CA1300538C - Maintenance of the viability of microorganisms for use in microbial inoculants - Google Patents

Abstract

ABSTRACT OF THE INVENTION

The invention pertains to the use of soluble, non-crosslinked polysaccharides, and in particular, algin-ate, as a stabilizing agent for microbial cells. The stabilized cells which result from the invention are useful as microbial inoculants in agriculture. A wide variety of microbial genera may be stabilized through the compositions of the invention.

Description

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Field of the Invention The invention pertains to a method for stabilizing bacterial cells for extended periods of time. This stabilization is achieved through the formation of a slurr~ containing the bacterial cells, and non-cross-linked agents such as polysaccharides. The invention further pertains to a composition of matter containing non-crosslinked sodium alginate and microorganisms in suspensionO The invention also pertains to the above composition which additionally contains oils in emulsions. Additionally, the invention pertains to the composition and use a dry powder which could be used to coat seeds. The invention further pertains to the use of such dry powders as microbial inoculants in agriculture. The invention also pertains to the use of sodium alginate and microorganisms in solution, dried to a powder and used to coat seeds directly or when suspended in oil.

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-Backqround The use of microbial inoculants (such as Rhizo-bium) has long been know to increase crop yields. The -utility of this approach has been limited by the availability of compositions capable of both affixing the microbial cells to the seed and maintaining the viability of the microbial inoculant. Burton, J.C.
(U.S. Patent 2,995,867) discloses the possibility of using water soluble gums such as methylcellulose as a seed coating. Through the use of such coatings, it is possible to keep microbial cells alive and effective for over two weeks.
The success of this work spurred others to at-tempt to identify alternate compositions which might be capable of maintaining bacterial viability for longer periods of time. A method was disclosed by Hamrin, B.S.A. (U.S. Patent 3,688,437), which involved the use of seed coatings comprising a gelatin-calcium alginate foam. Dommergues, Y.R., et al. ~U.S. Patent 4,155,737) disclosed the possibility of embedding the microbial inoculant in a polyacrylamide seed coating.
The possiblity of stabilizing bacterial cells in a microbial inoculant through the use of a polysaccha-ride matrix was shown by ~ugnier, J., et al. (French Patent 2,469,861). This patent discloses the ability to stabilize a suspension of bacterial cells by embed-ding them in an alginate gel. The gel disclosed in this patent is formed by the addition of calcium chlo-ride to a solution containing a suspension of bacter-ial cells and sodium alginate. The addition o~ cal-cium chloride results in the precipitation and cross-linking of the previously soluble sodium alginate mixture.

A3fi.8 050686 ~3~53~3 A similar alginate composition is disclosed by Jung, G. (European Patent 17,565 and U.S. Patent 4,434,231). These patents disclose the possibility of crosslinking or precipitating solutions of sodium al-ginate that contain suspensions of bacterial cells.
The Jung patents disclose the use of calcium sulfate to convert a sodium alginate suspension into a gel.
This crosslinking reaction can be delayed through the addition of sodium phosphate to the suspension. 3ac-terial cells treated in this manner remained viable for greater than 60 days. These patents further dis-close the use of such embedded microorganisms as mic-robial inoculants in agriculture. Although the ease of formation o~ crosslinked alginate ~ls commended their use as an embedding matrix for a m~crobial inoc-ulant, such matrices were found to be inEerior to em-bedding matrices of xanthan gums in that embedding matrices composed of xanthan gums were found to re-lease microorganisms into the soil more readily than matrices composed of alginates. ~Jung, G., et al., Plant and Soil, 65:219-231 t1982)).
Jung, et al. (European patent no. 83267) disclose compositions containing microbial cells and poly~eric gels which may be dried and used to inoculate plant seeds. The patent further discloses the use of a crosslinked alginate suspension of microbial cells as a microbial inoculant.
One concern reflected in the prior art is that suitable embedding agents should provide an environ-ment strong enough to resist external abrasion and adverse forces yet be pliable enough to allow release of internal components and permit breakdown of the A~;.fS 050686 ~L3~531C:~

