Patent ID: 12201110

DESCRIPTION OF EMBODIMENTS

The application of microorganisms for any purpose at commercial scale strongly depends on the reliability of the microbial preparation. Particularly, when microbial agents are intended to be applied as additive e.g. in coatings for seed treatment processes, the requirements for the formulation are high. Apart from the general desiccation stress in dry formulations, microorganisms are additionally exposed to physical, thermal as well as to detrimental chemicals, e.g., pesticidal agents. To circumvent said problems with the stability of microbial strains, mostly Gram-positive bacteria with spore-forming ability are currently used.

Conventionally, microbial agents are produced by cultivating microbial cells in liquid media to a certain cell number, harvesting, mixing with appropriate carriers and drying. The encapsulation of cells in biopolymers (e.g., sodium alginate) prior to drying is also commonly used (Bastian et al., 1986). To evoke longevity of the formulated microbes, several strain-specific protective compounds are applied. In case of multi-strain preparations, the single strains are usually cultivated individually before they are formulated together. Sensitive Gram-negative bacteria possess a great potential for biological plant protection. Despite much attention on the stabilization of microorganisms in formulations suitable for large scale applications, no appropriate solutions are available.

The invention discloses methods for formulation and fermentation to improve the viability of microbial cells during storage and application. In some embodiments, methods described herein comprise reducing pre-culture inoculation densities. In some embodiments, methods described herein comprise a step of in-bead cultivation of encapsulated microbial cells. Methods disclosed herein are suitable for all microorganisms including those for agricultural purposes, for food and feed purposes, as well as for medical purposes.

As used herein, the terms “microorganism” or “microbial” cover prokaryotic and eukaryotic, unicellular or multicellular organisms, and combinations thereof, which can be propagated and manipulated in a laboratory. In some embodiments, a microorganism includes, but is not limited to, eubacteria and archaea, protozoa, fungi and algae. In the present disclosure, the term microorganism typically denotes a live microorganism, e.g., capable of propagation, e.g., capable of having metabolic activity.

As used herein, the terms “bacteria” and “eubacteria” are used in reference to unicellular organisms, including recombinant organisms, lacking that lack a nucleus and other membrane bound organelles. Bacteria can function and reproduce as individual cells or multicellular aggregates.

As used herein “archaea” are single-cell organisms that lack nuclei and have archaeal membranes, specifically made of ether lipids.

As used herein, “fungi” is used in reference to eukaryotic organisms such as the molds and yeasts, including dimorphic fungi, fungi found in soil, recombinant fungi and any fungi capable of growing on plants.

As used herein, the term “strain” refers in general to a distinct genotype of organisms of the same species. More particularly, the term “strain” refers to members of a microbial species, wherein such members, i.e., strains, have different genotypes and/or phenotypes. Herein, the term “genotype” encompasses both the genomic and the recombinant DNA content of a microorganism and the microorganism's proteomic and/or metabolomic profile and post translational modifications thereof. Herein, the term “phenotype” refers to observable physical characteristics dependent upon the genetic constitution of a microorganism. As one skilled in the art would recognize, microbial strains are thus composed of individual microbial cells having a common genotype and/or phenotype. Further, individual microbial cells may have specific characteristics (e.g., a specific rep-PCR pattern) which may identify them as belonging to their particular strain. A microbial strain can comprise one or more isolates of a microorganism.

As used herein, the term “reference method” refers to an alternate method of microbial stabilization other than the inventive BFC technology described herein, e.g., an alternate method that does not include stabilizing microorganisms by shifting them into a dormancy-like state through targeted in-bead cultivation). A “reference composition” refers to a composition generated from an identical pre-culture material by application of a differing stabilization methodology. By way of a non-limiting example, stability of a microbial cells encapsulated in polymeric particles using a method of the present invention (e.g., stabilizing microorganisms by shifting them into a dormancy-like state through targeted in-bead cultivation) is compared to a reference composition wherein the reference composition was generated using a different method, such that differences in stability of the end-product microbial cells are attributable to differences in the method of production.

As used herein, the term “kingdom” refers to the general taxonomic category, e.g. fungi and bacteria.

As used herein, “CFU” refers to a colony forming unit.

As used herein, the term “inoculum” is intended to mean any form of microbial cells, or spores, which is capable of propagating in a culture medium.

Culture medium as used herein is defined as a mixture which supports the growth of microbial cells, such as disclosed microorganism, said mixture may contain for example ingredients such as peptone, soy peptone, molasses, potato starch, yeast extract powder, glucose, mannitol, skim milk or combinations thereof. Culture medium can be solid culture medium or liquid medium.

According to the invention there are provided microorganisms, such as for example, but not limited to, biotechnologically, agriculturally or medically useful microorganisms, microorganisms useful for food and teed purposes, which are encapsulated in biodegradable polymers.

Microorganisms of the invention can either be of natural origin or produced by recombinant methods.

As used herein, the term “biodegradable polymer” refers to polymer-based matrices that show at least some biodegradability. Non-limiting examples of suitable biodegradable polymers include collagen, gelatin, alginate, polycaprolactone, and poly lactic-co-glycolic acid (PLGA).

In some embodiments, the biodegradable polymer may be an unsaturated biodegradable polymer (i.e. a biodegradable polymer containing at least one unsaturated carbon-carbon bond, such as a double or a triple bond). Such unsaturated polymers may be cross-linkable in situ. Non-limiting examples of unsaturated biodegradable polymers include poly-propylene fumarate (PPF), poly(ζ-caprolactone-fumarate), and mixtures and co-polymers thereof.

In various embodiments, the polymer particles of the present invention may be hydrogel particles (e.g., alginates, fibrins, and gelatins), natural or synthetic biodegradable particles (e.g., particles derived from or coated with poly lactic-co-glycolic acid (PLGA)), biodegradable porous particles (e.g., silicon porous particles), and biocompatible vesicles (e.g., liposomes and/or micelles).

Particularly suitable biodegradable polymers for agriculturally suitable microorganisms are, amongst others, agarose, alginates, agar, gelatin, polyacrylamide, kappa-carrageenan, furcellaran, 2-methyl-5-vinyl-pyridine-methylacrylate, ethyl succinylated cellulose, chitosan, polyvinyl alcohol, polygammaglutamic acid, ethyl cellulose and other biodegradable polymers known to one of skill in the art.

