Title of Invention	STABLE ORGANIC-CARRIER-BASED MICROBIAL INOCULANTS AND METHOD FOR PRODUCING THE SAME
Abstract	The present invention relates to methods for producing microbial preparations for crop production and microbial preparations produced through such methods. The formulations contain microbial inoculants like Azospirillum spp. which are grown in a complex medium, lyophilized with powdered milk and further supplemented with minerals and vitamins, starch and inert substances such as talc, chalk and diatomaceous earth. The products manufactured by such methods have shelf-lives of more than 3 years. They are used to inoculate seeds, seedlings and planting materials among different agriculture and forestry species for enhancing root, root hair an shoot formation, enabling plants to efficiently use soil nutrients, supplying plants with nitrogen from the air, tolerating drought and protecting plants from root pathogenes.

Title of Invention

STABLE ORGANIC-CARRIER-BASED MICROBIAL INOCULANTS AND METHOD FOR PRODUCING THE SAME

Abstract

The present invention relates to methods for producing microbial preparations for crop production and microbial preparations produced through such methods. The formulations contain microbial inoculants like Azospirillum spp. which are grown in a complex medium, lyophilized with powdered milk and further supplemented with minerals and vitamins, starch and inert substances such as talc, chalk and diatomaceous earth. The products manufactured by such methods have shelf-lives of more than 3 years. They are used to inoculate seeds, seedlings and planting materials among different agriculture and forestry species for enhancing root, root hair an shoot formation, enabling plants to efficiently use soil nutrients, supplying plants with nitrogen from the air, tolerating drought and protecting plants from root pathogenes.