matrix at the appropriate time. Two different methods have been disclosed in order to accomplish this goal.
Redenbaugh, M.K., et al. (P.C.T. Publication wo85/02972) disclosed the use of crosslinked algin-ate-bacterial cell suspensions which are surrounded by an external membrane as a microbia:L inoculant.
Fravel, D.~., et al. (Phytopatholoq~, 75:774-777 ~1985)) disclosed the use of crosslinked alginate-bacterial cell suspensions which contain clays or other materials, added as bulking agents. The process disclosed by Fravel, et al produces solid pellets of relatively uniform size.
Thus, the prior art teaches the value of pre-in-~oculating s eds with microbial suspensions in order to enhance their germination and growth. A wide variety of different embedding matrices have been disclosed in the prior art. Especially significant are matrix com-positions which employ crosslinked or precipitated alginates or very thick, gum-like compositions. Such compositions have been successful in sustaining bacte-rial cell viability for extended periods of time, while simultaneously possessing the desired properties of rigidity and support. The preparation of gels, however, requires additional steps and both gels and gums involve handling difficulties.

Summary of the Invention The present invention relates to an improved pro-cess for maintaining cell viability and cellular in-tegrity under conditions ttemperature, pH, enzymatic reactions, etc) under which cell viability and integ-rity would normally deteriorate. The invention is useful in the formation of microbial inoculants in agriculture.

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The invention utilizes the properties of a liquid system wherein the cells are protected from environ-mental conditions, but freely accessible to substrates and co-factors, which may be added at any time to the system. The liquid system described by the invention may have varying degrees of fluidity, but does not have semi-solid, viscous, gel- or gum-like properties.
The invention is based on the discovery that non-crosslinked polysaccharide solutions stabilize the viability of non-dormant microbial cells. Such solu-tions can also, in accord with the present invention, be used to preserve dormant microbial cells for subse-quent use.
In detail, the invention pertains to a method for maintaining the viability of a microorganism which comprises: maintaining a suspension oE the micro-organism in a free-flowing composition which comprises a water dispersible, substantially soluble, non-cross-linked polysaccharide.
Additionally, the invention pertains to a method for inoculating a plant seed with a microorganism which comprises: providing to the plant seed a free flowing composition which comprises ti) a suspension of the microorganism and (ii) a water dispersible, su~stantially soluble, non-crosslinked polysaccharide.
The invention is also directed to a method for maintaining the viability of a microorganism which comprises: drying a free-flowing composition, the composition comprising a suspension of the micoorgan-ism and a water dispersable, substantially soluble, non-crosslinked polysaccharide to form a dried compo-sition.

~3~.8 050~86 ~3(~53~3 The invention is further directed to a method for inoculating a plant seed with a microorganism which comprises:
(a) drying a free-flowing composition, the compo-sition comprising a suspension of the microorganism and a water dispersible, substantially soluble, non-crosslinked polysaccharide, to form a dried composi-tion, and (b) providing the dried composition (a) to the plant seed.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to suspensions con-taining polysaccharides, and the use of such suspen-sions as a means oE stabilizing or preserving micro-bial cells.
The invention further relates to drying a solution which comprises a non-crosslinked polysaccharide, and a microorganism to produce a powder and using the pow-der obtained after drying either directly or when dis-persed in oil.
As used herein, the term "microbial cells" refers to any of a wide variety of microorganisms. Examples of such microorganisms are Pseudomonas, Serratia, Arthrobacter, Azospirillum, Rhizobium and ~acillus, but the invention can be used to maintain the viabil-ity of any microorganism which has a beneficial effect on the growth, emergence, yield, etc. of plants.
The term "polysaccharide," as used in this inven-tion is meant to refer both to polysaccharides which must be externally added to the suspension of micro-A36.8 050686 ~;~U(~53~3 bial cells, and to polysaccharides produced directlyby a microorganism under appropriate culture condi-tions, such as for example by pe~mitting cultures of ~icroorganisms to enter growth limiting conditions (e.g. nitrogen exhaustion) with an excess of carbon, or energy source. (Sutherland, I.W., ~licrobial Poly-saccharides and Polysaccharases, (Berkeleyl Gooday, and Ellwood, eds.) Acad. Press NY (1979)). An example of a polysaccharide included in the invention is the polysaccharide obtained from algae. Such polysaccha-rides are collectively referred to as "alginates."
The invention is specifically directed to the use of polysaccharides which are substantially soluble in an aqueous solution. Examples of such polysaccharides are alginates, celluloses, or starches. The solublt polysaccharides of the present invention are further restricted to those which are non-crosslinked.
As used in this invention, a polysaccharide is considered to be "non-crosslinked" if the polysaccha-ride is either free of intermolecular crosslinks, or if the ~egree of crosslinking is of such minor char-acter so as to permit the polysaccharide to remain soluble in an aqueous solution. The concentration of polysaccharide in these solutions is described as its concentration relative to that of the total volume of the solution (i.e. as a weight/volume percentage).
~nless otherwise indicated the alginate-microorganism mixtures of the present invention are prepared by dis-solving alginates in water and then adding the algi-nate solution to a culture broth. The degree of crosslin~ing is less than 10%, preferably less than 5~.

