The ability to preserve labile biological material for extended periods of time in an active and/or viable condition is important in a wide range of medical, agricultural and industrial applications. Current methods of preservation are energy-intensive and generally require cold storage.

Furthermore, activity and/or viability levels after preservation are often unsatisfactory.

Freeze-drying is often used for preservation and storage of biomaterial. The subject to be preserved is placed in solution and the solution is then frozen. The frozen solid is then exposed to a vacuum under conditions where it remains solid and the water and any other volatile components are removed by sublimation. Although freeze-drying is widely used, freeze-dried bacteria are unstable at ambient temperatures and require refrigeration. Significant reductions in viability are observed and the approach is generally time-and energy-intensive and thus expensive.

Other preservation methods, such as ambient temperature drying, spray drying, liquid formulations and freezing with cryoprotectants, also lead to significant reductions in activity/viability.

Addition of glass forming bioprotectors, such as trehalose or hydroxyectoine, prior to drying, has been shown to stabilize gram negative microorganisms to the extent observed in true anhydrobiotic organisms (Garcia de Castro et al (2000) Appl Environ Microbiol 66 4142-4144, Manzanera et al (2002) Appl Environ Microbiol 68 4328-4333). These stabilizers form glasses around the microorganism which dramatically slow the rate of chemical reactions. (Franks F; Biophysics and biochemistry at

low temperatures; Cambridge University Press 1985). Further improvements in stability are observed by osmotically preconditioning cells in a medium which stimulates intracellular trehalose, prior to vacuum drying in a trehalose solution (Bullifent et al (2001) Vaccine 1239-1245, Garcia de Castro et al (2000) supra). However, the stability of biomaterials in organic glass can be compromised by exposure to environmental factors such as humidity, oxygen and high temperature.

The present inventors have recognised that biomaterial in a dried state is resistant to organic solvents and can be encapsulated in an active form in solid plastics materials, such as polystyrene. Furthermore, cells encapsulated in this way have been found to remain in an active and viable form for long periods at ambient temperature. Biomaterial encapsulated as described herein is resistant to physical, chemical and environmental stress and can be moulded and subjected to manufacturing processes without significant reductions in activity and/or viability.

The present invention, in various aspects, provides methods and means for the stabilisation and preservation of active biomaterials. These methods may be useful, for example, in protecting biomaterial from humidity, oxidation and other stresses such as oxidation that would otherwise compromise activity and/or viable. Biomaterial preserved as described herein is stable for extended periods and may be suitable for long-term storage.

One aspect of the present invention provides a method of preserving biomaterial in an active condition comprising; drying active biomaterial in the presence of a glass forming stabiliser, admixing the dried biomaterial with a plastics material liquid, and;

causing said plastics material to solidify to encapsulate the biomaterial therein.

The biomaterial may be dried to below about 10% residual moisture. Under these conditions, the stabiliser coalesces to form a non-crystalline, vitreous, solid physical state (i. e. a glass). The particles of organic glass which are formed by drying the stabiliser encase the biomaterial and provide stability by dramatically slowing the rate of chemical reactions. The dried biomaterial thus comprises active biomaterial encased in the organic glass which is formed by the stabiliser.

The formation and use of organic glass is well known in the art (see, for example Bullifent et al (2001) Vaccine 1239-1245, Garcia de Castro et al (2000) Appl Environ Microbiol 66 4142- 4144, Manzanera et al (2002) Appl Environ Microbiol 68 4328- 4333). Glass-forming stabilisers suitable for use in methods of the invention are preferably insoluble in organic solvents and soluble in water.

Examples of glass-forming stabilisers include non-reducing carbohydrates, such as trehalose, hydroxyectoine, maltitol (4-O- ss-D-glucopyranosyl-D-glucitol), lactitol (4-O-ss-D- galactopyranosyl-D-glucitol), palatinit [a mixture of GPS (oe- D-glucopyranosyl-1-6-sorbitol) and GPM (a-D-glucopyranosyl-1-0-6-mannitol)], and its individual sugar alcohol components GPS and GPM and hydrogenated maltooligosaccharides and maltooligosaccharides. Non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other straight chain polyalcohols such as neotrehalose, lactoneotrehalose, galactosyl-trehalose, sucrose, lactosucrose, raffinose, stachyose and melezitose may also be used.

Other suitable glass-forming stabilisers include amino acids such as hydroxyectoine.