Full Text	STABLE ORGANIC-CARRIER-BASED MICROBIAL INOCULANTS AND METHOD FOR PRODUCING THE SAME DESCRIPTION OF THE INVENTION Field of Invention The present invention relates to microbial preparations for crop production. Background Art The present invention relates to microbial preparations for crop production, speciTicaliy microbial inoculants incorporated in carriers that provide protection for the colonizing microorganisms and the method for producing the same. In theory, microbial inoculants, without human intervention, have a low sun/ival rate and efficiency in their natural soil environment because of the insufficient colony forming units per gram soil. Sln The present invention Is a carrier-based biofertiiizer that promotes growth of plants of any type of terrestrial biome, using special biological Inoculants, such as plant growth promoting rhizobacteria. The invention induces extensive root and root hair formation, enable plants to efficiently use soil nutrients, supply plants with nitrogen from the air, increase drought tolerance and protect the plants from pathogentt. Further, the inoeulant can be mixed with an insecticide or fungicide without any appreciable decrease in effectivity. The closest prior art are reported in WO 2007/014974 {'Aseptic Mycorrhization Inoculant and In vitro and Ex vitro Application Methods'), W01^/006732 {'Method for Obtaining a Wettabia Powder Inoculant for Use with Leguminous C/t>ps). WO 2006/070061 {'Stable Microbial Inoculants and Methods for Production of Theirf), WO 2002/015702 {'A Thermostable Bkhmatrin, and WO 2000/034440 {'Microorganisms for Improving Plant Productive/). According to WO 2007/014974 {'Aseptic l\Aycorrhlzatlon Inoculant and In vitro and Ex vitro Application Methods'), a low-viscosity, light, semi-solid agar-type base culture medium is used to facilitate the penetration of the root in the inoculant, as well as to potentiate and preserve the infectiousness of arbuscular myconrhizal fungi, such as those of the present invention. WO 2007/014974 sought to obtain the advantage of being as effective in in vitro as well as ex vitro inoculation by using a semi-solid inoculant. However, it is gel-based and has a short shelf-life, which the present Invention seeks to address. WO 1994/006732 {'Method for Obtaining a Wettable Powder Inoculant for Use with Leguminous Crop^ reports a method for obtaining a wettable powder inoculant fomnulation for use with leguminous crops, comprising the industrialization of an inoculant for leguminous from a centrifugated biological mass of Rhizobium spp twice immersed in a cryogenic protective solution rich in sugar and protein which is subjected to a iyophilization method. Lil(e the present invention, it is fbnnulated with substances rich in polysaccharides and associated with a cellular protector which substitutes the previously used peat support. It also has a long shelf life of up to 25 months even when stored at room temperature. However, WO 1994/006732 can only be used for leguminous crops. Further, the formulation of the canler uses sugar and protein while the present invention uses miil( and glycerin. Also, WO 1994/006732 does not teach that the powder inoculant with a pest protectant such as an insecticide. WO 2006/0700ei {'Stabh Microbial Inooulonta and Metttoda for Productlort of Thorrf) teaches away from the present invention. It describes water containing microbial inoculants in paste fonri with shelf life of at least 3 months, preferably 12 months. It reports that drying, which is a method utilized in the present invention is complicated, expensive and may even be impossible. WO 2002/015702 ("A nBmo-stable Bi(HnatrbC) reports a method for producing a thernio-stable biodegradable medium for storage of biological materials wherein the biological material includes: a pesticide; a viricide; a bacteriadde; a fungicide; and a combination of these. While the method Is similar In that the biological material can be safely rhixed with potentially degradative substances such as pesticides, viricides, bacteriacides, fungicides or a combination of these, the medium Is structurally different tsecause it is gel-based and contains a blopolymer setecteid from the group of xanthan gum, acacia gum, guar gum, geltan, starch or a combination thereof, unliice the present invention which Is in powder form. Thus, it is no surprise that WO 2002/015702 discloses a maximum shelf life of only eighteen (18} months, which is considerably shorter.than that of the present invention which allows storage of at least three (3) years. WO 2000/034440 ^Microorganisms for Improving Plant Productivity) is directed to microbes which includes the microorganism Azo^irillum brasBensa, which belongs to the same genus as some of those in the present invention. As with the present invention, two specific strains of microbial inocuiant of WO 2000/034440 is effective for increasing the productivity of both nonlegume and legume plants as well as vegetable plants over a wide variety of soil types and climates. However, the WO 2000/034440 is narrowly directed only