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The present invention relates to the use of "water dispersible," "free flowing" compositions. These terms are meant to refer to compositions which have low viscosity, and are pourable. These terms are spe-cifically intended to exclude compositions which are solids, gels or gums at ambient (4C-30C) tempera-tures.
As used herein, the terms "dried" or "drying" is intended to describe the removal of water. Any means capable of achieving the removal of water may be em-ployed. Suitable techniques including air drying, spray drying, lyophilizing, etc. are well known in the art.
As used herein, the term "oil" refers to any of a large class oE compounds hich are soluble in hydro-phobic solvents such as ether or alcohol, but are sub-stantially insoluble in water. The oils suitable for use in the present invention are liquids at ambient temperatures. Examples of suitable oils are: Mineral oil, or vegetable oils such as soya oil, canola oil, corn oil, etc.
As mentioned above, an oil is substantially in-soluble in water. Thus a water/oil mixture will tend, upon storage, to spontaneously separate (on the basis of density) into two substantially pure solutions, one of which contains the oil and the second of which con-tains the water. Although the present invention can be practiced with a water/oil mixture, it is prefer-able to add an "emulsifier" or 'lemulsifying agent" to the mixture. An emulsifier is a substance which is capable of enhancing the dispersion of the aqueous phase into the oil. Emulsifiers are disclosed by A36.8 050686 ~L3~i3~

g--Huettinger, R~, (Seif., Oele, Fette, Wachse, 109:455-479 ~1983)). Examples of suitable emulsifying agents are Arlacel 8~ 1, Arlacel 186 , and Tween al*
Arlacel* is manufactured by ICI Chemical Corp.
The present invention provides a means for stabi-lizing the viability of microbial cells and may be used to preserve microorganisms for a wide variety of applications. In pa~ticular the present invention may be used to maintain viability of microorganisms in inoculants for agricultural and industrial applica-tionsO According to the invention cells may be sta-bilized to provide a suitable storage period and then applied easily to seeds in the form of a liquid, free-flowing coating to produce a variety of effects in-cluding enhancement of emergence, growth and yield.
The applied microorganisms may function in diverse manners to produce the desired response and include members of the genera Rhizobium, Pseudomonas, Bacil-lus, Arthrobacter, Serratia and ~zospirillum.
The invention can be performed using a large num-ber of possible non-crosslinked polysaccharides, such as starch, or xanthan gum, however it is preferable to use alginate polysaccharides. Although the invention can be practiced using polysaccharide solutions which range from 0.01-20%, it is preferable to use polysac-charides solutions of between 0.5-2.5~, with about 1.0-2.0% being most preferable for alginate.
To produce the polysaccharide-bacterial cell sus-pensions of the invention, aliquots of a fresh, heal-thy culture of cells containing a cell concentration hi~h enough to provide any desired biological or in-* Trade-mark ,,~1 ~ i .,. ~

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dustrial effect, but not so high as to negatively in-terfere with such effect, and preferably between 1-50 grams of cells (dry weight) per liter, are mixed with equal ali~uots o~ sterile solutions containing a solu-ble polysaccharide. ~acterial cultures treated in this manner may then be stored at any temperature wherein the bacteria remain viable, such as, e.g., between 4-30 C. The degree of bacterial stabilization has been found to be independent of the sample storage temperature.
The polysaccharide solution may contain nutrients or growth factors such as, for example, sugars, amino acids, proteins, salts, etc. In addition, the poly-saccharide solutions may contain osmoregulatory agents, such as buEfers, which may be required in order to practice the invention.
In one embodiment of the invention, the bacterial cells are suspended in a polysaccharide solution which may then be applied to seeds, by any of a variety of means, such as, for example, dipping, spray drying, etc. Such techniques are disclosed by Cull, S.W., et al. (European Patent 97,459); Mugnier, J., et al.
tFrench Patent 2,469,861); Jung, G. (U.S. Patent 4,434,231); and Redenbaugh M.K., et al. (PCT No. W085/
02972).
In a preferred embodiment of the invention, the bacterial cells are suspended in a non-crosslinked polysaccharide solution and then incorporated into an oil emulsion. This composition may then be used as an agricultural inoculant, for example, by diluting it with water and using it as a liquid spray or by coat~
ing it directly onto seeds. The polysaccharide/cell ~Ofi86 s~