The amount of glass-forming stabiliser used in the present methods will depend on several variables, most particularly, the nature of the stabiliser and the biomaterial that is being stabilized and the method of drying. For example, hydroxyectoine may be used at between 0.5M and 2M and trehalose at between 0. 1M and 1.5M.

Biomaterial dried with the glass forming stabiliser is resistant to the plastics material liquid (i. e. it retains activity). Biomaterial which is not dried with the glass-forming stabiliser is not resistant to the plastics material liquid.

The dried biomaterial may be admixed with the plastics material liquid in a particulate form i. e. particles of biomaterial may be mixed with and encapsulated by the plastics material liquid.

In other embodiments, the dried biomaterial may be in a non- particulate form and may, for example, be shaped, moulded, or formed into a solid 3 dimensional object such as a block, tablet, ingot, nugget, patch, sheet or ball of dried biomaterial. Such objects may be generated, for example, by compressing the dried biomaterial using standard techniques.

Objects comprising or consisting of dried biomaterial may be admixed with the plastics material liquid by immersing, suspending and/or mixing the objects in the plastics material liquid or by applying the plastics material liquid to the object, for example as a coating, in order to encapsulate the dried biomaterial. A coating may be applied by any convenient technique, including spraying or brushing.

Plastics materials suitable for encapsulating biomaterial include synthetic, semi-synthetic and natural organic polymers.

In some embodiments, the plastics material may be non- biodegradable, for example polystyrene, polyvinylchloride polycarbonate, polyethylene, Mylar, cellophane, polyacrylates,

ethylene-vinyl acetate polymers and other acyl substituted cellulose acetates and derivatives thereof, non-erodible polyurethanes, polyvinyl fluoride, poly (vinyl imidazole), chlorosulphonated polyolifms, polyethyleneoxide, polyvinyl alcohol, or nylon.

In some embodiments, the plastics material may be biodegradable or bioerodible, for example poly (lactide) (PLA), poly (glycolic acid) (PGA), poly {lactide-co-glycolide) (PLGA), and other poly (alpha-hydroxy acids), poly (caprolactone), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polyhydroxyalkanoates, polycyanoacrylates, degradable polyurethanes and other organic matrices, such as gums, latex/rubber and resins. Suitable plastics may, for example, be isolated from natural sources, including bacteria.

The plastics material is preferably soluble in an organic solvent. The plastics material liquid preferably comprises a plastics material dissolved in an organic (i. e. non-aqueous) solvent. The biomaterial, in the form of organic glass particles, is then admixed with this solution. The organic solvent is preferably a non-polar organic solvent, such as hexane, acetone, chloroform, toluene or xylene. Polar solvents such as ethanol may also be useful for some applications.

In order to solidify or fix the plastics material and encapsulate the biomaterial in the organic glass, the organic solvent may be progressively removed from the plastics material solution. This may be achieved by any convenient means, for example air-, freeze-, spray/freeze-or vacuum-drying. As the organic solvent is progressively removed, the plastics material solidifies and encapsulates the cells that are contained therein. In other embodiments, the plastics material liquid may be solidified by other means such as cooling, photo- polymerisation, heat-induced polymerisation, or chemical-induced polymerisation.

After preservation as described above and, optionally storage for an extended period, a method may comprise disrupting the plastics material to expose the encapsulated biomaterial. The plastics material may be physically disrupted, for example by piercing or tearing the material or by eroding or abrading its surface, or chemically or biochemically disrupted, for example by reaction with one or more external agents. External agents, may, for example, include organic solvents which dissolve the plastics material. Biodegradable plastics materials such as PLGA may be gradually dissolved or"dismantled"by biological agents, e. g. enzymes, phagocytic cells or micro-organisms.

Exposed biomaterial may then be placed in an appropriate medium or carrier and optionally combined with other constituents, for example an adjuvant, prior to use. Thus, the invention includes methods of reconstituting biomaterial preserved by the methods described herein. The nature and amount of medium or carrier used for reconstitution will depend upon the biomaterial as well as its intended use.

In other embodiments, biochemical degradation of the plastics material may occur in si tu and no further steps are required.

For example, the plastics material may degrade within the body to expose active biomaterial with therapeutic properties.

Active biomaterial may comprise or consist of viable cells, for example viable unicellular organisms or multi-cellular lower eukaryotes, such as nematodes; and/or other membrane structures, including lipid or phospholipid membrane structures such as liposomes. In some embodiments, active biomaterial may comprise active viruses and viral particles.