to the Azospliillum brasilense, a microorganism not covered by the present application. Further, it does not teach an inventive canler nor the method of production that prolongs its shelf life. The present invention, the microbes, method and product, are absolutely different and improve over other Icnown methods and products and have considerable advantages over the nature and quality of the methods and products, particularty in the storage life of the microbial preparation. Disclosure of the Invention The present invention relates to microbial preparations for crop production such as seed, seedling and other planting material inoculants have been the subject of many researches in the past 25 years in the Philippines. They have been shown to oe useful in increasing and protecting crop yields. However, they have not been put into commercial production because of their short shetf-lives which limit their effective distribution. This is also tnie of microbial inoculants for processing foods. Prior art discloses a shelf life of a maximum of only eighteen (18) months. This invention is intended to solve this problem of short shelf-lives and allow for the viable commercialization of microbial inoculants. The invention is expected to produce, products with a huge market since biochemical catalysts includirig microbial preparations have agricultural, food and forestry applications. The invention covers dried, semi-solid and related fonnulations of microbial inoculants like Azospirlllum spp. and Bacillus pumilus. These bacteria are used to inoculate seeds and seedlings of different agriculture and. forestry species to promote among others, the extensive root and root hair formation, enable plants to efficiently use soil nutrients, supply plants with nitrogen from the air, increase drought tolerance and protect the plants from root pathogens. These formulations are prepared in the following manner The microbes such as Azospirlllum spp. And Bacillus pumllus are grown infermentors usirig special media which contain substrates including high protein feed ingredients such as fish meal/bone meal/blood meal/soya meal, table sugar and tablet salt at SO^C. The baterial cells are han^ested by methods like filtration or centrifugation, added with powdered milk/glycerol to wet the cells and the AzoapMlum mixture lyophilized. The products a(& formulated under sterile conditions with different proportions of components like the bacterial preparations, minerals, milk, vitamins and flour with or without talc/chalk/diatomaceouB earth using standard mechanical devices. The dried and semi-solid products are packed or the tablets are made usually under sterile conditions. The products as invented have extended the shelf-lives of the biochemical catalysts/microbial preparations by at least 3 years way beyond .the 3 months observed with the non-lyophilized or other inoculants now available in very limited amounts in the market. The special media used to grow the bacteria improve the tolerance of the bacteria to lyophilization. f^ilk not only improves the tolerance of the bacteria to lyophilization; it also provides some vitamins, carbohydrates and minerals for a good start of the bacteria with inoculated plants. Bacillus pumllus need not be lyophilized because it is a spore fomier and can resist adverse conditions for many years. The added vitamins, minerals, nour and talc/chalk/diatomaceous earth also provide additional nutrients to selectively enrich the growth of the desired bacteria and the inoculated plants in problem soils with limiting micronutrients. Characteristics of the Mlcro^irganiama Azospihilum Is made up of curved rod cells, 1 mm in diameter with varying sizes and shapes, it can have polar and peritrichous flagella and undeigoes encystment. Its pinkish, whitish orange colonies vary in shape and surface characteristics. . Cart>on sources include organic acids and sugars. The nitrogen sources are amino acids, Hz, NH/ and NO3. Respiration can t>e aerobic, mlcroaerophilic or anaerot)ic. It Is capable cif aerotaxis, chemotaxis and can form clumps. Bacillus pumllus consists of rod-shaped cells, which can form endospores that are very resistant to many adverse conditions. Sporulatlon is not repressed by exposure to air. It is gram positive, or positive only in the early stages of growth, or negative. The flagellation Is peritrichous or degenerately peritrichous. It can be aerobic or fecuitatively anaerobic. Oxygen is the tenninal electron acceptor. Colony morphology and size are very variable on some media. It exhibits a wide diversity of physiological ability; psychrophilic to thennophilic; acidophilic to alkaliphilic. Some strains are salt tolerant while others have a specific requirement for salts. Catalase is fonned but may be oxidase positive or negative, it requires blotln and some strains also require amino acids. Spores formed occur ubiquitously The mole % G ■»■ C of the DNA ranges from 39.0-46.9 for 37 strains. .ONA-DNA hybridization studies indicate that the species is genetically homogenous. The depositoiy institution for