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effective for use in bioreactors, they may not be desirable in open environmental applications. The pore size of the support applied to a bioreactor process should be much smaller than the encapsulated cell to avoid or minimize cell release and washout from the bioreactor. Thus, cells remain inside the support while substrates and nutrients can diffuse in and products can diffuse out. In field applications, however, supports with high porosity are desirable because they provide high cell mass loading as well as high diffusional mass transfer rates. Therefore, the use of natural polymers with relatively larger pore size and greater degree of biode-gradability may be suitable for some subsurface applications. It may be desired to release the encapsulated cells after positioning them in the target zone, and after adaptation to the surrounding environment (35). In general, natural polymers are recommended for use in soil. The characteristics of encapsulated inoculants and their advantages over free cells for use in soil have been discussed comprehensively in earlier reports (36,37).

Emulsion techniques have been utilized for the encapsulation of viable cells within micro- (0.1-1 mm) and macrospheres (1-3 mm) of natural thermotropic gel polymers such as agar (48), agarose (48,49), k-carrageenan (48,50), and gellan gum (51). The sphere formation process involves the dispersion of two immiscible liquid phases resulting in a water-in-oil (w/o) emulsion. A suspension of viable cells in an aqueous solution of the polymer (dispersed phase) is emulsified in a hydrophobic phase such as a vegetable oil (continuous phase). The gelation of the small droplets of the dispersed phase is subsequently initiated by decreasing the emulsion temperature below the sol-gel transition temperature.

Other researchers have used emulsion techniques, termed as emulsification-internal gelation, to encapsulate biocatalysts (52,53), and DNA (54) in alginate microspheres. The gelation of alginate droplets was triggered by gentle acidification (to pH 6.5) of the water-oil dispersion through adding an oil-soluble acid and releasing soluble calcium ions from a salt complex.

This monograph outlines an emulsification-internal gelation method for encapsulation of gasoline-degrading bacteria in gellan gum microspheres. Calcium is used as the gelling agent and is available inside gellan gum sol droplets to induce an internal gelation.

2. Materials 2.1. Reagants

1. Microorganisms: A mixed bacterial culture in frozen or freeze dried state, capable of degrading gasoline hydrocarbons, for instance pre-isolated from a gasoline-contaminated soil and enriched in a mineral salts medium containing gasoline as a single source of carbon.

2. Mineral salts medium (MSM): an aqueous medium containing in g per liter: KH2PO4 0.87, K2HPO4 2.26, (NH4)2SO4 1.1, and MgSO4 0.047, and supplemented with 1 mL/liter trace metal solution composed in g/liter of: Co(NO3)2 ■ 6H2O 0.291, AlK(SO4)2 ■ 12H2O 0.474, CuSO4 0.16, ZnSO4 ■ 7H2O 0.288, FeSO4 ■ 7H2O 2.78, MnSO4 ■ H2O 1.69, Na2MoO4 ■ 2H2O 0.482 and Ca(NO3)2 ■ 4H2O 2.36. The pH of trace metal solution is lowered to about 2.0 by adding a small amount of a 2 N HCl for complete dissolution of minerals. Mineral salts medium is sterilized at 121°C for 20 min. The final pH of the medium is 7.0 ± 0.1.

3. Gasoline: an unleaded product with an octane number of 87 purchased from a commercial fuel distributor.

4. Gellan Gum: Gellan gum (Kelcogel®, CP Kelco US, Inc., San Diego, CA) is an extracellular polysaccharide produced through aerobic fermentation processes by the microorganism Sphingomonas paucimobilis ATCC 31461 (55), earlier referred to as Pseudomonas elodea (56,57), Auromonas elodea (58), and Sphingomonas elodea (59). The chemical structure of gellan gum (see Fig. 1) is made up of repeating tetrasaccharide units consisting of a linear sequence of D-glucose, D-glucuronic acid, D-glucose, and L-rhamnose (60,61).

The mechanism of gelation and texture of gellan gum suggests a strong similarity with agar and carrageenans. However, gellan gum gel has superior rheo-logical properties to agar and carrageenan gels at equivalent concentrations (62), and therefore, it can be used at substantially lower concentrations. The gel has been stable over the wide pH range of 2.0 to 10.0 (63), suggesting its suitability for use in both acidic and basic environments. The application of gellan gum for encapsulation of viable cells has been addressed at considerably lower concentrations of both gel and gelling agent compared to K-carrageenan, agar, and alginate (48,64). Unlike some other ion-sensitive gelling polysaccharides such as alginate and K-carrageenan, the reactivity between gellan gum and ions is non-

Gellan Biotechnology

-3).p.D.Glcp.(l-4).p.D.GlcpA.(l-4).p.D.Glcp.(l-4).a.L.Rhap.(l-

Fig. 1. Gellan gum repeating unit (60,61).

-3).p.D.Glcp.(l-4).p.D.GlcpA.(l-4).p.D.Glcp.(l-4).a.L.Rhap.(l-

Fig. 1. Gellan gum repeating unit (60,61).

specific and gels can be formed with a wide variety of cations including alkaline and alkaline-earth cations (65,66).