Alginate is a naturally occurring anionic polymer typically obtained from brown seaweed. Due to its biocompatibility, low toxicity, relatively low cost, and mild gelation by addition of divalent cations such as Ca2+it has been extensively used for many biomedical applications. Commercially available alginate is typically extracted from brown algae (Phaeophyceae). Alginate with more defined chemical structures and physical properties can be obtained from seaweed-derived alginate. Bacterial alginate can be produced fromAzotobacterandPseudomonas.

Alginate is a mix-polysaccharide composed of D-mannuronic acid (M) L-guluronic acid (G). The main alginate series include sodium alginate, potassium alginate, calcium alginate, ammonium alginate etc. In some embodiments sodium alginate is used.

For medical purposes, the biodegradable polymer is selected from materials which are considered safe to the human body and degraded in the body for a certain period of time. Examples of biodegradable polymers considered safe include albumin, collagen, gelatin, fibrinogen, casein, fibrin, hemoglobin, transferrin, chitin, chitosan, hyaluronic acid, heparin, chondroitin, keratin sulfate, alginate, starch, dextrin, dextran, polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, polyhydroxybutyric acid, polycaprolactone, polyanhydride and polyalkylcyanoacrylate.

Individual polymeric particles may be produced when specified hydrogel polymers are complexed in solutions of biologically active substrates. An alginate solution, for example, will form a gel when a complexing agent is added. Calcium chloride (CaCl2) is generally used; however, lanthanum chloride, ferric chloride, cobaltous chloride and calcium hydroxide are also acceptable.

In order to obtain alginate beads which include microbial cells, the microbial cells are dispersed in a sodium alginate solution. The obtained solution is dropped into a crosslinking solution containing divalent cations, e.g. Ca2+. Solidification of the droplets starts immediately on the droplet surface by ionotropic gelation. Ca2+reacts with the negatively charged polymer chains to form a three-dimensional rigid structure.

In one embodiment of the invention, the microbial cells are individually pre-cultured according to conventional methods up to a cell density of about 109to 1010CFU/ml. The individual pre-cultures are suspended in a biodegradable polymer solution up to a cell density of about 10° to 108CFU/ml. Thus, in one embodiment two pre-cultures of microorganism of different origin are suspended in the biodegradable polymer solution, e.g., in an alginate solution. In some embodiments, 3, 4, 5, 6, or more microorganism of different origin are suspended in an alginate solution. Optionally, the polymer solution contains additionally carriers and/or additives.

If 2 microorganisms of different origin are provided, they may be present in a ratio of 1:5 to 5:1, or 1:2 to 2:1, or 1:1. If 3 microorganisms of different origin are provided, they may be present in a ratio of 1:1:5 to 5:5:1, or 1:2:2 to 2:1:1, or 1:1:1, etc.

Various additives such as adherents, dispersants, surfactants, and nutrient and buffer ingredients, can also be included. Trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum, or zinc may be added. Other conventional additives including, but not limited to, coating agents, wetting agents, buffering agents, and polysaccharides may be added. At least one agriculturally acceptable carrier can be added to the formulation such as water, solids or dry powders. The dry powders can be derived from a variety of materials such as calcium carbonate, gypsum, fluency agent, vermiculite, talc, clay, humus, activated charcoal, and various phosphorous compounds. These additives are generally added to the suspension in an amount of 0.001% to 10% of the polymeric substance solution.

Suitable carriers include, but are not limited to mineral earths such as silica, silica gels, silicates, talc, kaolin, attaclay, limestone, chalk, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea and urea, products of vegetable origin such as cereal meals, tree bark meal, wood meal and nutshell meal, cellulose powders, attapulgites, montmorillonites, mica, vermiculites, synthetic silica and synthetic calcium silicates, or compositions of these.

Bead formation is performed for example by dropping the suspension, which comprises the pre-cultured microbial cells and a polymeric substance, into a multivalent ion solution bath using a vibrating nozzle. In some embodiments, the nozzle diameter may be in the range of about 1 μm to 500 μm. In some embodiments the nozzle diameter is within the range of 70 to 100 μm. In some embodiments the nozzle diameter is about 75 μm. In some embodiments the nozzle diameter is about 100 μm. In some embodiments the nozzle diameter is about 200 μm and the obtained wet beads have an average diameter of about 100 to 500 μm.

Bead diameters of less than 500 μm and preferably less than 100 μm may be advantageous because they allow optimal growth of bacteria across the entire bead and, thus, use full capacity of bead volume to achieve a maximum of bacterial load. A prerequisite for uniform growth is uniform allocation of nutrients and oxygen. The polymer matrix acts as a diffusion barrier limiting the transfer of compounds within the beads. It can be assumed that the concentration of nutrients and oxygen in particular, decreases from the outer layer to the bead core. The larger the diameter of the beads the lower the concentration of growth compounds in the inner parts, e.g., in the core of the beads. Insufficient availability of nutrients would impair bacterial growth and cause barren zones. In consequence, the larger the diameter of the beads the lower the volumes in which bacteria are able to multiply in relation to the total volume of the bead. The achievable cell number per gram polymeric particles is lower in beads with an average diameter greater than 500 μm, compared to beads with an average diameter less than 500 μm.

After a washing step, the obtained beads are transferred to a system for cultivation of the encapsulated microorganisms. The incubation time is of about 12 to 120 hours, preferably of about 48 hours. Suitable systems for in-bead cultivation of encapsulated microorganisms are, for example: fluidized bed reactors. Due to process-specific cultivation, the encapsulated microorganisms' stability is enhanced via stress adaptation. The growth rates and maximal cell counts resemble those from non-encapsulated cultures but can be slightly higher. Additionally, microbial cells that are allowed to multiply in a solid matrix format may induce biofilm-like substances and demonstrate improved shelf-life of the particle as described herein.

Batch fermentation processes represent closed systems, in which bacterial cultures pass through different growth phases. In the moment, the conditions become unfavorable e.g., nutrients are exhausted and waste products are enriched, bacteria populations enter the stationary phase characterized by stable cell numbers over a certain period of time. Such conditions induce a general stress-response in bacterial cells and an adaptation of their metabolic activity in preparation of a starvation period. Stress-mediated response involves changes in cell membrane structure, formation of biofilms and synthesis of compounds protecting essential molecules such as proteins and DNA. Stress-adapted cells possess a higher tolerance towards adverse conditions and a higher survivability.

