Sep 3, 1991 - pentachlorophenol (PCP), was grown in a defined mineral salts medium with sodium glutamate as the carbon and energy source. When cells ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1992, p. 727-730
Vol. 58, No. 2
0099-2240/92/020727-04$02.00/0 Copyright © 1992, American Society for Microbiology
Preparation of Encapsulated Microbial Cells for Environmental Applicationst KEITH E. STORMO AND RONALD L. CRAWFORD* Department of Bacteriology and Biochemistry and Center for Hazardous Waste Remediation Research, Food Research Center, Room 202, University of Idaho, Moscow, Idaho 83843 Received 3 September 1991/Accepted 14 November 1991
An improved method for the encapsulation of bacteria into microspheres of alginate, agarose, or polyurethane is described. Cell suspensions were passed through a low-pressure nozzle into an aqueous phase where matrix polymerization or gelation yielded beads 2 to 50 ,um in diameter. Trials with a chlorophenoldegrading Flavobacterium species showed that cells entrapped by these procedures were as catabolically active as free cells. These types of beads should have numerous applications in the fields of environmental science and engineering.
Immobilization of bacteria, plant or animal cells, and enzymes (1, 7, 8, 10, 21) in a variety of matrices such as calcium alginate (28), K-carrageenan (29), and polyurethane (19, 23, 33, 34) has been shown to provide advantages over the use of free cells or enzymes in various biotechnological applications. For example, immobilization may increase the cell loading capacity (16, 17, 30) and/or increase rates of production of microbial products (2, 13, 22) in bioreactors. A serious constraint to high productivity of immobilized aerobic cells, however, has been the limitations on activity caused by reduced mass transfer of oxygen into the interior of the immobilization matrix. This is particularly true for spherical matrices, where most of the interiors of large beads may be anaerobic (2, 4, 5). Such limitations can be overcome by decreasing the diameter of the beads. Chen and Humphrey (6) have discussed the phenomenon of critical particle diameter for optimal respiration by bacteria within beads. With rapidly metabolizing immobilized cells, active cells may be found to a depth of only 50 to 200 ,um into a bead (3-6, 21, 24, 25, 31), so that as much as 80% of the volume of a 2-mm bead may contain inactive or dead cells. Obviously, preparing beads that contain 100% active cells offers great advantages in terms of the speed and the cost of immobilized-cell processes. We are interested in promoting oxygen transfer into spherical immobilization matrices that have been loaded with aerobic pollutant-degrading bacteria. These bacteria, entrapped as active cells within polymeric beads, are to be introduced into subsurface environments to degrade specific toxic chemical contaminants. This will require the use of very small beads (9); unfortunately, current methods of entrapping unharmed bacteria in microspheres make it difficult to consistently prepare beads with diameters of less than about 0.5 mm. Emulsion techniques have been used as a principal method of entrapping biocatalysts within very small beads. For example, cells can be added to molten agarose held at 45°C, and the resulting suspension can be stirred into oil held at 45°C, forming an emulsion of agarose beads. Cooling of
the emulsion produces beads 10 to 100 ,um in diameter (3, 11, 12, 18, 30, 32). Unfortunately, producing large quantities of beads and washing them free of oil are both difficult with emulsion techniques. Here we describe a method of producing large quantities of small beads of a consistent diameter that does not require separating and washing the prepared beads. High cell loadings (>50%, wt/vol) of virtually 100% active cells within beads 2 to 50 ,um in diameter are possible. Flavobacterium sp. strain ATCC 39723, a gram-negative aerobe that degrades a variety of chlorinated phenols such as pentachlorophenol (PCP), was grown in a defined mineral salts medium with sodium glutamate as the carbon and energy source. When cells reached mid-logarithmic phase, PCP (50 mg/liter) was added to induce the catabolic enzymes responsible for degradation of chlorinated phenols. When the PCP had been degraded, the cells were harvested by centrifugation (19, 20). Harvested cells were suspended at 20% (wet weight) per volume of either 2 or 4% sodium alginate (Sigma) in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) immobilization buffer (20), 1.5 to 5.0% molten agarose (Bethesda Research Laboratories) held at 45°C, or a pure polyurethane prepolymer. The polyurethane prepolymer was synthesized in our laboratory. It was designed to minimize the release of carbon dioxide during the curing process once the prepolymer aerosol contacted an aqueous phase. It was prepared by reacting polyethylene glycol (100 meq of OH-) with toluene diisocyanate (200 meq of cyanate) and various metabolizable carbon compounds (e.g., glucose or dextrin at 5 meq of OH-) as the cross-linking agents. Cell suspensions were pumped through a low-pressure nozzle (Sonic Development Corp., Parsippany, N.J.) that passed through a rubber stopper and was arranged so that it introduced a fine aerosol of cell suspension into a glass carboy containing a stirred aqueous phase. The apparatus was equipped with a vent tube and a trap to remove any particles that passed out of the system in the exhaust phase (Fig. 1). A drawing of the apparatus appears in the review by Levinson et al. (14). Gas pressures through the nozzle were varied between 5 and 20 lb/in2, with nitrogen supplied from standard pressure bottles. The aqueous phase for the collection of microspheres was 50 mM CaCl2 for the cells in
* Corresponding author. t Publication no. 91515 of the Idaho Agricultural Experiment Station.
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FIG. 1. Immobilization apparatus.
alginate suspension or cold (4°C) buffer for the cells in agarose or prepolymer suspensions. The low-pressure nozzle apparatus produced an aerosol of very small spheres (2 to 50 p.m in diameter) irrespective of the type of cell suspension passed through the system. The spheres of alginate polymerized into bacterium-loaded microbeads upon contact with the CaCl2 collection fluid (Fig. 2). The agarose spheres hardened into microbeads upon contact with cold buffer. Cells in microbeads were acridine orange stained, and observation by epifluorescence micros-
copy showed that nearly all beads had stained cells inside. The polyurethane prepolymer polymerized into polyurethane microbeads upon contact and reaction with water. Typically, 23.5 ml of beads per 27.5 ml of suspension passed through the bead-making apparatus was produced. Bead sizes were determined with a multichannel Coulter Counter (model TA II; Coulter Electronics, Inc., Hialeah, Fla.) equipped with a population accessory that analyzed in 16 channels between 1.59 and 50.8 um in diameter. The counter used a 140-pum orifice and was calibrated with 9.82-,umdiameter standard latex beads; 50% of the beads (by volume) were in size fractions of