these isolates Is the UP'-NSRI Culture Collection at Diliman, Quezon City File numbers of some culture deposits are stated below while others will be submitted when already available. Azospirillum halopraefemns - UPCC1370 Azospirfllum llpoferum - UPCC 1371 Bacillus pumllus - UPCC1372 Azosplrillum SC/PH - UPCC1373 Azospirillum AH/EH - UPCC1374 Shelf Life Extensive iiteratur attwcte that such dried dormant microbial colls can remain viable for years. To further protect the product from environmental assault, nutrients that sen/e as buffer against changes in temperature and other environmental shock as well as provide the Initial growth factors for the microbes to grow once the product gets wet and the cells start metabolizing. However, the product is vulnerable to fungal growth and/or grain beetles which considerably shorten its shelf life. Experiments were then conducted to test the inclusion in the product of fungicides and insecticides. Two commercial fungicidal preparations and two insectiddal preparations(organic and synthetic) were added to the microbial preparation following manufecturer's recommended dosages. Liquid organic pesticides were absort>ed in flour and added to the microbial preparation. These products were kept in storage for 6 months - 3 years. All products added with fungickies and insectickles dkj not show any infection nor infestation within the 3 years that these were kept in storage. Ail products tested showed the expected effect of a viable, healthy microbe: reduced inorganic fertilizer requirement In all the crops tested (rice, com, pepper, onions, garlic, el al), protection from bacterial leaf blight infection in rice and fungal root rot in onion, early maturity in rice, robustness and greenness of treated crops. In an alternative embodiment, a pesticide, a viriclde, a bacteriacide, a fungicide, or a combination of these may be added to the micnsbial inoculant to further prolong its shelf life. Protection is being sought for preparation of biochemical catatysts/biofertiiizers characterized by the 1) addition of vitamins and minerals among others, 2) harvesting by methods including centrifugation/fiitration, and 3) lyophlllzation accompanied by skim milk with myo-inositol or glycerol or b) skim milk with honey, 4) the mixing of plant growth promoting rtiizobacteria like AzospiriUum heAopmeferens, AzosplrVlum lipoferum, Azosplrillum SC/PH or Azosplrillum AH/EH cells with carriers like sugar, flour, talc/chalk/diatomaceous earth 5) an insecticide, a viriclde, a fungicide, or a combination of these and 6) in powder, semi-solid, liqukJ or other fonrts, the products have extended shelf-lives of more than 3 years. These innovations make these products different fronl other formulations. They can induce changes like extensive root proliferation, drought tolerance, increased crop growth, yield and farmers' net incomes, beside being environment-friendly. Claims We claim: 1. A method for producing and formulating microbial inoculants with a prolonged shelflife, comprising: isolating a microbial colony from a culture of a bacterial microorganism; growing the colony in an organic medium; harvesting the microbial cells from the organic medium; mixing the harvested cells with milk and glycerol; drying the microbial cells by lyophilization; grinding the dried microt>ial cells into powder; and mixing the microbial powder with nutrients, anti-contaminant and a dessicant. 2. The method according to Claim I wherein the organic medium consists of a mixture of high protein and vitamin-supplemented feed formula. 3. The method according to Claim 1 wherein the harvesting of the microbial cells is done by filtration or centrifugation. 4. The method according to Claim 1 wherein the anti-contaminant agent is an anti¬fungal agent. 5. The method according to Claim I wherein the pest protectant agent is an anti-viral agent. 6. The method according to Claim 1 wherein the nutrients mixed with the microbial powder consists of vitamins, minerals and flour and the dessicant is selected from talc, chalk or diatornaceous earth. 7. The method according to Claim 1 wherein the organic medium consists of a mixture of high protein and vitamin-supplemented feed formula. 8. The -method according to Claim 1 wherein the bacterial microorganism is Azospirlllum halopraeferens. 9. The method according to Claim I wherein the bacterial microorganism is Azospirillum lipoferum. 10. The method according to Claim 1 wherein the bacterial microorganism is Bacillus punxilus. 11. The method according to Claim I wherein the bacterial microorganism is Azospirillum SC/PH. 12. The method according to Claim I wherein the bacterial microorganism is Azospirillum AH/^H. 13. A biologically pure culture of Azospirillum SC/PH treated according to the method described in any of Claims 1 to 7. 14. A biologically pure culture of Azospirillum AH/EH treated according to the method described in any of Claims 1 to 7. 15. The microbial inoculant of any of claims 1-14, wherein a processed microbial inoculant has a shelf life of at least 36 months or 3 years.