Thus, the duration of the cultivation process of bacteria within the beads is not determined by the end of the multiplication (begin of stationary phase) with the aim to obtain the maximum of cell numbers. As described herein, the inventors made the novel and unexpected discovery that by prolonging in-bead cultivation the stationary phase is subsequently maintained enabling bacteria to activate mechanisms and to adapt to stress conditions, and to exhibit the so-called stationary phase phenotype.

The time a culture needs to accomplish stress-adaptation and the underlying mechanisms are strain-specific, they have to be adapted to the respective microbial strains. For the majority of microorganisms a time period between 48 and 72 hours is sufficient for in-bead cultivation.

A biofilm is a well-organized community of microorganisms that adheres to surfaces and is embedded in a matrix of extracellular polymeric substances (EPSs). EPSs are a complex mixture of high-molecular-mass polymers (>10,000 Da) generated by the bacterial cells, cell lysis and hydrolysis products, and organic matter adsorbed from the substrate. EPSs are involved in the establishment of stable arrangements of microorganisms in biofilms. Biofilm formation is one of the mechanisms bacteria use to survive in adverse environments. Bacteria living in a biofilm usually have significantly different properties from free-floating (planktonic) bacteria of the same species, as the dense and protected environment within the biofilm allows them to cooperate and interact in various ways. One benefit of this environment is increased resistance to detergents and antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community.

Bashan (1986) discloses the immobilization and multiplication ofAzospirillum brasilenceCd in alginate heads. In Bashan (1986),A. brasilencewas pre-cultured in nutrient broth to a final concentration of 109CFU/ml, then encapsulated in alginate beads. The beads were washed and incubated for 24 to 48 h in fresh nutrient broth medium to allow multiplication inside the beads. The beads were either kept in hermetically sealed flasks or were lyophilized and were stable for about 12 weeks (Bashan (1986), page 1095,FIG.5B). However, if the particles were air dried, only a very small number of bacteria survive (Bashan (1986), page 1095,FIG.5A).

In contrast, the beads obtained according to the present invention exhibit significant post-drying stability and may be dried at ambient temperatures and still are stable for at least 35 weeks at 30° C. as shown inFIG.1A. For example, the beads are dried at 20 to 35° C. in a static bed or drum dryer, or at 25 to 30° C. The appropriate drying time is of about 15 to 48 h, or 22 to 36 h, or 24 to 30 h.

In contrast to Bashan (1986), the polymeric particles according to the invention can be stored at room temperature for several weeks without substantially reducing the number of viable microorganism cells.

A further embodiment of the invention relates to the polymeric particles comprising at least one microorganism, wherein at least 70% of the microbial cells in the beads are viable upon storage of at least 35 weeks at 30° C. In some embodiments at least 75%, 80%, 85%, 90%, or 95% of the microbial cells in the beads are viable upon storage of at least 35 weeks. In some embodiments at least 75%, 80%, 85%, 90%, or 95% of the microbial cells in the beads are viable upon storage of at least 35, 40, 45, 50 or 52 weeks at 30° C.

A further embodiment of the invention relates to the polymeric particles comprising at least two microorganisms of different origin, wherein at least 70% of the microbial cells in the beads are viable upon storage of at least 35 weeks at 30° C. In some embodiments at least 75%, 80%, 85%, 90%, or 95% of the microbial cells in the beads are viable upon storage of at least 35 weeks at 30° C. In some embodiments at least 75%, 80%, 85%, 90%, or 95% of the microbial cells in the beads are viable upon storage of at least 35, 40, 45, 50 or 52 weeks at 30° C.

in various embodiments the bacteria or fungi are agriculturally and/or horticulturally useful, for example, the bacteria are pesticidal and/or insecticidal, and/or supports plant growth and/or development, or any combination thereof.

In various embodiments the one or more bacteria may compriseSerratia(for example,Serratia entomophiliaorSerratia proteomaculans),Xanthamanas, Pseudomonas, Rhizobium, Bifidobacterium, Lactobacillus, Streptococcus(Enterococcus),Yersinia(for example,Yersinia entomophaga),Pseudomonas, Bacillus, Pasteuria, Azobacter, Enterobacter, Azospirillum, Cyanobacteria, Paecilomyces, Streptomycetes, Chromobacterium, Rhanella, Burkholderia, Paenibacillus, Collimonas, Sinorhizobium, Pantoea, Erwinia, Pediococus, Leuconostoc, Aeromonas, Neptunomonas, Klebsiella, Ponchonia, Brevibacillus, Acinetobacter, Paraburkholderia, Herbaspirillum, Bradyrhizobium, Methylobacterium, Ensifer, Sphingomonas, Azobacter, Lysobacter, Stenotrophamonas, or any combination of two or more thereof.

In various embodiments the one or more fungi may compriseBeauveria, Penicillium, Metarhizium, Trichoderma, Gliocladium, Coniothyrium, Paecilomyces, Verticillum, Sclerotinia, and mycorrhizae or any combination of two or more thereof.

Bacterial and fungal microorganism suitable for use in the present invention preferably are selected from one or more genera ofAzotobacter, Bacillus, Stenotrophomonas, SerratiaandPseudomonas, in addition to specific bacteria and fungi such asPseudomonas fluorescens, Azotobacter, Bacillus polymyxa, Stenotrophomonas rhizophila, Serratia plymuthica, Trichoderma herzianum, Trichoderma viriderespectively, or any combination thereof.

Using the present formulation method, the viability of microbial cells during storage and field application is improved compared to conventional method (seeFIGS.1A and1B). The method is particularly suitable for sensitive microorganisms, such as Gram-negative bacteria but also fungi. Microbial cells formulated by that methods show survival rates during storage and when applied to seeds superior to other known methods. Moreover, due the compatibility with a broad range of strains the method allows a simultaneous formulation and cultivation of more than one microbial strain in a single preparation. Thus, the costs for the production of multi-strain preparation is reduced compared to individual cultivation approach.

