Enhanced shelf life and on seed stabilization of liquid bacterium inoculants

Abstract

The present invention includes a method for producing a liquid inoculant containing a desiccant. The method can improve survival and stability of bacteria in liquid inoculants in pack and on seeds. The method includes providing a liquid inoculant of a bacteria grown to a substantially stationary phase. A desiccant treatment containing a desiccant is added to the liquid inoculant to form a partially desiccated inoculant product. The partially desiccated inoculant product can be packaged and stored. The partially desiccated inoculant product can also be applied to seeds.

1 . A method for preparing a partially desiccated inoculant product, the method comprising:
 providing a liquid inoculant of a bacteria grown to a substantially stationary phase; and   adding to the liquid inoculant, in an amount sufficient to partially desiccate the liquid inoculant, a desiccant treatment comprising a desiccant to form the partially desiccated liquid inoculant product, wherein the desiccant comprises from about 5% to about 50% by weight/volume of the partially desiccated liquid inoculant product.   
     
     
         2 . The method according to  claim 1  wherein the bacteria are from one or more of the genera  Rhizobium, Bradyrhizobium, Pseudomonas, Serratia, Bacillus, Paenibacillus, Pasteuria, Azotobacter, Enterobacter, Azospirillum, Methylobacterium  and  Cyanobacteria.    
     
     
         3 . The method according to  claim 1  wherein water activity of the partially desiccated inoculant product is less than about 0.990. 
     
     
         4 . The method according to  claim 1  wherein the water activity of the partially desiccated inoculant product is less than about 0.980. 
     
     
         5 . The method according to  claim 1  wherein the liquid inoculant is provided by:
 introducing bacteria to a liquid nutrient medium to create a bacterial culture; and 
 incubating the bacterial culture to allow the bacteria to grow to a substantially stationary phase, thereby creating a liquid inoculant. 
 
     
     
         6 . The method according to  claim 5  wherein the incubating of the bacterial culture is conducted from about 2 to about 7 days. 
     
     
         7 . The method according to  claim 1  wherein the desiccant comprises one or more of a non-reducing sugar and a sugar alcohol. 
     
     
         8 . The method according to  claim 1  wherein the desiccant comprises one or more of trehalose, sucrose, glycerol, triethylene glycol, and mannitol. 
     
     
         9 . The method according to  claim 8  wherein the desiccant comprises a mixture of two or more of trehalose, sucrose, glycerol, triethylene glycol, and mannitol. 
     
     
         10 . The method according to  claim 8  wherein the desiccant comprises trehalose. 
     
     
         11 . The method according to  claim 10  wherein the desiccant is trehalose. 
     
     
         12 . The method according to  claim 11  wherein the trehalose is from about 10% to about 40% by weight/volume of the partially desiccated inoculant product. 
     
     
         13 . A method according to  claim 12  wherein the trehalose is from about 20% to about 30% by weight/volume of the partially desiccated inoculant product. 
     
     
         14 . The method according to  claim 9  wherein the desiccant comprises a mixture of trehalose and glycerol. 
     
     
         15 . The method according to  claim 14  wherein the trehalose comprises from about 5% to about 40% by weight/volume of the partially desiccated inoculant product and the glycerol comprises from about 1% to about 10% by weight/volume of the partially desiccated inoculant product. 
     
     
         16 . The method according to  claim 15  wherein the trehalose comprises about 20% by weight/volume of the partially desiccated inoculant product and the glycerol comprises about 5% by weight/volume of the partially desiccated inoculant product. 
     
     
         17 . The method according to  claim 1  wherein the method further comprises packaging the partially desiccated inoculant product. 
     
     
         18 . The method according to  claim 17  wherein the method further comprises storing the partially desiccated inoculant product. 
     
     
         19 . The method according to  claim 1  wherein the method further comprises applying the partially desiccated inoculant product to a seed. 
     
     
         20 . The method according to  claim 19  wherein the bacteria are from one or more of genera  Rhizobium, Bradyrhizobium, Pseudomonas, Serratia, Bacillus, Paenibacillus, Pasteuria, Azotobacter, Enterobacter, Azospirillum, Methylobacterium  and  Cyanobacteria.    
     
     
         21 . The method according to  claim 19  wherein the desiccant comprises one or more of a non reducing sugar and a sugar alcohol. 
     
     
         22 . The method according to  claim 19  wherein the desiccant comprises one or more of trehalose, sucrose, glycerol, triethylene glycol, and mannitol. 
     
     
         23 . The method according to  claim 22  wherein the desiccant comprises trehalose. 
     
     
         23 . The method according to  claim 23  wherein the desiccant is trehalose. 
     
     
         24 . The method according to  claim 23  wherein the trehalose comprises from about 10% to about 40% by weight/volume of the partially desiccated inoculant product. 
     
     
         25 . The method according to  claim 25  wherein the trehalose comprises from about 20% to about 30% by weight/volume of the partially desiccated inoculant product. 
     
     
         27 . The method according to  claim 19  wherein the seed comprises a seed for a leguminous plant. 
     
     
         28 . The method according to  claim 19  wherein the number of bacteria on the seed after about 10 weeks on the seed exceeds about 1×10 5 . 
     
     
         29 . A method according to  claim 19  wherein the method further comprises applying an extender to the seed after the partially desiccated inoculant product is applied. 
     
     
         30 . A method for preparing a dry flowable inoculant formulation, the method comprising:
 providing a liquid inoculant of a bacterium grown to a substantially stationary phase; and   adding to the liquid inoculant, in an amount sufficient to partially desiccate the liquid inoculant, a desiccant treatment comprising a desiccant to form the partially desiccated liquid inoculant product; wherein the desiccant comprises from about 5% to about 50% by weight/volume of the partially desiccated liquid inoculant product and wherein the partially desiccated liquid inoculant product is applied to a dry carrier to form a dry flowable inoculant formulation.   
     
     
         31 . The method according to  claim 30  wherein the bacteria are from one or more of the genera  Rhizobium, Bradyrhizobium, Pseudomonas, Serratia, Bacillus, Paenibacillus, Pasteuria, Azotobacter, Enterobacter, Azospirillum, Methylobacterium  and  Cyanobacteria.    
     
     
         32 . The method according to  claim 30  wherein the dry carrier is peat. 
     
     
         33 . A method for preparing a partially desiccated liquid inoculant product, the method comprising:
 first providing a liquid inoculant of a bacterium grown to a substantially stationary phase;   second adding to the liquid inoculant, in an amount sufficient to partially desiccate the liquid inoculant, a desiccant treatment comprising a desiccant to form the partially desiccated liquid inoculant product, wherein the desiccant comprises from about 5% to about 50% by weight/volume of the partially desiccated liquid inoculant product;   third allowing the partially desiccated liquid inoculant product to equilibrate for a predetermined amount of time before applying the partially desiccated liquid inoculant product to a seed; and   fourth applying the partially desiccated liquid inoculant product to a seed.   
     
     
         34 . The method of  claim 33  wherein the predetermined amount of time is about 7 days. 
     
     
         35 . The method according to  claim 35  wherein the bacteria are from one or more of the genera  Rhizobium, Bradyrhizobium, Pseudomonas, Serratia, Bacillus, Paenibacillus, Pasteuria, Azotobacter, Enterobacter, Azospirillum, Methylobacterium  and  Cyanobacteria.