Full Text

STABLE ORGANIC-CARRIER-BASED MICROBIAL INOCULANTS AND METHOD FOR PRODUCING THE SAME
DESCRIPTION OF THE INVENTION Field of Invention
The present invention relates to microbial preparations for crop production.
Background Art
The present invention relates to microbial preparations for crop production, speciTicaliy microbial inoculants incorporated in carriers that provide protection for the colonizing microorganisms and the method for producing the same.
In theory, microbial inoculants, without human intervention, have a low sun/ival rate and efficiency in their natural soil environment because of the insufficient colony forming units per gram soil. Sln The present invention Is a carrier-based biofertiiizer that promotes growth of plants of any type of terrestrial biome, using special biological Inoculants, such as plant growth promoting rhizobacteria. The invention induces extensive root and root hair formation, enable plants to efficiently use soil nutrients, supply plants with nitrogen from the air, increase drought tolerance and protect the plants from pathogentt. Further, the inoeulant can be mixed with an insecticide or fungicide without any appreciable decrease in effectivity.

The closest prior art are reported in WO 2007/014974 {'Aseptic Mycorrhization Inoculant and In vitro and Ex vitro Application Methods'), W01^/006732 {'Method for Obtaining a Wettabia Powder Inoculant for Use with Leguminous C/t>ps*). WO 2006/070061 {'Stable Microbial Inoculants and Methods for Production of Theirf), WO 2002/015702 {'A Thermostable Bkhmatrin, and WO 2000/034440 {'Microorganisms for Improving Plant Productive/).
According to WO 2007/014974 {'Aseptic l\Aycorrhlzatlon Inoculant and In vitro and Ex vitro Application Methods'), a low-viscosity, light, semi-solid agar-type base culture medium is used to facilitate the penetration of the root in the inoculant, as well as to potentiate and preserve the infectiousness of arbuscular myconrhizal fungi, such as those of the present invention. WO 2007/014974 sought to obtain the advantage of being as effective in in vitro as well as ex vitro inoculation by using a semi-solid inoculant. However, it is gel-based and has a short shelf-life, which the present Invention seeks to address.
WO 1994/006732 {'Method for Obtaining a Wettable Powder Inoculant for Use with Leguminous Crop^ reports a method for obtaining a wettable powder inoculant fomnulation for use with leguminous crops, comprising the industrialization of an inoculant for leguminous from a centrifugated biological mass of Rhizobium spp twice immersed in a cryogenic protective solution rich in sugar and protein which is subjected to a iyophilization method. Lil(e the present invention, it is fbnnulated with substances rich in polysaccharides and associated with a cellular protector which substitutes the previously used peat support. It also has a long shelf life of up to 25 months even when stored at room temperature. However, WO 1994/006732 can only be used for leguminous crops. Further, the formulation of the canler uses sugar and protein while the present invention uses miil( and glycerin. Also, WO 1994/006732 does not teach that the powder inoculant with a pest protectant such as an insecticide.
WO 2006/0700ei {'Stabh Microbial Inooulonta and Metttoda for Productlort of Thorrf) teaches away from the present invention. It describes water containing microbial inoculants in paste fonri with shelf life of at least 3 months, preferably 12 months. It reports

that drying, which is a method utilized in the present invention is complicated, expensive and may even be impossible.
WO 2002/015702 ("A nBmo-stable Bi(HnatrbC) reports a method for producing a thernio-stable biodegradable medium for storage of biological materials wherein the biological material includes: a pesticide; a viricide; a bacteriadde; a fungicide; and a combination of these. While the method Is similar In that the biological material can be safely rhixed with potentially degradative substances such as pesticides, viricides, bacteriacides, fungicides or a combination of these, the medium Is structurally different tsecause it is gel-based and contains a blopolymer setecteid from the group of xanthan gum, acacia gum, guar gum, geltan, starch or a combination thereof, unliice the present invention which Is in powder form. Thus, it is no surprise that WO 2002/015702 discloses a maximum shelf life of only eighteen (18} months, which is considerably shorter.than that of the present invention which allows storage of at least three (3) years.
WO 2000/034440 ^Microorganisms for Improving Plant Productivity) is directed to microbes which includes the microorganism Azo^irillum brasBensa, which belongs to the same genus as some of those in the present invention. As with the present invention, two specific strains of microbial inocuiant of WO 2000/034440 is effective for increasing the productivity of both nonlegume and legume plants as well as vegetable plants over a wide variety of soil types and climates. However, the WO 2000/034440 is narrowly directed only to the Azospliillum brasilense, a microorganism not covered by the present application. Further, it does not teach an inventive canler nor the method of production that prolongs its shelf life.
The present invention, the microbes, method and product, are absolutely different and improve over other Icnown methods and products and have considerable advantages over the nature and quality of the methods and products, particularty in the storage life of the microbial preparation.
Disclosure of the Invention
The present invention relates to microbial preparations for crop production such as seed, seedling and other planting material inoculants have been the subject of many

