EDITOR
`
`ELEFTHERIOS T. PAPOUTSAKIS
`
`A WILEVJNTERSCIENCE PUBLICATION
`
`qv WILEY
`
`I'll!’/l\hL'I\ mm 1' IHH7
`NEW VORK / CHICHESTEH / BRISBANE / TORONTO ./ SINGAPORE
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`BIBIAU 39 (4) (1992)
`
`ISSN ooos-3592
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`&BI()TECHNOLOGY
`BIOENGINEERING
`
`Eleftherios T. Papoutsakis
`Northwestern University, Evanston, Illinois
`Associate Editors
`
`Douglas S. Clark, University of California
`Berkeley, California
`C. P. Leslie Grady, Jr., Clemson University
`Clemson, South Carolina
`Maria-Regina Kula, Heinrich-Heine Universitat Diisseldort
`in der KFA Julich, Germany
`Daniel l.C. Wang, Massachusetts Institute of Technology,
`Cambridge, Massachusetts
`
`Founding Editor
`
`Elmer L. Gaden, Jr., University of Virginia,
`Charlottesville, Virginia
`Editorial Board
`
`James E. Bailey
`Harvey W. Blanch
`Arnold L. Demain
`lsao Karube
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`Malcolm D. Lilly
`Michael L. Shuler
`Daniel Thomas
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`Scope: The Editors of B&B will consider for publi-
`cation original articles and mini reviews that deal
`with all aspects of applied biotechnology. These
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`- applied aspects of cellular physiology, metabo-
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`enzyme reactors, purification, and applied as-
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`including aerobic
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`in-
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`The editors will consider papers for publications
`0 plant-cell biotechnology;
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`transport
`including
`° biochemical
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`in-
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`including coal biotech-
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`0 mineral biotechnology,
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`sources engineering; and
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`Biotechnology and Bioengineering (ISSN:0006-
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`Kuo-Ying Amanda Wu and Keith D. Wisecarver*
`Department of Chemical Engineering, The University of Tulsa,
`Tulsa, Oklahoma 74704
`
`Received July 78, 7997/Accepted July 26, 79.97
`
`A new cell immobilization technique is described in which
`polyvinyl alcohol
`is crosslinked with boric acid, with the
`addition of a small amount of calcium alginate. The pres- V
`ence of the calcium alginate improves the surface proper-
`ties of the beads, preventing agglomeration. A pure culture
`of phenol-degrading Pseudomonas was immobilized in the
`PVA-alginate beads. Phenol was successfully degraded in a
`fluidized bed of the beads, indicating that cell viability was
`maintained following the immobilization procedure. The
`PVA-alginate beads proved to be very strong and durable,
`with no noticeable degradation of the beads after 2 weeks
`of continuous operation of the fluidized bed.
`Key words: immobilized cell - PVA - polyvinyl alcohol - flu-
`idized bed
`
`INTRODUCTION
`
`Immobilization of living cells has become an established
`technique for increasing the productivity of biochemi-
`cal engineering processes. One of the most \videly used
`techniques for cell immobilization is cell entrapment, in
`which the living cells are enclosed in a polymeric matrix
`which is porous enough to allow the diffusion of sub-
`strates to the cells and of products away from the cells.
`Materials which have been successfully used for cell
`entrapment include agar, agarose, kappa—carragennan,
`collagen, alginates, chitosan, polyacrylamide, polyure-
`thane, and cellulose.°’I-Iowever, each of these polymers
`has drawbacks, such as poor mechanical strength and
`durability (agar, agarose, kappa—carragennan, collagen,
`alginates, chitosan),
`toxicity to microorganisms (poly-
`acrylamide, polyurethane), or high cost.”
`Recently, the use of polyvinyl alcohol (PVA) for cell
`immobilization has been investigated. Ariga et al.’ used
`the technique of iterative freezing and thawing of PVA
`to form a gel suitable for cell immobilization. They found
`that this technique produced a low—Cost material with
`a rubber—like elasticity and high strength. Hashimoto
`and Furukawa3 used a simpler and less energy—intensive
`method for PVA immobilization. The crosslinked the
`PVA using a boric acid solution, producing a monodiol—
`type PVA-boric acid gel lattice.“ Activated sludge was
`successfully immobilized using this technique, with no
`apparent loss of biological activity. The beads thus pro-
`duced proved to be very durable, outlasting beads pro-
`
`* To whom all correspondence should be addressed.
`
`duced by other cell immobilization techniques such as
`polyacrylamide and calcium alginate.‘
`The PVA-boric acid technique provides an easy and
`low—cost method of cell immobilization, producing elas-
`tic beads of high strength and durability} There are two
`potential problems with this technique, however. The
`saturated boric acid solution used to crosslink the PVA
`
`is highly acidic (pH of approximately 4) and thus could
`cause difficulty in maintaining cell viability. Hashimoto
`and Furukawal hypothesized that activated sludge could
`be successfully immobilized without loss of biological
`activity due to the presence of extracellular polymer in
`the sludge, which enables the microorganisms to endure
`changes in cultivation conditions, and cast doubt on
`whether the technique could be applicable for other
`types of cells. To date, the use of the PVA-boric acid im-
`mobilization technique for other types of microorgan-
`isms has not been demonstrated. In addition, PVA is an
`extremely sticky material; PVA beads, therefore, have a
`tendency to agglomerate. This is particularly a problem
`in applying PVA-immobilized cells to fluidized bed
`reactors. The elasticity and high strength of the PVA
`beads are ideally suited to the high shear stresses en-
`countered in fluidized beds,’ but the tendency of PVA
`beads to agglomerate can make fluidization difficult or
`impossible.
