o f yeast cells o n m a g n e t i c s u p p o r t s gives b e t t e r results than those obtained in the presence of glutaraldehyde. These results can be used for scale-up optimizat i o n o f t h e c o n t i n u o u s p r o c e s s in f l u i d i z e d - b e d b i o r e a c t o r s u t i l i z i n g m a g n e t i c fields. REFERENCES 1. Hailing, P . J . and Dunnill, P.: Improved non-porous magnetic supports for immobilized enzymes. Biotechnol. Bioeng., 21, 393-416 (1979). 2. Robinson, P. J., Dunnill, P., and Lilly, M. D.: The properties of magnetic supports in relation to immobilized enzyme reactor. Biotechnol. Bioeng., 14, 597-603 (1973). 3. Leemputten, van E. and Horisberger, M.: Immobilization of enzymes on magnetic particles. Biotechnol. Bioeng., 16, 385-396 (1974). 4. Chaplin, M . F . and Kennedy, J . F . : Magnetic immobilized derivatives of enzymes. Carbohydr. Res., 50, 267-274 (1975). 5. Horisberger, M.: Immobilization of protein and polysaccharide on magnetic particles: selective binding of microorganisms by Concanaval A--magnetite. Biotechnol. Bioeng., 18, 1647-1651 (1976). 6. DunnUl, P. and Lilly, D.: Purification of enzymes using magnetite bioaffinity materials. Biotechnol. Bioeng., 16, 987-989 (1974).

1.06

0.87

0.63

0.51

0.38

7. Sada, E., Katon, S., and Terashima, M.: Performance of an enzyme reactor utilizing a magnetic field. Biotechnol. Bioeng., 22, 243-246 (1980). 8. Sada, E., Katon, S., Shiozawa, M., and Fukui, T.: An enzyme reactor using magnetite-containing beads. J. Chem. Eng. Japan, 14, 6, 496-497 (1981). 9. Sada, E., Katon, S., and Terashima, M.: Enhancement of oxygen absorption by magnetite-containing beads of Immobilized glucose oxidase. Biotechnol. Bioeng., 21, 1037-1044 (1981). 10. Sada, E., Katon, S., Shiozawa, M., and Fukui, T.: Performance of fluidized bed reactor utilizing magnetic field. Biotechnol. Bioeng., 21, 2561-2567 (1981). 11. Sada, E., Katon, S., Shiozawa, M., and Matsui, I.: Rates of glucose oxidation with a column reactor utilizing a magnetic field. Biotechnol. Bioeng., 25, 2285-2292 (1983). 12. Hailing, P. J. and Dnnnill, P.: Magnetic supports for immobilized enzymes and bioaffinity adsorbents. Enzyme Microb. Technol., 2, 1-10 (1980). 13. Larsson, P . O . and Mosbach, K.: Alcohol production by magnetically immobilized yeast. Biotechnol. Lett., 1, 501-509 (1979). 14. Birnbaum, S.S. and Larsson, P.O.: Application of magnetic immobilized microorganisms. Ethanol production by Saccharomyces cerevisiae. Appl. Biochem. Biotechnol., 7, 55-60 (1982). 15. Tsnng-Tbing, H. and Jian-Jone, W.: Study on the characteristics of a biological fluidized bed in a magnetic field. Chem. Eng. Res. Des., 65, 237-242 (1987). 16. Parosheva, A.: Polymer Materials, 119 (1984) (in bulgarian) 17. Somogyi, M.: Notes on sugar determination. J. Biol. Chem., 196, 19-23 (1952).