EXAMPLES

The Examples which follow are set forth to aid in the understanding of the invention but are not intended to, and should not be construed to limit the scope of the invention in any way. The Examples do not include detailed descriptions of conventional methods, e.g., cultivation of microorganisms. Such methods are well known to those of ordinary skill in the art.

Example 1

Shelf-Life of Microorganisms Formulated by Bacterial and Fungal Conservation (BFC) Technology

Results from strains with strain-specifically optimized fermentation and formulation parameter.

The potential of the stabilization formulation technology is exemplary demonstrated for selected plant-beneficial strains. The viability of microbial cells (CFU/g) produced by the invented technology was compared to conventionally encapsulated cells according to Bashan et al. (1986). Depending on the initial objective of a study, preparations were stored for different periods of time at 30° C..

Microbial cells propagated and formulated by the methods described herein show survival rates during storage and when applied e.g. to seeds superior to other known methods. Moreover, the methods described herein allow a simultaneous cultivation of more than one microbial strain in a single device. The invention involves two main concepts: i) a combined formulation and fermentation approach that enhances shelf-life stability and elevates the tolerance of formulated microbial cells towards desiccation and adverse effects during storage and application and ii) a method to cost-effectively cultivate more than one microbial strains in a single device. In a first step, individual pre-cultures from bacteria or fungi are obtained and suspended with a polymer solution that may contain additional carriers and additives. In some embodiments, the polymer solution is a 1.5-5% solution. In some embodiments, the polymer solution is an alginate solution. In some embodiments, the alginate solution is sodium alginate. In some embodiments, the sodium alginate solution is a 1.5-5.0% solution. Depending on the strain(s) to be inoculated the cell density is adjusted to 10° to 108CFU/ml. In some embodiments, inoculation cell density is adjusted to about 104to 108CFU/ml. In some embodiments, inoculation cell density is adjusted to about 103to 107CFU/ml. In some embodiments, inoculation cell density is adjusted to about 102to 106CFU/ml. In some embodiments, inoculation cell density is adjusted to about 101to 105CFU/ml. In some embodiments, inoculation cell density is adjusted to about 100to 104CFU/ml. In some embodiments, inoculation cell density is adjusted to about 105to 108CFU/ml. In some embodiments, inoculation cell density is adjusted to about 104to 107CFU/ml. In some embodiments, inoculation cell density is adjusted to about 103to 106CFU/ml. In some embodiments, inoculation cell density is adjusted to about 102to 105CFU/ml. In some embodiments, inoculation cell density is adjusted to about 101to 104CFU/ml. In some embodiments, inoculation cell density is adjusted to about 100to 103CFU/ml. In some embodiments, inoculation cell density is adjusted to about 106to 108CFU/ml. In some embodiments, inoculation cell density is adjusted to about 104to 106CFU/ml. In some embodiments, inoculation cell density is adjusted to about 102to 104CFU/ml. In some embodiments inoculation cell density is adjusted to about 100to 102CFU/ml. In some embodiments, inoculation cell density is adjusted to 101CFU/ml. In some embodiments, inoculation cell density is adjusted to 102CFU/ml. In some embodiments, inoculation cell density is adjusted to 103CFU/ml. In some embodiments, inoculation cell density is adjusted to 104CFU/ml. In some embodiments, inoculation cell density is adjusted to 105CFU/ml. In some embodiments, inoculation cell density is adjusted to 106CFU/ml. In some embodiments, inoculation cell density is adjusted to 107CFU/ml. In some embodiments, inoculation cell density is adjusted to 108CFU/ml.

Alginate bead formation was realized by dropping the solution into a CaCl2solution bath using a vibrating nozzle. After washing to remove remaining CaCl2solution, beads were transferred to a fermenter-like system for the in-bead cultivation which may be described as a fluidized bed reactor. Growth rates and maximal cell counts resembled those from planktonic cultures, but could be slightly higher. After 24 to 72 h of cultivation, the beads were washed again. Washed beads may, optionally be, treated with solutions containing osmoprotective compounds. The last step involved drying under ambient temperatures in appropriate drying devices (including, but not limited to a static bed in a drying chamber or drum dryer) for 24-30 hours.

Depending on the microorganism and the applied protocol, the cell density in the final product ranged from 109to 1012CFU/g dry weight. Methods described herein enhance the ability of the formulated (stabilized) microbial cells to outlast desiccation and starvation for the time of storage and application. The inventors made the novel and unexpected discovery that that the combination of reduced inoculation density and subsequent in-bead cultivation is critical for final stability and long shelf life of the beads. In contrast to planktonic fermentation approaches, cells produced by the methods described herein are forced to multiply as colonies within the polymeric bead. Thereby, the cells produced by the methods described herein form biofilm-like structures including the generation of matrices made of extracellular polymeric substances. In fact, forming of and living in biofilms is the typical lifestyle of microorganisms in nature. Naturally, in biofilms embedded microorganisms survive adverse conditions due to the protective properties of the biofilm matrix. In one aspect of the invention, the genetic potential of any microorganism to form protective biofilms is artificially induced through a surprising combination of reduced inoculation density and in-bead cultivation, resulting in long-term improved survivability. Methods described herein are also suitable for cost-effective multi-strain fermentation. In some embodiments, only pre-culturing is performed in individual reactors, whereas the main cultivation is conducted in one single device. Moreover, the post-cultivation steps are simplified by processing only one preparation.

Example 2

Characterization of Stability of Compositions Produced Using BFC Technology

A series of experiments were performed to show the contribution of steps in the BFC technology.Stenotrophomonas rhizophilaSPA-P69 (SPA-P69) was prepared using the following default parameters: SPA-P69 was grown in 0.5× Nutrient Broth II (SIFIN, Germany), and suspended in 1.5% sodium alginate solution to obtain a pre-culture cell density of 106CFU/ml. The composition of 0.5× Nutrient Broth II is 1.75 g/l casein peptone, 1.25 g/l meat peptone, 1.25 g/l gelatin peptone, 0.75 g/L yeast extract, and 2.5 g/l sodium chloride. Alginate beads were formed by dropping the solution into 150 mM CaCl2, using a vibrating nozzle having nozzle diameter of 100 μm. The newly formed beads were washed to remove excess CaCl2, and beads were transferred to a fluidized bed reactor, for secondary in-bead cultivation of encapsulated microorganisms at 22° C. for 48 hours. After cultivation the beads were washed before drying at ambient temperature in a static bed in a drying chamber for 24-30 hours. For these experiments these default parameters were modulated as described in the following table, and the viability of the encapsulated strains prepared using these experimental parameters were compared after various lengths of time in storage.