solution may alternatively be dried to a powder to provide successful storage either alone or t pref~r-ably, in combination with agrichemicals. This powder may be applied directly to seeds, or may be dissolved in water or dispersed in oil to prod~ce a liquid which can be sprayed onto seeds.
The previously ci~ed techniques applying polysac-charide/bacterial cell suspensions to seeds can be readily adapted by one of ordinary skill in the art so as to enable their application to the compositions of the present invention. It is, however, preferable to dry the suspensions at 30C by blowing air over their surfaces. This is preferably accomplished by placing the suspension in an incubator and blowing sterile, filtered air across tha surface of the suspension.
The dried composition may then be coated onto seeds by mixing.
Having now generally described this invention, the same will be better understood by reference -to certain specific examples which are included herein for pur-poses of illustration only and are not intended to be limiting of the invention, unless specified.

Example 1: Growth of Microbial Strains Rhizobium ~aponicum cultures where maintained on YEM agar plates tlOg mannitol; 0.5g (Difco) yeast ex-tra~t; 0-5g K2HP04; 0.2g MgS04.7H20; O.lg NaCl; 20g agar; water to lL. 100 ml liquid cultures were obtain-ed by inoculating lOOml of medium A (lOg sucrose, 0.5g (Veeprex*B800) yeast autolysate; 0.22g R2~P04; O.lg MgS04.7H20; 0.04g CaC12; 0.02g FeC13; water to lL) with a single colony from a YEM agar plate. The li-* Trade-mark ~- ~ n~n~86 ~3V~53~

quid cultures were incubated at 30C for 7 days on a orbital shaker at 140 RPM. One liter cultures were obtained by transferring a fresh 100ml culture into lL
of medium A and incubating for 5-7 days at 30C on an orbital shaker at 140 RPM.
Unless otherwise indicated, all other cultures were maintained on PAF agar plates (38g (Gibco) Pseu-domonas agar F; 10g glycerol; 20g agar; water to lL) liquid cultures were obtained by inoculation of TSs (3Q g (Gibco) tryptic soy broth; water to lL) with a single colony from a PAF agar plate and incubating at 30C for 48 hours on an orbital shaker at 140 RPM.

Example 2: Effect of Temperature on ,tability of_ Rhizobium iaponicum Rhizo~ium iaponicum USDA 138 was grown as describ-ed in Example 1. 50ml of fresh culture medium was mixed with an equal amount of sterile solutions of 1%
and 2% non-crosslinked sodium alginate to produce samples having .5~ or 1~ alginate. Samples containing a final alginate concentration of 1~ were stored at 4C, room temperature, and 30C. The sample contain-ing 0.5% alginate was stored at room temperature.
Periodically, the mixtures were well agitated and samples removed for viable cell culture counts. The result of this experiment is shown in Table 1. This experiment demonstrated that the stability of R.
japonicum cells in an alginate slury was essentially unaffected by storage temperature.

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T~B$E 1 Log viable count/mL
Sample to lwk 4wks lOwks 21wks 26wks 1%; 4C8.8~ 8.45 7.99 9.06 g.59 ---1~; RT9.61 8.93 9.86 9.44 8.64 ---1%; 30C9.428.70 8.85 9.06 ~.53 8.17 0.5~; RT 10.42 9.68 9.62 9.29 8.86 ---Example 3: Effect of Polysaccharide_ on Stability of Rhizonbium iapon _ m Rhizobium japonicum USD~ 138 was grown as describ-ed in Example 1. Samples of the liquid cultures were removed and mixed with the non-crosslinked polysac-chari.des sls.wn in Table 2. Mixtures were stored at room temperature and sampled periodically for viable cell counts following vigorous agitation. This exper-iment showed that a wide variety of soluble polysac-chrides could be used to sustain the viability of bac-terial cells.