`In this article, a new cell immobilization technique is
`described which eliminates the agglomeration problem
`of the PVA-boric acid method by the addition of a small
`amount of calcium alginate. The technique was demon-
`strated for a pure culture ofPseudom0nas. The resulting
`beads were utilized in a three—phase fluidized bed biore-
`actor to demonstrate viability of the immobilized cells.
`
`MATERIALS AND METHODS
`
`The microorganisms used in this work was a pure strain
`of phenol-degrading Pseudomomzs isolate (strain #1101)
`obtained from Microbe Masters Inc., Baton Rouge, LA.
`The organisms had been acclimated to 500 ppm phenol
`as a sole source of carbon before shipment. The me-
`dium used for growth of the culture is given in Table I;
`phenol was the sole carbon source in all experiments.
`Cultures were grown at a temperature of 30°C to an
`
`Biotechnology and Bioengineering, Vol. 39, Pp. 447-449 (1992)
`© ‘I992 John Wiley & Sons, Inc.
`
`CCC 0006-3592/92/O40447—O3$04.00
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`Table 1. Composition of stock nutrient medium.
`
`Component
`
`Concentration
`
`CaClg - 2HgO
`wgso,-7H3o
`MHSO4 ‘ H30
`FCSO4 ’
`Trace elements“
`
`K;HPO4/KH3PO4 buffer, pH 7.6 to 9.0
`
`Variable
`
`2.13 g/L
`1.00 g/L
`0.6 g/L
`0.02 g/L
`0.02 g/L
`
`i5muL
`
`“ Trace elements include (mg/L): zinc, 0.01; copper, 0.001; sele-
`nium, 0.001.
`
`OD (460 nm) of approximately 1.1, a period of approxi-
`mately 24—30 h, at which time they were centrifuged at
`1000 rpm for 10 min.
`The cell immobilization process is illustrated in Fig-
`ure 1. Water was added to 43.7 g of polyvinyl alcohol
`(100% hydrolyzed, average MW 77,000 to 79,000) to ob-
`tain 330 mL of solution. The solution was then carefully
`heated to a temperature of 60°C to completely dissolve
`the PVA. A solution of 3.5 mL of 2% sodium alginate
`(low viscosity, approximately 250 cps) in water was pre-
`pared by gently stirring for 30 min and added to the PVA
`solution. The PVA-alginate solution was then cooled to
`a temperature of 35°C.
`The centrifuged cells (45.6 g wet weight) and 35 mL
`of distilled water were mixed with 6.3 mL of the growth
`medium (Table I) to supply nutrients for the microbial
`
`Concentrated suspension
`of bacteria
`
`Aqueous solution
`-13% PVA at 60 "C
`
`~0.02% sodium alginate
`1
`Cool to 35 °C
`/
`mix and drop into solution of saturated
`boric acid with 2% calcium chloride
`
`1
`
`allow to harden in
`solution for 24 hours
`
`1
`rinse with distilled water
`
`1
`3 mm diameter PVA-immobilized beads
`~10% PVA
`
`cells during the solidification process. This mixture
`was then added to the PVA solution and mixed thor.
`oughly. The final suspension of cells contained 10.8%
`(w/v) wet cells (total cell density of 6.4 X 10” cells/cm3),
`10.4% (w/v) PVA, 0.02% (w/v) sodium alginate, and
`growth medium diluted to 1.5 parts per 100.
`Beads were formed by crosslinking the PVA with boric
`acid: the PVA-alginate cell suspension was extruded via
`a peristaltic pump through a thin needle into 10 L of so-
`lution consisting of saturated boric acid and 2% CaCl2 -
`2H 3O at room temperature. The beads were kept gently
`stirred in this solution for 24 h to complete the solidi-
`fication, and then rinsed with distilled water to remove
`any excess boric acid. Approximately 420 cm} of beads
`containing the immobilized cells were thus formed. The
`diameter of the beads was approximately 3 mm.
`Cell viability and bead integrity were tested in an 8-L
`fluidized bed column. The 10.16 cm ID column was
`
`sparged with air at a rate of 1.4 L/min. Growth medium
`(Table I), containing varying concentrations of phenol,
`was fed continuously from the top of the column, and
`effluent withdrawn at the column bottom, at a rate of
`2.5 L/h. The fluidized bed experiments were conducted
`at room temperature.
`the samples in this
`Phenol concentrations of all
`work were analyzed by gas chromatography" (Hewlett-
`Packard model 5890A with flame ionization detector).