A viable cell is a cell that is capable of performing normal cell functions, including replication and cell division. A cell may be from a naturally occurring or wild type organism.

Alternatively, the cell may have been treated, engineered or mutated prior to preservation as described herein. For example, the cell may have been rendered transfection-competent or may contain recombinant nucleic acid. Cells encapsulated as described herein may be homogeneous, e. g. identical cells of a particular species, strain or isolate, or heterogeneous, e. g. a library of cells each containing a different recombinant nucleic acid.

Cells suitable for preserving in accordance with the present invention include prokaryotic cells, such as bacterial cells and eukaryotic cells including yeasts, in particular desiccation tolerant yeasts (e. g. brewers'yeast and bakers'yeast) and mammalian cells, such as tissue culture cells, organ cultures, sperm and egg cells.

Preferably, the cell is a non-anhydrobiotic cell (i. e. a cell which is sensitive to desiccation). Non-anhydrobiotic prokaryotic organisms are generally non-sporulating (i. e. do not form spores) and include, for example, gram-negative bacteria such as E. coli, S. typhimurium and P. putida.

Other examples of suitable organisms include Salmonella spp for live vaccines, Rhizobium spp. for nitrogen fixation,; Pseudomonas spp or Rhodococcus spp for biodegradation/bioremediation, and Lactobacillus spp. or Bifidobacterium spp for probiotics and dairy use.

Multi-cellular lower eukaryotes may include nematode worms, which may, for example, be useful as biopesticides.

Optionally, stability of a microorganism may be improved by culturing under conditions that increase the intracellular concentration of trehalose or other stabiliser prior to drying in the presence of glass forming stabiliser. For example, conditions of high osmolarity (i. e. high salt concentration)

stimulate intracellular trehalose production in bacteria such as E. coli.

A method may comprise determining the viability of a cell before or after encapsulation in a plastics material.

Cell viability may be determined using any of a number of techniques known in the art, including for example, a plate assay for colony forming units (CFU). Viability may for example, be determined after disruption of the plastics material but before application and/or administration of the preserved cells.

Biomolecules may also be preserved using the techniques described herein.

A method of preserving an active biomolecule may comprise; drying the biomolecule in the presence of a glass-forming stabiliser, admixing the biomolecule with a liquid plastics material, and; solidifying the plastics material to encapsulate the biomolecule therein.

In other embodiments, a biomolecule may include nucleic acids, polysaccharides, lipids, vitamins, cofactors and small organic molecules which have a biological activity, for example a pharmacologic, immunogenic, and/or physiologic effect by local and/or systemic action. Biomolecules may include DNA or RNA containing genes or parts of genes or genetically active sequences, and small bioactive molecules including drugs.

Biomaterial and biomolecules according to some embodiments may specifically exclude polypeptides.

Active biomolecules may be therapeutic agents and may include anti-infectives such as antibiotics and antiviral agents; analgesics and analgesic combinations; local and general

anesthetics; anorexics; antiarthritics; antiasthmatic agents; anticonvulsants; antidepressants; antihistamines; anti- inflammatory agents; antinauseants; antineoplastics ; antipruritics; antipsychotics; antipyretics; antispasmodics; cardiovascular preparations (including calcium channel blockers, beta-blockers, beta-agonists and antiarrythmics); antihypertensives; diuretics; vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostics; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressives; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized or recombinantly produced); and nucleic acid molecules (polymeric forms of two or more nucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) including both double-and single-stranded molecules, gene constructs, expression vectors, anti-sense, sense and RNAi molecules).

An active biomolecule may be comprised within a lipid membrane.

Active biomolecules are commonly labile and preservation in accordance with the present methods allows the activity of the biomolecule to be maintained for extended periods without the need for expensive storage conditions.

The steps of drying the biomolecule, admixing and solidifying are described above.

Before solidifying, the plastics material liquid may be placed on a support, for example a glass slide. In some preferred embodiments, the liquid is placed in a mould or applied as a coating. Moulded plastics materials and plastics material coatings comprising active biomaterial (i. e. active biomolecules or cells or both) have a range of applications.

For example, biomaterial encapsulated in a plastics material may be applied as a coating to a biosensor component or seed.

The invention encompasses a seed coated with a plastics material comprising encapsulated biomaterial, in particular viable cells such as rhizobacteria, as described herein, and a method of coating a seed with a plastics material comprising producing a plastics material solution which comprises such biomaterial as described herein, contacting a seed with the solution, and solidifying or fixing the plastics material on the seed to form a coating of encapsulated biomaterial.

