Production and cryopreservation of high concentration inocula
The subject invention provides standardized, high concentration inocula in solid form for use as seed cultures in scaled-up cultivation. Further embodiments include methods of producing standardized inocula, as well as methods of preserving and storing the inocula for convenient use over extended periods of time.
1. A method of producing standardized high concentration inocula comprising:
obtaining a first composition by mixing alginate and agar;
obtaining a purified reference culture of a microorganism;
mixing the reference culture with the first composition to form a second composition;
depositing aliquots of the second composition as droplets directly into a CaCl 2 solution that has a temperature of −15° C. to 4° C.;
adding a sterilized cryoprotectant to the CaCl 2 solution; and
placing the CaCl 2 solution having the second composition and the cryoprotectant therein, into a refrigerator at −10° C. to 4° C., wherein each droplet forms an individual inoculum pellet,
removing the individual inoculum pellets from the refrigerator and placing them directly into a freezer for storage at a temperature between −80° C. and −10° C.,
after storage, transferring one or more of the inoculum pellets directly from the freezer into a fermentation reactor having liquid fermentation broth therein, and
cultivating the one or more inoculum pellets.
2. The method of claim 1 , wherein the reference culture comprises a bacteria, yeast, fungi, archaea, virus, protozoan or propagules thereof.
3. The method of claim 2 , wherein the propagules are vegetative cells, reproductive spores, endospores or exospores.
4. The method of claim 2 , wherein the reference culture comprises one or more yeasts and/or fungi selected from Starmerella, Pichia , and Mycorrhiza.
5. The method of claim 2 , wherein the reference culture comprises a Bacillus bacteria selected from B. subtilis, B. licheniformis, B. firmus, B. laterosporus, B. megaterium, B. amyloliquefaciens and Bacillus coagulans GBI-30 (BC30).
6. The method of claim 1 , carried out in a plastic or glass container selected from a tube, flask, cylinder and dish.
7. The method of claim 1 , wherein each inoculum pellet comprises 1×10 1 to 1×10 6 cells or propagules of the microorganism.
8. The method of claim 1 , wherein the cryoprotectant is glycerol.
9. The method of claim 1 , wherein the alginate and agar are heated prior to or concurrently with mixing.
10. The method of claim 1 , wherein the cryoprotectant is mixed with the CaCl 2 prior to depositing the aliquots into the CaCl 2 .
11. The method of claim 1 , wherein each inoculum pellet has a volume that is between 10 μL and 5 ml.
12. The method of claim 1 , wherein the fermentation broth comprises a germination enhancer.
13. A method of producing standardized high concentration inocula comprising:
obtaining a first composition by mixing 1.5% (w/v) alginate and 1% (w/v) agar;
heating the alginate and agar prior to or concurrently with mixing;
obtaining a purified reference culture of a microorganism;
mixing the reference culture with the first composition at a ratio of 1:0.5 (first composition to culture) to produce a second composition;
depositing aliquots of the second composition as droplets directly into a 2% (w/v) CaCl 2 solution that has a temperature of −15° C. to 4° C.;
adding sterilized 5-25% (v/v) glycerol to the CaCl 2 solution; and
placing the CaCl 2 solution having the second composition and glycerol therein, into a refrigerator at −15° C. to 4° C., wherein each droplet forms an individual inoculum pellet,
removing the individual inoculum pellets from the refrigerator and placing them directly into a freezer for storage at a temperature between −80° C. and −10° C.,
after storage, transferring one or more inoculum pellets directly from the freezer into a fermentation reactor containing liquid fermentation broth, wherein the fermentation reactor has a volume of 1 L to 5,000 L, and cultivating the one or more inoculum pellets.