5. Canola oil A food-grade product purchased from a local food distributor, sterilized at 121°C for 20 min.

6. Sorbitan monoleate, Span®80.

7. Polyoxyethylene [20] sorbitan monooleate, Tween®-80, A 0.1% (v/v) solution is prepared in distilled water sterilized at 121°C for 20 min.

8. Calcium chloride: A 0.1% (w/v) solution is prepared in distilled water sterilized at 121°C for 20 min.

9. Sodium hydroxide: A 0.1 N solution is prepared in distilled water.

10. Hydrogen chloride: A 2 N solution is prepared in distilled water.

11. De-ionized water sterilized at 121°C for 20 min.

12. Ice/water bath.

2.2. Equipment

1. Round-bottomed cylindrical reaction vessel, 10-cm diameter x 1 L capacity, equipped with a standard four-blade baffle

2. Quarter-circular paddle impeller, 5-cm diameter.

3. High shear mixer, 5000 rpm (e.g. ,T-line Laboratory Stirrer, model 102, Talboys Engineering Corp., Montrose, PA).

4. Photo/contact digital tachometer.

5. Hot plate/magnet stirrer.

6. Magnet stirrer.

7. Graduated plastic 50-mL centrifuge tubes with screw caps.

8. Centrifuge and rotor suitable for 50-mL centrifuge tubes.

9. Shaker/incubator.

10. Tap water vacuum ejector with connector tube and plastic conical tip.

11. 1000 mL Erlenmeyer flasks.

12. 100 and 1000 mL Graduated cylinders.

14. All glassware and equipment in contact with bacteria are sterilized at 121°C for 20 min.

3. Methods

See Note 1.

3.1. Preparation of Enrichment Culture

1. A given amount of mixed bacterial culture is suspended in 250 mL mineral salts medium supplied with gasoline at 100 to 150 mg/L, as the sole source of carbon, in a 1000-mL Erlenmeyer flask. The initial number of cells may range from108 to 109 colony forming units (CFU)/L of medium.

2. The enrichment can be done through multiple successive transfers before harvesting the cells for encapsulation.

3.2. Cell Harvesting

1. The cells are harvested by transferring the enriched culture into centrifuge tubes and centrifugation at 12,000g for 15 min at 4°C. Cell pellets are collected and re-suspended in 2 mL calcium chloride solution.

3.3. Preparation of Gellan Gum Pregel Solution (Dispersed Phase)

1. Gellan gum is dispersed in 100 mL de-ionized water at a concentration of 0.75% (w/v).

2. The dispersion is heated to between 75°C and 80°C while being stirred on a hot plate/magnet stirrer to dissolve gellan gum.

3. Calcium chloride is added to the solution at a concentration of 0.06% (w/v).

4. The resulting pregel solution is then left at room temperature to cool to 45°C.

3.4. Incorporation of Bacteria

1. The pH of pregel solution is adjusted between 6.9 and 7.2 with 0.1 N NaOH before incorporation of bacteria.

2. The suspension of bacteria in calcium chloride solution (see Subheading 3.2.) is then added to the pregel solution.

3. The mixture is agitated for 2 to 3 min for complete homogenization.

4. The final concentration of bacteria may range from 109 to 1010 CFU/mL (see Note 2).

3.5. Preparation of Canola Oil (Continuous Phase)

1. Span® 80 is admixed with 300 mL canola oil at 0.1% (w/w) in the reaction vessel (see Note 3).

2. The oil is heated to 45°C on a hot plate while being stirred using the high shear mixer.

3. The impeller is adjusted at one-sixth of the liquid height from the reactor bottom.

4. The impeller speed is set at 4500 rpm by means of a tachometer and re-adjusted during operation if necessary.

3.6. Emulsification

1. Homogenized bacteria suspension (see Subheading 3.4.) is emulsified in canola oil (see Subheading 3.5.). The resulting w/o emulsion is stirred for 3 min to achieve homogeneity (see Notes 4-6).

3.7. Gelation

1. The gelation of gellan gum is triggered by cooling the reaction vessel to about 15°C using the ice bath, leading to formation of gellan gum-encapsulated cell microspheres.

3.8. Separation of Microspheres

1. The oil-microsphere dispersion (see Subheading 3.7.) is transferred with gentle mixing into 500 mL of calcium chloride solution.

2. The oil is removed by aspiration using the tap water ejector after partitioning of microspheres into the aqueous phase.

3. The microspheres are rinsed repeatedly with the Tween®-80 solution , and stored in calcium chloride solution at 4°C.

4. Notes

1. Even though the materials and methods described in this monograph are used for the encapsulation of gasoline-degrading bacteria, they may be applicable to encapsulation of other microbial degraders in macro- or microspheres subject to the employment of proper emulsification conditions.

2. Concentration of emulsifier, Span® 80, in the continuous phase may vary from 0 to 0.15% (w/w) to obtain different final particle size distributions.

3. Emulsification-internal gelation method described in this monograph allows encapsulating bacteria at a cell loading range of, but not limited to, 109 to 1010 CFU/mL microsphere.

4. Emulsion stirring speed may vary from 1000 to 5000 rpm to obtain different final particle size distributions.

5. Dispersed phase volume ratio (the ratio of dispersed phase volume to total emulsion volume) may be adjusted between 0.077 and 0.25 to obtain different final particle size distributions.

6. Emulsification time may be set between 1 and 10 min to obtain different final particle size distributions.

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