For example, a seed may be immersed in a plastics material solution which comprises dried cells of a beneficial prokaryotic organism, for example a root colonising organism such as P. putida or a nitrogen fixing organism such as Rhizobium spp. The seed is then removed from the solution and dried to remove the organic solvent and fix the plastics material on the seed to produce a coating comprising the encapsulated cells of the organism.

The present methods also allow the use of biomaterial in manufacturing processes for which the biomaterial would otherwise be too unstable. In particular, the invention encompasses extrusions and mouldings comprising preserved biomaterial and methods of producing such products.

A method of producing a moulding containing active biomaterial may comprise providing a plastics material solution which comprises biomaterial as described above, adding the solution to a mould and solidifying the plastics material, for example by drying to remove the solvent.

After the plastic material has solidified to fix the shape of the moulding, the mould may be removed.

The use of moulds to shape a range of plastics materials is well known in the art. For example, standard injection moulding techniques may be used. The mould may be of any shape, for example a particle, tablet, pill, block, sheet or strip, depending on the application to which the moulding is to be put.

For example a plastics material moulding comprising encapsulated active biomaterial may be useful as a component of a biosensor.

For example, a biosensor may comprise bacteria which are capable of detecting specific toxic compounds, or pathogens, or general toxicity, for example by modulating their respiration and/or proliferation e. g. the baroxymeter (Tzoris et al (2002) Anal.

Chim. Acta 460 257-272).

Moulded plastics materials comprising encapsulated active biomaterial may be useful as foodstuffs or food supplements (e. g. neutraceuticals). For example, probiotic bacteria such as Lactobacillus spp. or Bifidobacterium spp. may be encapsulated within a plastics material such as PLGA (biodegradable) and ingested directly by an individual. Degradation of the plastics material within the body exposes the bacteria to produce the beneficial effect. The plastics material comprising the probiotic cells may be moulded into a pill or tablet to facilitate ingestion. In other embodiments, the plastics material may be disrupted and the exposed cells used to supplement other foodstuffs prior to ingestion.

The encapsulation of biomaterial in moulded plastics materials may be useful in the formulation of biopharmaceuticals and vaccines. For example, immunogenic bacterial cells such as M. bovis BCG, Salmonella spp. or Lactococcus spp. (vaccine vectors) may be encapsulated within a plastics material such as described above allowing the storage of the immunogenic biomaterial for extended periods without the need for specialised or expensive storage conditions. The plastics material may be broken or pierced to expose the immunogenic biomaterial prior to or during

administration or the plastics material may degrade within the body to expose the immunogen.

Biomaterial encapsulated as described herein in a biodegradable plastics material such as PLGA may be conveniently administered transdermally to an individual using needle-less pneumatic delivery devices which are well known in the art (see for example US6475181, US6063053, US5899880, US5630796, US6004286) If the biomaterial is to be used as a vaccine (i. e. an immunogenic agent) an adjuvant may be added in an amount sufficient to enhance the immune response to the immunogen. The adjuvant may be added to the biomaterial before desiccation and preservation or may be separately reconstituted along with the biomaterial prior to use.

Suitable adjuvants are well known in the art. The precise choice of adjuvant depends in part on the stability of the vaccine in the presence of the adjuvant, the route of administration, and the regulatory acceptability of the adjuvant, particularly when intended for human use. For instance, alum is approved by the United States Food and Drug Administration (FDA) for use as an adjuvant in humans.

A pharmaceutical composition comprising encapsulated biomaterial may comprise one or more added materials such as carriers, vehicles, and/or excipients."Carriers,""vehicles"and "excipients"are substantially inert materials which are non- toxic and do not interact with other components of the composition in a deleterious manner. These materials can be used to increase the amount of solids in particulate pharmaceutical compositions. Examples of suitable carriers include water, silicone, gelatin, waxes, and like materials. Examples of normally employed"excipients, "include pharmaceutical grades of dextrose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol, dextran, starch, cellulose, sodium or calcium

phosphates, calcium sulfate, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycols (PEG), and combinations thereof. In addition, it may be desirable to include a charged lipid and/or detergent in the pharmaceutical compositions. Such materials can be used as stabilizers, anti- oxidants, or used to reduce the possibility of local irritation at the site of administration. Suitable charged lipids include phosphatidylcholines (lecithin). Detergents will typically be a nonionic, anionic, cationic or amphoteric surfactant. Examples of suitable surfactants include, for example, Tergi and Triton, surfactants (Union Carbide Chemicals and Plastics, Danbury, Conn. ), polyoxyethylenesorbitans, e. g., TWEEN. surfactants (Atlas Chemical Industries, Wilmington, Del.), polyoxyethylene ethers, e. g. , Brij, pharmaceutically acceptable fatty acid esters, e. g. lauryl sulfate and salts thereof (SDS), and like materials.