TABLE 1Default and varied parameters and conditionsStenotrophomonas rhizophilaSPA-StrainP69Alginate concentration1.5% (default)CaCl2concentration150 mM (default)Nozzle diameter100 μm (default)75 μmInoculation density1 × 105CFU/ml1 × 106CFU/ml (default)1 × 107CFU/ml1 × 108CFU/mlCultivation time12 h24 h36 h48 h (default)Cultivation mediumNutrient broth II (default)Cultivation temperature22° C. (default)

In a first experiment, the effect of the time spent in secondary cultivation was assayed. SPA-P69 was formulated by the BFC technology methodology described above except that the in-bead cultivation times were varied between samples.

Encapsulated samples of SPA-P69 were subjected to in-bead cultivation of 0 hours, 12 hours, 24 hours, 36 hours and 48 hours and CFU/g dry mass was assayed immediately after the cultivation step (tc), after the drying step (t0), 14 days after storage at 30° C., or 42 days after storage at 30° C. Longer periods of in-bead cultivation resulted in increased survival of cells, this was especially noticeable when comparing samples that were stored for 42 days at 30° C. EncapsulatedS. rhizophilaSPA-P69 that was prepared with an in-bead cultivation step of 48 hours resulted in a 0.65 log reduction in CFU/g dry biomass after 42 days of storage at 30° C. relative to the CFU per g dry biomass recorded immediately after cultivation. It is also noteworthy that samples that underwent in-bead cultivation of 36 or more hours showed enhanced survival ofS. rhizophilaSPA-P69 in samples that had been dried relative to samples assayed immediately before the drying step. Results are shown in Table 2 andFIG.14, images of exemplary beads are shown inFIG.15.

TABLE 2Effect of cultivation time on cell counts before, after drying and 42 days after storage at 30° C.After in-beadAfter 14 days storageAfter 42 days storagecultivation (tc)After drying (t0)at 30° C. (t14)at 30° C. (t42)CFU/gCFU/gΔ log10CFU/gΔ log10% Δ log10CFU/gΔ log10% Δ log10Hoursdry massdry massCFU/gdry massCFU/gCFU/gdry massCFU/gCFU/gin-beadwetdrieddry massdrieddry massdry massdrieddry massdry masscultivationbeadsbeads(t0 − tc)beads(t14 − tc)(t14 − tc)beads(t42 − tc)(t42 − tc)07.790.00−7.790.000.000.000.000.000.00129.400.00−9.400.000.000.000.000.000.002411.4610.31−1.158.78−2.68−25.987.90−3.57−31.123611.7312.000.2711.48−0.26−2.1410.13−1.61−13.714811.5311.880.3511.52−0.01−0.0910.88−0.65−5.62

In a second experiment, the effect of pre-cultivation inoculation density was assayed. SPA-P69 was formulated by the BFC technology methodology using varying inoculation densities (105CFU/mL, 106CFU/mL, 107CFU/mL). CFU/g dry mass was assayed after encapsulation but before in-bead cultivation (te), immediately after the 48 hour in-bead cultivation step (tc), after the drying step (t0), after 14 days of storage at 30° C. (t14), and after 42 days of storage at 30° C. (t42). The lower inoculation densities resulted in smaller log losses of CFU/g dry mass after storage relative to CFU/g dry mass detected immediately after the in-bead cultivation step. Results are shown in Table 3 andFIG.16, images of exemplary beads are shown inFIG.17. Table 4 describes characteristics of colonies formed within the beads prepared using the BFC technology methodology with differing inoculation densities.

TABLE 3Effect of pre-cultivation inoculation density on cell counts before, after drying and 42 days after storage at 30° C.AfterAfter 14 days storageAfter 42 days storageencapsulationAfter 48 hour in-at 30° C. (t14)at 30° C. (t42)(te)bead cultivation (tc)After drying (t0)%%CFU/gCFU/gΔ log10CFU/gΔ log10CFU/gΔ log10Δ log10CFU/gΔ log10Δ log10dry massdry massCFU/gdry massCFU/gdry massCFU/gCFU/gdry massCFU/gCFU/gInoculationwetwetdry massdrieddry massdrieddry massdry massdrieddry massdry massdensitybeadsbeads(tc − te)beads(t0 − tc)beads(t14 − tc)(t14 − tc)beads(t42 − tc)(t42 − tc)1057.0311.474.4511.920.4511.490.020.1510.92−0.55−4.821067.7911.533.7411.880.3511.52−0.01−0.0910.88−0.65−5.621078.8411.832.8911.78−0.0511.50−0.33−2.8410.63−1.20−10.161089.8511.972.1211.76−0.2111.24−0.74−6.2510.13−1.84−15.41

TABLE 4Characteristics of colonies formed within the beads prepared usingthe BFC technology methodology with differing inoculation densitiesInoculation density1.0E+51.0E+61.0E+71.0E+8Number of12.5125125012500colonies/beadColony diameter16.429.335.58n.d.(μm)Colony volume1.89E+43.37E+31.29E+3n.d.(μm{circumflex over ( )}3)CFU/colony1.14E+052.02E+47.74E+3n.d.CFU/bead1.42E+062.53E+69.68E+6n.d.

Example 3

Metabolic Profiling of Microorganisms Formulated by Bacterial and Fungal Conservation (BFC) Technology Using High Resolution Mass Spectrometry

Preparation of Strains Using BFC and Reference Methods

Stenotrophomonas rhizophilaSPA-P69,Sphingomonas sanguinis, Pseudomonas brassicacearumL 13, andSerratia plymuthica3Re were each individually formulated using the BFC methodology. To generate reference alginate encapsulated planktonically grown cells (“Alginate Planktonic”), cells in liquid culture were pelleted and resuspended in 1/10 the initial volume using 0.9% NaCl. The 10 fold concentrated cell suspension was mixed with alginate solution such that cell densities in the resulting alginate beads were similar to densities obtained after in-bead cultivation. Immediately following bead formation, Alginate Planktonic beads were air dried and stored at 30° C. Reference alginate encapsulatedStenotrophomonas rhizophilaSPA-P69,Pseudomonas brassicacearumL 13, andSerratia plymuthica3Re were also prepared using the method of Bashan (1986).