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~Le 2 Log viable count/ML
Sample to lwk 4wks lOwks 30wks

2.5% Dextran ~ 7.7610.48 9.148.788.00 0.25% W~terLok B100 * 7.929.788.37 8.52 6.60

3.5 Corn starch 7.839.56 9.9310.549.29 0.5~ Carbo~ymethyl-cellulose 7.889.33 9.2610.848.18 0.5% Gum ghatti 7.638.51 8.658.368.31 0.5~ Xanthan gum 8.19 9.729.399.13 8.79 1% Gum arabic 7.779.26 9.4710.217.89 0.5% Gelatin 7.739.42 9.599.04 - -O.5~ Alginate/0.5M
sorhitol 7.889.55 9.0310.999.23 0.5% Alqinate/0.5M sucrose 7.36 7~979.949.00 9.15 0.5% Alginate/0.5% saline7.62 8.437.44 ~ - O
0.5% Alginate/0.5% yeast autolysate 7.818.49 7.295.31 0 O.5% Alginate/0.5%
activated charcoal 7.799.68 8.878.148.03 0.5% Alginate/14% beet molasses 8.018.51 6.615.82 0 1.5% Carbcxymethyl-cellulose 7.619.44 9.499.267.56 2.5% Alginate 7.718.74 9.08 - - -0.5% Alginate 7.749.38 9.008.317.85 0.05~ Alginate 7.778.87 9.348.33 0.5% Alginate + 0.15%
B-cyclodeKtrin 7.718.97 9.248.617.94 * ~rade-mark ~3~3~

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Example 4: StabilitY of Pseudomonas and Bacillus Cultures in Alqinate Slurries 50ml of fresh healthy cultures of Pseudomonas fluorescens 17-34, Pseudomonas putlda :L-104, Pseudo-monas sp 55-14, Pseudomonas sp. 67-4, Bacillus sp.
86-64 and Pseudomonas putida G2-8 were grown as described in Example 1 and mixed with equal aliquots of sterile 1% non-crosslinked alginate solutions. The mixtures were stored at room temperature and periodi-cally agitated and sampled for viable cell counts.
The results of this experiment are shown in Table 3.
This experiment shows that 1% al.ginate slurries are successful in stabilizing a variety of bacterial strains.

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TrBL~ 3 Log viable count/ML
Orqanism To lwk 4wks 8w~s 10w~s 34wks P fluorescens 17-34 11.88 11.83 11.02 8.16 7.43 7.00 P Eutida 1-104 10.19 11.33 10.32 8.27 7.45 7.40 Pseudomonas sp 55--14 11.80 11.08 9.62 8.76 6.61 7.22 Pseudomonas sp.
67-4 11.~5 12.04 10.27 ~.22 7.21 8.29 Eacillus sp.
86-64 9.49 9.86 9.36 7.79 7.03 8.20 P. putida G2-8 10.41 11.73 11.48 8.26 7.25 8.26 h36.8 050686 ~3~3~

Example 5: Effect of Storaqe in Sealed Vials on the Stability of Pseudomonas and Bacillus Strains in Alqinate Solutions Aliquots of fresh healthy cultures of Pseudomonas fluorescens 17-34, Pseudomonas putida 1--104, Pseudo-monas sp. 55-14, Pseudomonas sp. 67-4, Bacillus sp.
86-64 and Pseudomonas putida G-2~ were grown as described in Example 1. Aliquots were mixed with equal volumes of a sterile 1% non-crosslinked alginate solution and dispensed into sterile 2.2ml glass vials.
The vials were completely filled and tightly capped, and were then stored at 30C or room temperature.
Periodically samples were agitated and viable cell counts were determined. The results of this experi-ment are shown in Table 4. ~his experiment shows that adequate gaseous exchange is required in order to maintain cell viability in alginate solutions.

A36.8 050686 ~3~(~S38 T~8L~ 4 Storage Log viable count/ML
Tempera-Organism ture _ ~ lwk 2wks 3~ks Swks P. fluorescens RT 12.03 9.79 5.32 2.71 3.79 17-34 3~C11.72 9.97 6.37 5~76 3.97 P. putida RT 11.44 -- 9.20 6.20 3.62 1-104 30C11.4S - 5.32 4.32 3.86 Pseudomonas sp. RT 11.80 11.64 5.70 4.72 3.97 55-14 30C11.88 11.67 8.15 1.79 0 Pseudomonas sp. RT 12.60 --3.34 3.46 3.32 67-4 30C12.84 -- 3.46 3.75 3.62 Bacillus sp. RT 9.32 9.46 9.17 5.72 5.35 56-64 30C9.32 9.57 5.71 4.67 2.98 P. putida G2-8 RT 12.41 ---5.46 5.45 5.31 30C12.45 - 5.62 4.25 3.32 n~n~;R~ ¦