`
`RESULTS AND DISCUSSION
`
`Initial attempts at cell immobilization were made using
`only PVA crosslinked with boric acid. However,
`the
`beads thus formed had a strong tendency to agglomerate
`into a mass of polymer which was very difficult to break
`up. The beads could, therefore, not be utilized in a flu-
`idized bed. This agglomeration problem appears to be
`due to the relatively slow crosslinking of the PVA by
`boric acid. Droplets of PVA, which have not been suffi-
`ciently crosslinked, tended to agglomerate into a mass.
`This problem persisted even with vigorous stirring of
`the boric acid solution to keep the beads suspended.
`Attempts were then made to use a combination of
`PVA—boric acid and calcium alginate, by hardening a
`mixture of PVA and sodium alginate with a mixed solu-
`tion of boric acid and calcium chloride. In this way, it
`was thought that the PVA might contribute durability
`and strength to the beads, while calcium alginate might
`improve the surface properties of the beads, reducing
`the tendency to agglomerate. The percentage of PVA ill
`the beads was kept in the range of 10% to 12.5%, 35
`recommended by Hashimoto and Furukawaf to main‘
`tain maximum bead strength. Varying ratios of dfl’
`PVA to 2% sodium alginate solution were attempted fOf
`the immobilization procedure. It was found that ag-
`glomeration of the beads was entirely prevented f0T
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`taneously when the sodium alginate solution contacts
`the calcium chloride solution, and that the resulting
`polymeric structure is sufficient to keep the beads from
`agglomerating during the PVA crosslinking process. The
`resulting beads were strong and highly elastic, and of
`nearly spherical shape.
`Fluidized bed experiments were conducted continu-
`ously over a period of 2 weeks. Inlet phenol concentra-
`tion was increased in steps from 250 to 1300 mg/L (inlet
`phenol concentrations of 250, 350, 450, 550, 650, 750,
`850, 950, 1000, 1100, 1200, and 1300 mg/L were used),
`with the bioreactor allowed to achieve steady state be-
`tween each step increase. This range was chosen to be
`close to the range of phenol concentrations to which the
`microorganisms had been acclimated. The steady-state
`outlet phenol concentration, measured by gas chro-
`matography, was found to be zero for each of these inlet
`phenol concentrations.
`Phenol removal due to stripping was measured by di-
`verting the off-gas from the column through a NaOH
`solution (pH 10) and measuring the absorbed phenol.
`The phenol removed by stripping was found to be less
`than 0.1% of the total phenol degradation. Bioadsorp—
`tion has been found by a number of investigators to be
`small in comparison with biodegradation.”'7 Therefore,
`it can be concluded that phenol removal in the fluidized
`bed was due almost entirely to biodegradation, indicat-
`ing that cell viability was maintained through the im-
`mobilization procedure.
`The bioparticles made from the PVA—alginate immo-
`bilization technique proved to be very durable, show-
`ing no sign of breakage or disintegration after 2 weeks
`of continuous operatioii of the fluidized bed. The PVA-
`alginate beads showed no tendency to agglomerate at
`
`CONCLUSIONS
`
`Immobilization of living cells of a phenol—degrading
`Pseudomonas isolate in a PVA-boric acid gel was dem-
`onstrated. Cell viability was indicated by degradation of
`phenol in a fluidized bed reactor of the beads. The pres-
`ence of a small amount of calcium alginate in the beads,
`as small as 0.02%, was shown to prevent agglomeration
`of the PVA beads. The resulting beads were highly elas-
`tic and durable; they were able to withstand high shears
`in a three—phase fluidized bed for 2 weeks of continu-
`ous operation with no noticeable breakage or shrinkage
`of the beads. The successful immobilization of a Pseu-
`
`domonas isolate by the PVA-boric acid method indi-
`cates that the technique might be applicable to a wide
`variety of microorganisms.
`
`References
`
`l. Ariga, O., Takagi, H., Nishizawa, H., Sano, Y. 1987. J. Ferment.
`Technol. 65: 651.
`
`2. Gaudy, A.F., Kincannon, D.F., Manickam, T.S. June 1982.
`EPA—600/2-82-075.
`
`3. Hashimoto, S., Furukawa, K. 1987. Biotechnol. Bioeng. 15: 52.
`4. Kincannon, D.F., Stover, E.L., Nichols, V., Medley, D. 1983.
`J. Water Pollut. Control Fed. 55: 157.
`
`5. Kuu, W.Y., Polack, J. A. 1983. Biotechnol. Bioeng. 25: 1995.
`6. Ochiai, H., Shimizu, S., Tadokoro, Y., Murakami, I. 1981. Poly-
`mer 22: 1456.
`
`7. Petrasek, A. C., Kugelman, I. J., Austern, B. M., Pressley, T. A.,
`Winslow, L. A., Wise, R. H. 1983.1 Water Pollut. Control Fed.
`55: 1286.
`8. Standard methods for the examination of water and waste-
`water, 16 ed. 1985. American Public Health Association, Wash-
`ington, DC.
`9. Tampion, J., Tampion, M. D. 1987. Immobilized cells: Principles
`and applications, Cambridge University Press, Cambridge, UK.
`
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