Preparation of Samples

For metabolite analyses, about 100 mg cell mass for each sample and replicate (3 per microbe/method) were collected. Cell material from planktonically grown bacteria were obtained by pelleting 1.9 ml of a liquid culture in 2 ml tubes at 13,500×g for 15 min at 4° C. and discarding the supernatant. Samples from bacteria immobilized in alginate beads were prepared by dissolving 200 mg of dry beads in 40 ml 50 mM. Na3C6H5O7in 50 ml tubes using an orbital shaker for 3 hours at room temperature. The bacterial pellet was obtained by two centrifugation steps. The first centrifugation step was done at 9,000×g for 20 min at 10° C. After discarding the supernatant, the remaining pellet was re-suspended in 1.0 ml 50 mM Na3C6H5O7followed by an additional centrifugation step at 13,500×g for 15 min at 4° C. and removal of the supernatant. The mass of alginate pellets were weighed.

For disrupting bacterial cells, the pellet was re-suspended in 1 ml −70° C. cold methanol, transferred to 2 ml screw-capped tubes containing 250 mg glass beads with a diameter of 0.25-0.5 mm and three glass beads with a diameter of 3 mm, and treated using the FastPrep-24 Instrument (MPBiomedicals, Illkrich, France) for 2×30 s at speed level 4 (lowest). Finally, to remove cell debris, the suspension was centrifuged at 13,500 for 15 min at 4° C. 500 μI of the supernatant was collected and stored at −70° C. until analysis. For preparing sample blanks of the cultivation medium (NBII), aliquots of 100 μI nutrient broth II were mixed with 1 ml methanol. Sample blanks for alginate beads derived samples were obtained by dissolving 2 g of wet, sterile alginate beads in 40 ml Na3C6H5O7(50 mM) and mixing 100 μl of the solution with 1 ml methanol.

The characteristics of the samples are additionally described in Table 5.

TABLE 5Average characteristics of samples prepared for metabolite profiling.Stenotrophomonas rhizophilaSPA-P69Sphingomonas sanguinisAlginateAlginateBFC (in-beadencapsulatedBFC (in-beadencapsulatedcultivation)PlanktonicplanktonicBashancultivation)PlanktonicplanktonicCell count/ml1.78E+112.96E+096.78E+109.11E+108.43E+092.65E+097.01E+09(g dry beads)Volume ml (mass g)2.00E−011.50E+002.00E−012.00E−012.00E−011.50E+002.00E−01of sampleWeight of pellet1.07E+029.78E+011.03E+021.02E+021.02E+029.61E+011.00E+02(mg)Calculated CFU/3.57E+104.45E+091.36E+101.82E+101.69E+093.97E+091.40E+09LC-MS Sample
Metabolic Profiling Using High Resolution Mass Spectrometry

For metabolite analysis, a Thermofisher, HPLC and Orbitrap (Q-Exactive) HPLC-MS-System was used with the following specifications: Column was an Atlantis dC18, 3 μm, 2, 1×100 mm; Flow was set at 0.3 ml/min; Gradient was 10% B (2 min)-50% B (5 min)-80% (15 min)-10% B (5 min), with a total run time of 40 min; Mobile Phase A was 0.1% Formic Acid/H2Odd; B: 0.1% Formic Acid/Acetonitril.

Specific mass spec parameters were as follows: Voltage. 3100; Capillary Temperature: 330; Positive, Negative Mode extra; Resolution: 70.000, AGC target: 1 e6, Maximum IT: 200 ms, Scan Range: 100 to 1500 m/z; MS2-Parameter: Resolution: 17.500.

Mass spec general settings were as follows: Precursor Selection: Use MS(n-1) Precursor; Use New Precursor Reevaluation: True; Use Isotope Pattern in Precursor Reevaluation: True; Store Chromatograms: False.

Spectrum Properties Filter settings were as follows: Lower RT Limit: 0; Upper RT Limit: 0; First Scan: 0; Last Scan: 0; —Ignore Specified Scans: (not specified); Lowest Charge State: 0; Highest Charge State: 0; Min. Precursor Mass: 100 Da; Max. Precursor Mass: 5000 Da; Total Intensity Threshold: 0; Minimum Peak Count: 1.

Scan Event Filter settings were as follows: Mass Analyzer: (not specified);—MS Order: Any; Activation Type: (not specified); Min. Collision Energy: 0; Max.

Collision Energy: 1000; Scan Type: Any; Polarity Mode: (not specified).

Peak filter settings were as follows: S/N Threshold (FT-only): 1.5.

Replacements for Unrecognized Properties settings were as follows: Unrecognized Charge Replacements: 1; Unrecognized Mass Analyzer Replacements: ITMS; Unrecognized MS Order Replacements: MS2; Unrecognized Activation Type Replacements: CID; Unrecognized Polarity Replacements: +; Unrecognized MS Resolution®200 Replacements: 60000; Unrecognized MSn Resolution®200 Replacements: 30000.

HPLC mass spec data analysis was performed using Compound Discoverer version 2.1.0,398 (Thermofisher) and an untargeted metabolomics workflow to find and identify the differences between samples. The workflow data analysis included retention time alignment, unknown compound detection, and compound grouping across all samples, predicted elemental compositions for all compounds, filled gaps across all samples, and hid chemical background (using Blank samples).

For grouping unknown compounds, Compound Consolidation settings were as follows: Mass Tolerance: 5 ppm, RT Tolerance [min]: 0.05. Fragment Data Selection used the following Preferred Ions: [M+H]+1; [M−H]−1. To fill gaps, the following General Settings were used: Mass Tolerance: 5 ppm, S/N Threshold: 1.5, Use Real Peak Detection: True. To normalize areas, QC-based Area Correction settings were as follows: Regression Model: Linear, Min, QC Coverage [%]: 50, Max. QC Area RSD [%]: 30, Max. # Files Between QC Files: 20. Area Normalization settings were as follows: Normalization Type: None, Exclude Blanks: True.