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Example 6: Survival of Rhizoblum ~aponicum 138 in Alginate Solutions and Oil Emulsions in sulk and When Coated onto Soyabeans Rhizobium japonicum cultures were grown as des-cribed in Example 1. 250 ml of a fresh, healthy R.
Japonicum culture was mixed with 250 ml of a sterile l~ non-crosslinked alginate solution. This mixture was designated solution A. 100 ml aliquots of solu-tion A were removed and mixed with Food and Cosmetic Blue Dye #l (final dye concentration = 1.0~) (to form mixture B) or with Food and Cosmetic Blue Dye #l (final dye concentration = 1.0~) and Tween*81 (final concentration = ~.0~) (to form mixture C). The mix-tures were i) added to the alginate prior to sterili-zation (blue dye) and ii) sterilized independently and added to the final mixture (Tween)*

4 ml aliquots of the above mixtures were then dis-pensed into sterile 4~5 ml glass vials. In addition, 0.8 ml aliquots of solution C were dispensed into vials containing 3.2 ml of sterile mineral oil (to form solution D). All vials were then tightly sealed and stored at room temperature. Vials were removed periodically and agitated vigorously prior to sampling for viable cell count analysis. Table 5 shows the effect of time on the viability of cells in mixtures A-D.
Additionallyt at the time of preparation 0.4ml samples of mixtures A-D were used to coat 10g samples of soybean seeds. Aliquots of coated seeds were re-moved periodically to allow the number of viable cells surviving on the seed coating to be determined. Fig-ure 6 shows the effect of time on the viability of *trade-marks ~,~

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cells of mixtures A-D coated onto soybeans. While viability of R. japonicum was maintained in all the mixtures, the alginate/cell/oil emulsion is preferred since this composition facilitated seed coating and enhanced the flowability of the seeds. Successful sur-vival on the seed coat also shows that the mixtures can be employed as seed inoculants.

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5~LE 5 Lcg viable count/ml To3 wks 7 wks10 wks16 wks 37 wks A10.4090589.33 9.26 8.11 7.75 B10.9310.939.819.39 7.71 7.99 C9.658.46 9.20 8.75 6.30 6.31 D10.5210.62lQ.189.92 8.70 8.20 T~LE 6 Log viable courlt/seed ~stored at room teTperature) To 2h 4h 6h 24h 1 wk A6.225.87 5.55 4.62 4.64 3.27 B6.616.35 6.43 4.66 5.24 3.65 C6.015.~7 ~.12 4.81 4.48 '2.73 D5.395.66 5.35 4.25 5.24 3.79 O ~cn~R~

~3~ S3~3 Example 7: Effect of Pol~saccharide on Stability of 8acillus sp. 86-64 Bacillus sp. 86-64 was grown as described in Example 1. 50 ml aliquots of a fresh, healthy culture were mixed with an equal amount of a solution contain-ing: 6% alginate; 4~ alginate; 2% alginate; 4% algin-ate and 1.8~ saline; 4~ alginate and l.OM sucrose; 4~
alginate and 1~ activated charcoal; 4% alginate and 1.53 cyclodextrin; 4% alginate and 2% corn starch; 4%
alginate and 2~ glycerol; 4% alginate and 1% gelatin;
or 4% dextran, to form the following mixtures: Mix-ture A - culture medium + 3~ alginate; Mixture B -culture medium + 2~ alginate; Mixture C - culture medium + 1 alginate; Mixture D - culture medium + 2 aiginate ~ 0.9% saline; Mixture E - culture medium ~
2% alginate + 0.5M sucrose; Mixture F - culture medium + 2% alginate + 0.5% activated charcoal; Mixture G -culture medium + 2% alginate + 0.75% cyclodextrin;
Mixture H - culture medium + 2% alginate + 1% corn starch; Mixture I ~ culture medium + 2~ alginate ~
glycerol; Mixture J - culture medium + 2% alginate +
0.5% gelatin; Mixture K - culture medium + 2% dextran.
All alginate solutions contained only non-crosslinked alginates. The mixtures were stored at room tempera-ture and sampled periodically to determine the viable cell count. The result, given in Table 7, shows that a variety of polysaccharide mixtures and alginate to-gether with various adjuvants enhanced the stability of Bacillus sp. 86-64.