researches in the past 25 years in the Philippines. They have been shown to oe useful in increasing and protecting crop yields. However, they have not been put into commercial production because of their short shetf-lives which limit their effective distribution. This is also tnie of microbial inoculants for processing foods. Prior art discloses a shelf life of a maximum of only eighteen (18) months. This invention is intended to solve this problem of short shelf-lives and allow for the viable commercialization of microbial inoculants. The invention is expected to produce, products with a huge market since biochemical catalysts includirig microbial preparations have agricultural, food and forestry applications.
The invention covers dried, semi-solid and related fonnulations of microbial inoculants like Azospirlllum spp. and Bacillus pumilus. These bacteria are used to inoculate seeds and seedlings of different agriculture and. forestry species to promote among others, the extensive root and root hair formation, enable plants to efficiently use soil nutrients, supply plants with nitrogen from the air, increase drought tolerance and protect the plants from root pathogens. These formulations are prepared in the following manner
The microbes such as Azospirlllum spp. And Bacillus pumllus are grown infermentors usirig special media which contain substrates including high protein feed ingredients such as fish meal/bone meal/blood meal/soya meal, table sugar and tablet salt at SO^C. The baterial cells are han^ested by methods like filtration or centrifugation, added with powdered milk/glycerol to wet the cells and the AzoapMlum mixture lyophilized. The products a(& formulated under sterile conditions with different proportions of components like the bacterial preparations, minerals, milk, vitamins and flour with or without talc/chalk/diatomaceouB earth using standard mechanical devices. The dried and semi-solid products are packed or the tablets are made usually under sterile conditions.
The products as invented have extended the shelf-lives of the biochemical catalysts/microbial preparations by at least 3 years way beyond .the 3 months observed with the non-lyophilized or other inoculants now available in very limited amounts in the market. The special media used to grow the bacteria improve the tolerance of the bacteria to lyophilization. f^ilk not only improves the tolerance of the bacteria to lyophilization; it also provides some vitamins, carbohydrates and minerals for a good start of the bacteria with inoculated plants. Bacillus pumllus need not be lyophilized because it is a spore fomier and can resist adverse conditions for many years. The added vitamins, minerals, nour and talc/chalk/diatomaceous earth also provide additional nutrients to selectively enrich the growth of the desired bacteria and the inoculated plants in problem soils with limiting micronutrients.

Characteristics of the Mlcro^irganiama
Azospihilum Is made up of curved rod cells, 1 mm in diameter with varying sizes and shapes, it can have polar and peritrichous flagella and undeigoes encystment. Its pinkish, whitish orange colonies vary in shape and surface characteristics. . Cart>on sources include organic acids and sugars. The nitrogen sources are amino acids, Hz, NH/ and NO3. Respiration can t>e aerobic, mlcroaerophilic or anaerot)ic. It Is capable cif aerotaxis, chemotaxis and can form clumps.
Bacillus pumllus consists of rod-shaped cells, which can form endospores that are very resistant to many adverse conditions. Sporulatlon is not repressed by exposure to air. It is gram positive, or positive only in the early stages of growth, or negative. The flagellation Is peritrichous or degenerately peritrichous. It can be aerobic or fecuitatively anaerobic. Oxygen is the tenninal electron acceptor. Colony morphology and size are very variable on some media. It exhibits a wide diversity of physiological ability; psychrophilic to thennophilic; acidophilic to alkaliphilic. Some strains are salt tolerant while others have a specific requirement for salts. Catalase is fonned but may be oxidase positive or negative, it requires blotln and some strains also require amino acids. Spores formed occur ubiquitously The mole % G ■»■ C of the DNA ranges from 39.0-46.9 for 37 strains. .ONA-DNA hybridization studies indicate that the species is genetically homogenous.
The depositoiy institution for these isolates Is the UP'-NSRI Culture Collection at Diliman, Quezon City File numbers of some culture deposits are stated below while others will be submitted when already available.
Azospirillum halopraefemns - UPCC1370
Azospirfllum llpoferum - UPCC 1371
Bacillus pumllus - UPCC1372
Azosplrillum SC/PH - UPCC1373
Azospirillum AH/EH - UPCC1374
Shelf Life
Extensive iiteratur* attwcte that such dried dormant microbial colls can remain viable for years. To further protect the product from environmental assault, nutrients that sen/e as buffer against changes in temperature and other environmental shock as well as provide the Initial growth factors for the microbes to grow once the product gets wet and the cells