Preservation of biomolecules as described herein may be useful in bioelectronics. For example, preserved fluorophores may be used in organic light-emitting diodes in which synthetic fluorophores based on the active centres of molecules such as GFP or phycoerythrin (You et al., Adv. Mater. 12,1678-1681, 2000), are embedded in a polymer layer, possibly together with a dye to act as an emissive centre. Encapsulated fluorescent biomaterials may also be used in products such as hair gel or skin paint, lettering or graphics, decorations for clothes, toys etc.

Moulded plastics materials comprising encapsulated biomaterial may also be useful in the development of medical devices, for example contact lenses containing pharmaceuticals; implants or patches containing drugs or vaccines; stents for repairing or maintaining blood vessel function, made from PLGA containing anticoagulants or molecules to discourage blood cell attachment; cervical rings containing, for example, antibiotics; and biodegradable plastic thread for surgical stitching containing

growth factors for tissue repair. Techniques for the production of medical devices from moulded plastics materials are well known in the art.

Anti-rejection drugs such as rapamycin and cyclosporin may also be incorporated into medical devices by the encapsulation techniques described herein. Conveniently, anti-rejection drugs are encapsulated in biodegradable plastics materials, such as PLGA to provide an active effect as the plastics moulding breaks down.

Release of biomaterial from the plastics material, may be instantaneous or controlled to provide delayed, gradient or staged release profiles. Multiple reagents may be laminated together for simultaneous or staged release. Release might be controlled in various ways, for example through erosion of biodegradable plastics (sustained or gradient release); through multiple layers of degradable or semi-porous plastics (staged release); through the above, plus mechanical disruption or other method of disruption.

For example, a pharmaceutical cell, particle or molecule may be released from a subcutaneous implant as the biodegradable plastics material casing breaks down. Initial and booster doses of biopharmaceutical may be provided by means of a composition comprising two or more layers of biodegradable plastics material. Examples of suitable materials are described above. A "dummy"layer between two"bioactive layers"comprising encapsulated biomaterial may be used to cause delayed release of the second booster dose of biomaterial.

Small drug molecules may be encapsulated as described herein in a plastics material moulded into a therapeutic patch.

Biodegradation of the patch where it contacts the skin allows the transdermal administration of the small drug molecule.

Therapeutics such as drugs or probiotic bacteria may also be

encapsulated in a gum matrix according to methods of the invention and delivered to the individual via chewing the gum.

Moulded plastics materials comprising encapsulated viable biomaterial or biomolecules may also be useful in other applications, for example in agriculture, ecology or bioremediation in which it is desirable to preserve and store active biomolecules or viable microorganisms for extended periods prior to use. For example, bioremediation bacteria such as Pseudomonas spp. or Rhodococcus spp. may be stored in an encapsulated form. In the event of a pollution event such as an oil spill, the encapsulated bacteria may be applied rapidly to the pollution, either by physically disrupting the encapsulating plastics material prior to application or by allowing the plastics material to degrade in si tu to release the bacteria.

Alternatively, beads of biodegradable plastics material containing bioremediation microorganisms may be ploughed into affected soils to provide steady release of organisms over time as the lamination breaks down. Microorganisms which accumulate heavy metals may be incorporated into the machine or device containing the pollutant, so that if it is disposed of incorrectly, micro-organisms are already present at site of disposal.

In agriculture, biodegradable plastic bags containing encapsulated micro-organisms may be used to store compostable waste materials to accelerate the composting process. In other embodiments, nutrients, fertilizer or biopesticides may be encapsulated in a plastics material which is extruded as a plastic sheet using conventional techniques. Suitable biopesticides may include, for example, Bacillus spp, Pseudomonas spp, or entomopathogenic eukaryotes such as nematodes. The sheet containing the embedded biomaterial may be useful in cultivating or mulching plants.

Preserved bacterial cells, for example Lactobacillus spp. , Bifidobacterium spp. , Streptomyces spp. may also be useful for example in the dairy industry, for example, for the production of cheese and other diary products.