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Log viable count/ml _to4 wks 10 wks 29 wks A 8.94 8.91 7.52 7.2~
B 8.68 8.38 7.52 7.51 C 8.61 8.06 6.09 7.98 D 8.75 8.78 7.33 6.50 E 8.71 8.77 7.52 S.59 F 8.6n 9.26 7.90 7.23 G 8.64 8.13 6.45 6.51 H 8.69 8.56 7.12 7.63 I 8.69 8.75 7.89 7.60 J ~.80 8.09 7.59 7.24 K 8.73 8.32 6.43 6.55 Example 8: ViabilitY of P. fluorescens 17-34, Pseudomonas sp. 55-14 and Bacillus sp.
84-64 in Alqinate Mixtures and Alqinate/Oil Emulsions 50 ml aliquots of fresh healthy cultures of Pseu-domonas fluorescens 17-34, Pseudomonas sp 55 14 and Bacillus sp. 86-64 were grown as described in Example 1. Cultures were grown in 250ml Erlenmeyer flasks.
50ml of the culture broth were mixed with equal ali-quots of the following non-crosslinked polysaccharide compositions: (A) 4~ alginate, ~B) 4% alginate plus 2%
Food and Cosmetic Blue Dye #l; and (C) 4~ alginate plus 2~ (final concentration) Food and Cosmetic Blue Dye #1 plus 2% activated charcoal. Additionally, 10ml n~nfiRfi 13~

culture aliquots were mixed with lOml aliquots of the above alginate/dye mixture and either (D) 80ml sterile soya oil; (E) 78ml sterile soya oil plus 2ml Arlacel 83* or (F) 78ml sterile soya oil plus 2ml Arlacel 186 *. All flasks were stoppered with cotton wool bungs and stored at room temperature. At various times, the mixtures were well agitated and a viable cell count determination was made.
The results, shown in table 8, indicates that incorporation of the alginate/cell solutions into an oil emulsion enhanced cell survival.

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Log viable count/ml Bacillus sp. 86-64 Pseudomonas sp. 55-14 P. fluorescens 17-34 To lmo 6mo To lmo 6~o To lmo 6no A7.16 7.99 8.00 9.96 8.10 7.23 11.057.81 8.01 B7.86 8.10 7.35 11.338.80 7.41 11.228.34 7.97 C7.62 8.32 6.71 11.068.61 7.68 11.048.35 7.93 D8.78 11.3311.81 8.35 11.92 10.96 8.53 11.99 11.57 E8.51 8.33 12.04 8.25 11.32 11.36 8.57 11.69 11.62 F8.46 -- 9.76 8.47 12.40 9.51 8.41 11.25 11.31 r ~ ~ n ~

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Example 9: Survival of Rhi~obacteria Coated onto Soyabeans and Canola trape) Seed Usinq an Alqinate/Oil Emulsion System Cultures of rhizobacterial strains were prepared as described in Example 1 Fresh, healthy cultures were mixed with equal vol-umes of sterile 4~ alginate. Aliquots of the culture/
alginate solutions were then incorporated into emul-sions with either canola or soya oil in a ratio of 30~
aqueous: 68~ oil: 2~ emulsifier (Tween 81) to pro-duce mixtures which were then coated onto canola seeds or soyabeans using 60 uI, of mixture/lOg of seed or 75uL of mixture/2Sg of beans. Seed samples were re-moved periodically to dete~nine the viable cell count.
The results, presented in Table 9, show the efficacy of the fonnulation as a microbial seed inoculant de-livery system.

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q~B 9 Log viable count/seed To 2h 24h 48h 9_ Coated A2ospirillum sp. 2.94 2.58 1.98 2.14 onto Arthro~acter sp. 44-9 2.55 2.55 2.22 2.35 c~lola P. fluorescens 34-13 3.15 3.10 2.91 3.20 seeds in canola emulsion Coated Bacillus sp. 86-64 5.31 4.92 4.56 - 3.81 onto A2ospirillum sp. 4.06 3.43 3.03 - 2.21 soyabeans P. putida 57-10 4.95 4.41 3.95 - 3.33 in soya P. fluorescens G20-18 4.31 3.95 3.43 - 3.38 oil Serratia liqu.ifaciens 1-102 3.93 3.75 3.02 - 2.16 emulsion A3hA~ n~n~