For unknown compound detection mass tolerance was set at 5 ppm, intensity tolerance at 30%, S/N threshold of 3, min. peak intensity of 1000000, ions:[2M+ACN+H]+1, [2M+ACN+Na]+1, [2M+FA−H]−1, [2M+H]+1, [2M+Na]+1, [2M+NH4]+1, [2M−H]−1, [2M−H+HAc]−1, [M+2H]+2, [M+3H]+3, [M+ACN+2H]+2, [M+ACN+H]+1, [M+ACN+Na]+1, [M+CI]−1, [M+FA−H]−1, [M+H]+1, [M+H+K]+2, [M+H+Na]+2, [M+H+NH4]+2, [M+H−H2O]+1, [M+H−NH3]+1, [M+K]+1, [M+Na]+1, [M+NH4]+1, [M−2H]−2, [M−2H+K]−1, [M−H]−1, [M−H+HAc]−1, [M−H−H2O]−1, Base Ions: [M+H]+1; [M−H]−1, Min. Element Counts: C H, Max. Element Counts: C90 H190 CI4 K2 N10 Na2 O15 P3 S5.

For peak detection, -the following settings were used: Filter Peaks: True, Max. Peak Width [min]: 0.5, Remove Singlets: True, Min, # Scans per Peak: 5, Min. # isotopes: 1. QC-based area correction and area normalization settings were as follow: QC-based Area Correction: Regression Model: Linear, Min. QC Coverage [%]: 50, Max. QC Area RSD [%]: 30, Max. # Files Between QC Files: 20; Area Normalization:

Normalization Type: None, Exclude Blanks: True. Composition prediction settings were as follows: Mass Tolerance: 5 ppm, Min. Element Counts: C H, Max. Element Counts: C90 H190 C14 N10 018 P3 S5, Min. RDBE: −1, Max. RDBE: 40, Min. H/C: 0.1, Max. H/C: 4, Max. # Candidates: 10, Max. # Internal Candidates: 200. Fragment matching settings were as follows: Use Fragments Matching: True, Mass Tolerance: 5 mmu, S/N Threshold: 3.

Compounds were identified using mzCloud (ddMS2) and ChemSpider (formula or exact mass). mzCloud search settings were as follows: Compound Classes: All, Match Ion Activation Type: False, Match Ion Activation Energy: Match with Tolerance, Ion Activation Energy Tolerance: 20, Apply Intensity Threshold: True, Precursor Mass Tolerance: 10 ppm, FT Fragment Mass Tolerance: 10 ppm, IT

Fragment Mass Tolerance: 0.4 Da, Identity Search: HighChem HighRes, Similarity Search: Similarity Forward, Library: Reference, Post Processing: Recalibrated, Match Factor Threshold: 50, Max. # Results: 10. Pattern matching settings were as follows: Intensity Tolerance [%]: 30, Intensity Threshold [%]: 0.1, S/N Threshold: 3, Min.

Spectral Fit [%]: 30, Min. Pattern Cov. [%]:90, Use Dynamic Recalibration: True.

ChemSpider used the following data sources: BioCyc,E. coliMetabolome Database, Human Metabolome Database, KEGG, MeSH, NIST Chemistry WebBook Spectra, PubChem, and the Yeast Metabolome Database.

Similarity searches were also performed for all compounds with ddMS2 data using mzCloud. Compounds were mapped to biological pathways using KEGG database. Map to KEGG pathway used the following settings: 1. By Mass Search Settings: Mass Tolerance: 5 ppm; 2. By Formula Search Settings: Max. # of Predicted Compositions to be searched per Compound: 3, and 3. Display Settings: Max. # Pathways in ‘Pathways’ column: 20.

QC-based batch normalization was applied when QC samples were available. Workflow also included differential analysis calculation (t-test or ANOVA), p-value determination, adjusted p-value determination, ratios, fold change, and CV.

Using ThermoFisher Compound Discoverer, comma-separated variable (CSV) files representing the Input Files Table and Expected Features Table were exported as described in the Thermo Compound Discoverer User Guide (available at thermofisher.com). All downstream processing and analysis was performed in the R statistical language R, version 3.5.1 (R Core Team, 2016. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. R-project.org/). Unless stated otherwise, all analyses were performed identically but separately for positive and negative ion datasets. Peak groups were constructed using the XCMS function do_groupChromPeaks_nearest with default parameters (Smith et al. (2006).XCMS: Processing mass spectrometry data for metabolite profiling using Nonlinear peak alignment, matching, and identification.

Analytical Chemistry, 78:779-787). Within each sample, intensities of peaks having identical m/z and retention time were summed. All sample datasets were aggregated into a single matrix, with a NA indicator representing the absence of detection of a particular peak in a particular sample. To increase dataset density, metabolites represented by NA in over half of the samples were removed. Remaining NA values were replaced with a minimum intensity value, defined as one-half of the smallest, nonzero intensity of the dataset. To normalize intensity levels across the samples, quantile normalization was performed using the “normalize.quantiles” function in the preprocessCore package Bolstad B (2018). preprocessCore: A collection of preprocessing functions. R package version 1.42.0, available at github.com/bmbolstad/preprocessCore). Normalized intensities were log-2 transformed for subsequent statistical analysis.

To reduce unnecessary multiplicity of statistical hypothesis testing, nonspecific filtering was performed using genefilter [Gentleman2018] to eliminate metabolites with a coefficient of variance less than 0.03 or an interquantile range of less than 0.5. Statistical t-testing was performed with the limma function ImFit (Ritchie et al. (2015). “limma powers differential expression analyses for RNA-sequencing and microarray studies.”Nucleic Acids Research,43(7), e47.), using an empirical Bayesian estimate for variance via the eBayes function, and using the topTable function to obtain a Benjamini and Hochberg p-value adjustment. To project metabolites onto known metabolic pathways and compound names, the mummichog algorithm (Li et al. Predicting Network Activity from High Throughput Metabolomics. Ouzounis C A, ed.PLoS Computational Biology.2013; 9(7):e1003123) was used via the implementation in MetaboAnalystR (Chong & Xia (2018) MetaboAnalystR: an R package for flexible and reproducible analysis of metabolomics data.Bioinformatics.2018 Jun. 28). The InitDataObjects was called with analysis type set to “mummichog”, followed by UpdateMummichogParameters with instrumentOpt set to “0.1”, ion polarity set to positive or negative, and pvalCutoff set to 1.0E-6. Finally, the PerformMummichog function was called with an appropriate prokaryote model, along with “fisher” and “gamma” parameters for enrichOpt and pvalOpt, respectively. Compound identifiers were mapped to the corresponding descriptive KEGG names (Kanehisa & Goto (2000) KEGG: kyoto encyclopedia of genes and genomes.Nucleic Acids Res.28(1):27-30). The log fold change was adjusted to reflect the relative CFU/LC-MS sample.