Encapsulated cells as described herein may also be useful in the biotechnology industry, for example for seeding bioreactors and fermentation vessels. Vessels may, for example, be seeded with tablets or wafers of encapsulated cells derived from a single original stock, providing uniformity and consistency. The surface of the tablet may be abraded by mechanical stirring or shaking to release the encapsulated cells and seed the medium.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. All documents mentioned in this specification are incorporated herein by reference in their entirety.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the tables set out below.

Table 1 shows the percentage survival of wet and dried cells in organic solvents.

Table 2 shows survival values (total viable cell numbers) of plastic encapsulated P. putida or spores of B. subtilis after treatment with different disinfectants.

Experimental Solvent Treatment of Dry Samples of Bacteria.

Chemicals were obtained from Sigma or Fisher, unless otherwise stated. High-grade trehalose was obtained from Alchemy International Ltd. , Hambrook, United Kingdom; hydroxyectoine [ (S)-2-methyl-5 bydroxy-1,4, 5,6 tetra-hydropyrimidine-4-

carboxylic acid] was purchased from Bitop GmbH (Witten, Germany).

P. putida KT2440 and E. coli MC4100 bacteria were grown in Luria Broth (LB) or in M9 minimal medium with glucose (20 mM) as the sole carbon source at 30°C for P. putida and at 37°C for E. coli as previously described (Garcia de Castro et al (2000) supra).

To generate HMM, NaCl was added to M9 at a final concentration of 0.4 M and 0.6 M for P. putida and E. coli, respectively (Manzanera et al. 2002 supra).

B. subtilis was grown in M9 with glycerol 0.5% (v/v) as sole C- source. To obtain spores from B. subtilis, 107-109 cells were collected by centrifugation. The pellets were washed with sterile distilled water and incubated at 72°C for 30 minutes.

Around 30% of spores per vegetative cell were obtained.

Viability of spores did not changed despite of being dried or not.

Aliquot volumes (15 ml) of cultures were harvested by centrifugation. Cell pellets were rinsed and then resuspended at 107 to 108 cells/ml in 100 gl of a solution of drying excipient (usually trehalose or hydroxyectoine) plus 1.5% (wt/vol) polyvinylpyrrolidone (PVP) as a viscosity enhancer; all manipulations were done at ambient temperature. Drying was performed in serum vials under vacuum without freezing in a modified freeze dryer (Dura-Stop ßP ; FTS Systems, Stone Ridge, N. Y. ) at 30°C shelf temperature and 15 mTorr (2 Pa; 2 x 10-5 atm) for 20 h followed by temperature ramping of 25°C/min with a 15- min pause after every increase of 2°C up to a maximum temperature of 40°C. Samples were sealed under vacuum and stored for variable periods at 30°C after which they were resuspended in LB to a total volume of 1 ml. For assessment of viability, serial dilutions (in LB) were plated on LB agar plates, incubated at 37°C for 24 hr, and counted to determine CFU/ml.

The tolerance to different pure organic solvents of dried samples of E. coli and P. putida in presence of trehalose and hydroxyectoine was compared with that of spores of B. subtilis.

Osmotically induced cells from the gram-negative microorganisms were vacuum-dried on trehalose (1M) plus PVP 1.5% (w/v), or hydroxyectoine (1M) plus PVP 1. 5% (w/v). Cells presented a standard tolerance to desiccation in the short term at values between 60% (for P. putida when dried in hydroxyectoine and 80% (for E. coli when dried in trehalose).

Fresh dried samples containing approximately 108 cells of E. coli and P. putida were mixed with 200 ßl pure acetone, chloroform, or ethanol, and incubated with these solvents for at least 5 min. To remove the solvents, a vacuum chamber at low pressure was used. The time of solvent removal varied from 25 min for samples with acetone, or 30 min for samples with chloroform, to 135 min for samples with ethanol. The survival of the organic solvent treated bacteria was compared with that of non-treated cells.

Surprisingly, the dried gram-negative samples showed extremely high tolerance to acetone and chloroform treatment. The survival rate to these treatments showed values above 70%. As a control of tolerance, spores from B. subtilis were treated in the same way and presented similar rates of survival to the bio-protected samples. (Table 1).

An analogous experiment was done using concentrated wet samples of cells of E. coli and P. putida. As expected, survival of wet samples of E. coli and P. putida was below detection levels, while viability of spores of B. subtilis were always above 80% (Table 1).

Bioprotectors against desiccation protect against pure organic solvent.

In order to test whether the protection against acetone and chloroform treatment was due solely to the lack of water or if the effect was caused by the presence of the bioprotectors, fresh dried samples of E. coli and P. putida in absence of any additive were treated as previously described.

A much lower survival rate was found in samples dried without bioprotection and this rate dropped dramatically with time although more than 105 cells were still viable at day one after desiccation.

When these samples were treated with pure solvents, no survival was observed even immediately after the treatment.

A similar experiment was performed using cells dried in PVP 1. 5% (w/v) alone, to identify any possible effect of PVP as bioprotector. Untreated samples showed values above 105 viable cells, and shelf life also dropped dramatically with time.

Samples dried in PVP did not survive any of the treatments with pure solvents (Table 1), even when solvent insult was applied immediately after desiccation.

Lamination of cells Approximately 107 cells of E. coli and P. putida strains dried as described herein were crumbled to powder and mixed with 1 ml pure chloroform, and 50 mg of polystyrene, in that order. The mixture was spread on top of a glass plate and allowed to air- dry for 15 min.

Plastic layers were shredded with a sterile blade razor and incubated at the same conditions previously mentioned. Growth was detected in less than 24 hours; the culture was pure and consisted of the same strains included in each plastic layer.

Serial dilutions of bacteria in the dried state Dried bacteria in trehalose were reduced to powder in a mortar.

"Dilutions"in the dried state was made by mixing 100 mg of powdered dried bacteria with 900 mg of powdered trehalose. This mixture was called dilution 10-1 and serial 10-fold"dilutions" were then made from this in trehalose. To determine if the number of cells was consistent with the dilution, 100 mg from each dilution were dissolved in 5 ml of LB and then 100gel plated to estimate the number of cfu/ml in each dried sample. In every case, numbers of bacteria corresponded to dilution level.

Moulding dried bacteria into tablets Tablets were prepared from a powdered mixture of dried cells and trehalose. 500 mg of a mixture of trehalose and 104 dried cells were compressed in a KBR press (Spectralab) at 10 atms pressure for 1-5 min. The resulting tablets were 3 mm in height and 14 mm in diameter. To check any possible deleterious effect of the pressure on the cells, 500 mg of same mixture (trehalose and dried-cells) were dissolved in 25 ml of sterile phosphate buffer before and after moulding. After plating 100 ßl of each suspension, a similar number of cfu was found. This result indicated that the pressure applied has no detrimental effect on viability of stable-dried cells.

Lamination of bacterial tablets with plastic A layer of polystyrene was applied (by paint-brushing) to tablets containing dry cells, prepared as described above. The plastic solution was prepared from 200 mg of polystyrene dissolved in 1 ml of a 1: 1 (v/v) mixture of chloroform and acetone. Laminated tablets were allowed to dry for 3 hours (touch dry after less than 15 min), providing a plastic layer coating the tablet. The waterproof nature of the polystyrene layer on the tablets was demonstrated as follows: laminated and non-laminated tablets were incubated in 25 ml of sterile phosphate buffer pH 7 in a 100 ml flask for 1 h at room temperature in a rotary shaker at 100 rpm. Only the laminated

tablet remained intact, while the non-laminated one dissolved in the buffer.

In a different experiment, non-laminated tablets and broken laminated tablets were incubated in phosphate buffer, as described before, after which 100 ßl of phosphate buffer from each flask were plated on LB-agar plates and incubated overnight at 30°C for cfu determination. Similar cfu values were found in both flasks, confirming that the lamination process involving the use of organic solvents does not affect cell viability.

Resistance of laminated bacteria to anti-microbial agents The tolerance of laminated bacterial cells against a variety of chemical insults was tested.

Laminated or non-laminated tablets were incubated in 25 ml of the following solutions: phosphate buffer pH 7,70% ethanol, 4% formaldehyde, 0.1 M K2MnO4, 1% bleach, 1/80 solution of seldine (commercial iodine solution), 10 mM NaOH (pH 12), 10 mM HC1 (pH 2). These solutions are well known as disinfectants (McDonnell & Russell, 1999, Clin. Microbiol. Rev. 12,147-179).

After 30 min incubation at 70 rpm (at room temperature), serial dilutions from each solution containing non-laminated tablets were made for colony counting. Laminated tablets were withdrawn from solution and washed twice with sterile milli-Q water. Then the plastic layer was disrupted and incubated in sterile phosphate buffer for another 30 min.

Survival data are shown in Table 2.