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Example 10: Survival of S. liquifaciens s~. 1-102 in Powder Produced by Dryinq Alqin-ate/Culture Solution Serratia liquifaciens 1-102 was grown as described in Example 1. An aliquot of fresh, healthy Serratia liquifaciens 1-102 culture was mixed with 1~ sterile non-crosslinked alginate. The resultant mixture was then dried by blowing sterile, filtered air (30C, liters/min) across the surface of 100 ml. aliquots of the mixture in 4~ Erlenmyer flasks. The material ob-tained was then stored in a sealed container and sampled periodically for viable cell count determina-tion. The results obtained, given in Table 10, show that this composition can be used to maintain viabil-ity of S. liquifaciens sp. 1-102.

~ABLE 10 Log viable count/g To 3 daYs 9 daYs 15 day~ 60 days 10.21 12.11 11.60 12.06 11.28 While the invention has been described in connec-tion with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia-tions, uses, or adaptations of the invention follow-ing, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be ap-plied to the essential features hereinbefore set forth and as follows in the scope of the appended claims~

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Claims (23)

THE EMBODIMENTS OF THE INVENTION, IN WHICH AN EXCLUSIVE
PRIVILEGE OR PROPERTY IS CLAIMED, ARE DEFINED AS FOLLOWS:

1. A method for maintaining the viability of a microorganism which comprises:

maintaining a suspension of said microorganism in a free-flowing composition, substantially free from semi-solid, viscous, gel-or gum-like properties, which comprises a water dispersible, substantially soluble, non-crosslinked polysaccharide.

2. The method of claim 1 wherein said composition additionally contains an oil.

3. The method of claim 1 or claim 2 wherein said composition additionally contains an emulsifying agent.

4. The method of claim 2 wherein said oil is selected from the group consisting of soya oil, mineral oil, and canola oil.

5. The method of claim 1 wherein said non-crosslinked polysaccharide is alginate.

6. The method of claim S wherein said alginate is present in said composition at a concentration of between 0.1% - 10%.

7. A method for maintaining the viability of a microorganism which comprises:

drying a free-flowing composition, said composition being substantially free from semi-solid, viscous, gel- or gum-like properties and comprising a suspension of said microorganism and a water dispersible, substantially soluble, non-crosslinked polysaccharide to form a dried composition.

8. The method of claim 7 wherein said composition additionally contains an oil.

9. The method of claim 7 or claim 8 wherein said composition additionally contains an emulsifying agent.

10. The method of claim 8 wherein said oil is selected from the group consisting of soya oil, mineral oil, and canola oil.

11. The method of claim 7 wherein said non-crosslinked polysaccharide is alginate.

12. The method of claim 11 wherein said alginate is present in said composition at a concentration of between 0.1 - 10% .

13. The method of claim 7 wherein said drying is accomplished by air drying at ambient temperature.

14. The method of claim 1, claim 2, claim 4, claim 5, claim 6, claim 7, claim 8, claim 10, claim 11, claim 12 or claim 13, wherein said microorganism is selected from the group consisting of Pseudomonas, Serratia, Arthrobacter, Azospirillum, izobium, and Bacillus.

15. A microorganism-containing composition useful as an inoculant for a plant seed, which comprises a free-flowing composition substantially free from semi-solid, viscous, gel- or gum-like properties, said composition comprising (i) a suspension of said microorganism and (ii) a solid composition comprising water dispersible substantially soluble, non-crosslinked polysaccharide.

15. The composition of claim 15 additionally containing an oil.

31

17. The composition of claim 15 or claim 16 additionally containing an emulsifying agent.

18. The composition of claim 16 wherein said oil is selected from the group consisting of soya oil, mineral oil, and canola oil.

19. The composition of claim 15 wherein said non-crosslinked polysaccharide is alginate.

20. The composition of claim 19 wherein said alginate is present in said composition as a concentration of from 0.1% - 10%.

21. The composition of claim 15, claim 16, claim 18, claim 19, or claim 20,wherein said microorganism is selected from the group consisting of Pseudomonas, Serratia, Arthrobacter, Azospirillum, Rhizobium, and Bacillus.

22. Use of the compositions prepared according to claim 1, as plant seed inoculants.

23. Use of the compositions according to claim 15, as plant seed inoculants.