Compounds increased in RFC formulatedSphingomonas sanguinisandStenotrophomonas rhizophilaSPA-P69 relative to reference compositions are shown in Table 6A. Compounds increased in BFC formulatedSphingomonas sanguinisrelative to reference composition Alginate Planktonic are shown in Table 6B. EPS refers to an extracellular polymeric substance. Exemplary compounds should be understood to include related compounds such as oxidized and reduced forms and derivatives.

TABLE 6ACompounds increased in BFC formulatedSphingomonas sanguinisandStenotrophomonas rhizophilaSPA-P69 relative to reference compositionsStenotrophomonasrhizophilaSphingomonasSPA-P69sanguinisTiter-Titer-adjustedTiter-adjustedadjustedRange offold-change offold-change offold-changetiter-adjustedBFC relative toBFC relative toof BFCfold-changereferencereferencerelative torelative tocompositioncompositionreferenceClasses orExemplaryreferenceAlginateAlginatecompositionFunctionsCompound(s)compositionsPlanktonicPlanktonicBashanOthernaphthazarin13.8-485.3485.3313.8375.04Vitamin/RedoxNAD+/NADH1.25-4.03.133.733.98Thiamine2.3-143.5143.489.592.26Adenine1.1-15.97.4615.951.07AA, protein,tryptophan1.7-13.913.892.773.79EPSphenylalanine1.3-4.44.401.281.58AA, non-proteinhomocysteine3.7-16.116.096.653.69Stabilizationtryptophan1.7-13.913.892.773.79Pathwayhomocysteine3.7-16.116.096.653.69cyclingerythrose 4-3.2-9.78.062.031.64phosphatepyridoxine3.1-5.34.401.281.58phosphatephenylalanine1.3-4.44.401.281.58

TABLE 6BCompounds increased in BFC formulatedSphingomonas sanguinisrelative to reference composition Alginate PlanktonicSphingomonas sanguinisTiter-adjusted fold-changeof BFC relative toClasses/Exemplaryreference compositionFunctionsCompound(s)Alginate PlanktonicOthernaphthazarin485.33Vitamin/RedoxThiamine143.48Nicotinamide8.12Adenine7.46NAD+/NADH3.13Stabilizationglutamic acid/glutamate55.44tryptophan13.89spermidine6.07Pyocyanin1.55Pathwayhomocysteine16.09cyclingadenosine10.18erythrose 4-phosphate8.06pyridoxine phosphate4.40phenylalanine4.40methionine4.16N-methylene-L-glutamate0.91Othermethylthioninium chloride1.82EPSglucosamine/chitosamine6.58DNA/RNA,Cytosine/adenine8.95EPSAntioxidantglutathione97.31citric acid8.21Pyocyanin1.55AA, protein,glutamic acid/glutamate55.44EPStryptophan13.89tyrosine6.58phenylalanine4.40methionine4.16lysine2.70proline2.63AA, non-homocysteine16.09protein

TABLE 6CCompounds increased in BFC formulatedStenotrophomonas rhizophilaSPA-P69 relative to reference compositionsStenotrophomonas rhizophilaSPA-P69Titer-adjustedfold-change ofTiter-adjustedBFC relative tofold-change ofreferenceBFC relative tocompositionreferenceClasses/ExemplaryAlginatecompositionFunctionsCompound(s)PlanktonicBashanOthernaphthazarin13.8375.04AA, non-proteinhomocysteine6.653.69AA, protein, EPStryptophan2.773.79phenylalanine1.281.58OsmolyteRhamnose1.482.38Pathwayhomocysteine6.653.69cyclingerythrose 4-phosphate2.031.64pyridoxine phosphate1.281.58phenylalanine1.281.58Stabilizationtryptophan2.773.79Vitamin/RedoxNAD+/NADH3.733.98Thiamine9.592.26Adenine15.951.07

Example 4

Colony Characteristics within BFC Produced Beads

Paraburkholderia caledonicaandEnterobacter cowaniiwere prepared by the BFC method using pre-culture inoculation densities of between 2E+7 and 4E+7 with differing times of in-bead cultivation, and forEnterobacter cowaniidiffering alginate percentages and nozzle diameters were used.

Paraburkholderia caledonicasamples were prepared using an 80 μm diameter nozzle and a 2% alginate solution. In-bead cultivation was performed for 47, 138, or 168 hours.Enterobacter cowaniisamples were prepared using 80 μm diameter nozzle and a 120 μm diameter nozzle and 2% and 3% alginate solutions. In-bead cultivation was performed for 50, 70, or 71 hours. Beads produced by the 80 μm diameter nozzle produced beads had an average diameter of 186 μm and a range of 134-235 μm when wet, and an average diameter of 78 μm and range of 50-103 μm after drying. The beads had approximately between 2 and 100 colonies per bead. The average, smallest and largest bead diameters are listed in Tables 7A and 7B. Colony diameter was measured at the widest point of the colony. Colony measurements were made in wet beds. Increasing in-bead cultivation time resulted in only moderate increases in colony diameter.

TABLE 7AColony size measurements ofBFC encapsulatedParaburkholderia caledonicaParaburkholderia caledonicaColony Size MeasurementsSmallestAverageLargestTimecolonycolonycolonyBatch(hr)(μm)(μm)(μm)180907-80 μM-2% Alginate47162135180907-80 μM-2% Alginate138152537180907-80 μM-2% Alginate168192741Avg:162438

TABLE 7BColony size measurements of BFC encapsulatedEnterobacter cowaniiEnterobacter cowaniiColony Size MeasurementsSmallestTimecolonyAverageLargestBatch(hr)(μm)(μm)(μm)180803-175-80 μM: 2% Alginate50213240180620-175-80 μM: 3% Alginate70263133180619-175-120 μM: 3% Alginate71243743Avg:243339