No surviving cells were found in any of the solutions tested (except for the phosphate buffer) in those flasks containing non-laminated tablets. On the contrary, a high degree of survival was obtained in those flask containing laminated tablets, regardless of the chemical used (except for the seldine

solution, where the plastic on the tablet was completely disrupted in the highly oxidising solution). For comparison, similar experiments were performed with B. subtilis spores to determine the degree of tolerance of natural spores to the chemicals used (Table 2). As expected, high survival values were obtained. From these experiments we conclude that the survival rates obtained in laminated tablets containing dried cells in trehalose are comparable to natural sporulant microorganisms.

Stable seed coating with P. putida Lamination of stable cells on the surface of sweet-corn seeds was used as a model to test the efficiency and applicability of this method. The soil strain P. putida was selected to be encapsulated around sweet-corn seeds because it is recognized as a very efficient root coloniser which has beneficial effects for plants (Campbell and Greaves, (1990) Anatomy and community structure of the Rhizosphere. In The Rhizosphere Lynch (ed) pll- 34 Chichester, Wiley & Sons Lugtenberg and Dekkers, (1999) Environ. Microbiol. 1 (1) 9-13).

To follow cell viability along the germination process, a bioluminescent P. putida was constructed by insertion of the lux operon from Photorhabdus luminescens into P. putida chromosomal DNA. The resulting strain, named P. putida MAX10, was used in the assay below described.

Corn seeds were sterilised by incubation in ethanol 70% (v/v) for 5 min. Followed by a second incubation in sodium hypochlorite 20% (v/v). A final set of 5 washes in sterile distilled water was performed to remove traces of ethanol or bleach. Finally, the seeds were air dried in a sterile flow cabinet Sterile sweet-corn seeds were coated with cells by immersion in a mixture containing dried P. putida MAX10, chloroform and

polystyrene (as previously described). After soaking for 2 minutes, the seeds were withdrawn from the mixture and allowed to air-dry for 10 min. Seeds were sowed onto sterile agar plates 1,30 and 90 days after coating and were incubated at 30°C in the dark. Root colonization was followed over time.

Root size was observed to reach several cm two days after germination.

Before seed germination, no light was observed. In contrast, light emission from the P. putida MAX10 cells was observed when seed germination occurred.

Plastic lamination of phospholipid structures Phosphatidyl ethanolamine (PE) labelled with the fluorescent compound nitrobenzoxadiazole (NBD) was used to test the applicability of plastic lamination to membrane systems.

NBD-PE was dissolved at 0.25 mg/ml in 1 M trehalose and a small volume dropped onto a microscope slide which was then placed on an aluminium heating block at 70°C overnight. The trehalose solution was observed to form a clear glass which incorporated many vesicular structures similar to that formed in aqueous solution.

When overlaid with polystyrene solution in chloroform, and the plastic allowed to set, the NBD-PE structures in the trehalose glass were observed to remain intact, showing that the phospholipid was protected from the organic solvent by the plastic lamination.

In contrast, when NBD-PE in trehalose solution was exposed directly to the plastic solution by mixing without prior trehalose glass formation vesicular structures were seen to be disrupted. Acetone Chloroform Ethanol P. putida Wet samples « 0. 001% « 0. 001% « 0. 001% Dried in 1M Trehalose 71.5% 95.6% 5. 0% Dried in 1M 94.4% >99% #0. 001% hydroxyectoine Dried in 1. 5% (w/v) « 0. 001% « 0. 001% #0. 001% polyvinylpyrrolidone E. coli Wet samples 0 0 0 Dried in 1M Trehalose 60.4% 77.6% 3.0% Dried in 1M 18.7% 53. 5% 0 hydroxyectoine Dried in 1.5% (w/v) 0 0 0 polyvinylpyrrolidone B. subtilis Dried w/o bp 92. 6% 83. 0% 99. 0%

Table 1 Media P. putida B. subtilis Non-laminated Laminated spores tablets tablets Phosphate 0. 7 x 104 0. 8 x 104 51 x 104 buffer 70% Ethanol N. D. 1.5 x 104 36 x 104 4% Formaldehyde N. D. 1.8 x 104 39x 104 0.1 mM K2MnO4 N. D. 0.3 x 104 28 x 104 1% Bleach N. D. 1.3 x 104 44 x 104 1/80 Seldine N. D. N. D. 13 x 104 lOmM NaOH pH12 N. D. 0.1 x 104 45 x 104 lOmM HC1 pH 2 N. D. 0.9 x 104 49 x 104

N. D: non detected.

Table 2