start metabolizing. However, the product is vulnerable to fungal growth and/or grain beetles which considerably shorten its shelf life. Experiments were then conducted to test the inclusion in the product of fungicides and insecticides.
Two commercial fungicidal preparations and two insectiddal preparations(organic and synthetic) were added to the microbial preparation following manufecturer's recommended dosages. Liquid organic pesticides were absort>ed in flour and added to the microbial preparation. These products were kept in storage for 6 months - 3 years.
All products added with fungickies and insectickles dkj not show any infection nor infestation within the 3 years that these were kept in storage. Ail products tested showed the expected effect of a viable, healthy microbe: reduced inorganic fertilizer requirement In all the crops tested (rice, com, pepper, onions, garlic, el al), protection from bacterial leaf blight infection in rice and fungal root rot in onion, early maturity in rice, robustness and greenness of treated crops.
In an alternative embodiment, a pesticide, a viriclde, a bacteriacide, a fungicide, or a combination of these may be added to the micnsbial inoculant to further prolong its shelf life.
Protection is being sought for preparation of biochemical catatysts/biofertiiizers characterized by the 1) addition of vitamins and minerals among others, 2) harvesting by methods including centrifugation/fiitration, and 3) lyophlllzation accompanied by skim milk with myo-inositol or glycerol or b) skim milk with honey, 4) the mixing of plant growth promoting rtiizobacteria like AzospiriUum heAopmeferens, AzosplrVlum lipoferum, Azosplrillum SC/PH or Azosplrillum AH/EH cells with carriers like sugar, flour, talc/chalk/diatomaceous earth 5) an insecticide, a viriclde, a fungicide, or a combination of these and 6) in powder, semi-solid, liqukJ or other fonrts, the products have extended shelf-lives of more than 3 years. These innovations make these products different fronl other formulations. They can induce changes like extensive root proliferation, drought tolerance, increased crop growth, yield and farmers' net incomes, beside being environment-friendly.

Claims
We claim:
1. A method for producing and formulating microbial inoculants with a prolonged
shelflife, comprising:
isolating a microbial colony from a culture of a bacterial microorganism;
growing the colony in an organic medium;
harvesting the microbial cells from the organic medium;
mixing the harvested cells with milk and glycerol;
drying the microbial cells by lyophilization;
grinding the dried microt>ial cells into powder; and
mixing the microbial powder with nutrients, anti-contaminant and a dessicant.
2. The method according to Claim I wherein the organic medium consists of a mixture of high protein and vitamin-supplemented feed formula.
3. The method according to Claim 1 wherein the harvesting of the microbial cells is done by filtration or centrifugation.
4. The method according to Claim 1 wherein the anti-contaminant agent is an anti¬fungal agent.

5. The method according to Claim I wherein the pest protectant agent is an anti-viral agent.
6. The method according to Claim 1 wherein the nutrients mixed with the microbial powder consists of vitamins, minerals and flour and the dessicant is selected from talc, chalk or diatornaceous earth.
7. The method according to Claim 1 wherein the organic medium consists of a mixture of high protein and vitamin-supplemented feed formula.
8. The -method according to Claim 1 wherein the bacterial microorganism is Azospirlllum halopraeferens.
9. The method according to Claim I wherein the bacterial microorganism is Azospirillum lipoferum.
10. The method according to Claim 1 wherein the bacterial microorganism is Bacillus punxilus.
11. The method according to Claim I wherein the bacterial microorganism is Azospirillum SC/PH.
12. The method according to Claim I wherein the bacterial microorganism is Azospirillum AH/^H.
13. A biologically pure culture of Azospirillum SC/PH treated according to the method described in any of Claims 1 to 7.
14. A biologically pure culture of Azospirillum AH/EH treated according to the method described in any of Claims 1 to 7.

15. The microbial inoculant of any of claims 1-14, wherein a processed microbial inoculant has a shelf life of at least 36 months or 3 years.

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Patent Number

272255

Indian Patent Application Number

7454/CHENP/2009

PG Journal Number

14/2016

Publication Date

01-Apr-2016

Grant Date

24-Mar-2016

Date of Filing

21-Dec-2009

Name of Patentee

HALOS, SATURNINA

Applicant Address

2027 MAGNOLIA ST., PLEASANT VILLAGE, COLLEGE, LAGUNA

Inventors:

#	Inventor's Name	Inventor's Address
1	HALOS, SATURNINA	2027 MAGNOLIA ST., PLEASANT VILLAGE, COLLEGE, LAGUNA
2	HALOS, PONCIANO	2027 MAGNOLIA ST., PLEASANT VILLAGE, COLLEGE, LAGUNA

PCT International Classification Number

C12N1/20

PCT International Application Number

PCT/PH07/00012

PCT International Filing date

2007-06-21

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA