Process for Inoculating a Bioreactor with Cyanobacteria

Abstract

A process is described for the creation of an inoculum for an organism capable of photosynthesis of a target compound within a closed bioreactor, involving steps of transfer of a sterile growth medium under pressure without exposure to atmosphere. The process is useful for the creation of inocula for the growth of cyanobacteria to produce target chemical products, such as ethanol, in closed photobioreactors.

We claim: 
     
         1 . A process for inoculating a closed vertical bioreactor of volume less than 25 liters with a genetically enhanced cyanobacteria comprising an inducible promoter for formation of a target molecule comprising the steps of
 a. placing the enhanced cyanobacteria, in an uninduced state, within an initial liquid culture of volume 0.1 liter to 1.0 liter into a first container of larger size;   b. adding sterile liquid growth media into the first container by transferring the sterile liquid growth media under pressure without exposure to atmosphere in an amount larger than the volume of the initial liquid culture medium, and less than the container size to enable a growth to an OD of 1.5 to 2.0 to form a first inoculum liquid; and   c. transferring the first inoculum liquid to the closed vertical bioreactor.   
     
     
         2 . The process of  claim 1  wherein the target molecule is ethanol. 
     
     
         3 . The process of  claim 1  wherein the transfer of the first inoculum liquid to the closed vertical bioreactor is done with a diaphragm pump. 
     
     
         4 . A process for inoculating a closed vertical bioreactor of volume less than 25 liters with a genetically enhanced cyanobacteria comprising an inducible promoter for formation of a target molecule comprising the steps of
 a. placing the enhanced cyanobacteria, in an uninduced state, within an initial liquid culture of volume of 0.1 liter to 1.0 liter and of OD 1.0 to 2.0 into a first container of volume approximately 10 times the volume of the initial liquid culture;   b. adding sterile liquid growth media into the first container by transferring the sterile liquid growth media under pressure without exposure to atmosphere in an amount larger than the volume of the initial liquid culture medium, to enable a growth to a final OD from 1.5 to 2.0 to form a first inoculum liquid; and   c. transferring the first inoculum liquid to the closed vertical bioreactor.   
     
     
         5 . The process of  claim 4  wherein the target molecule is ethanol. 
     
     
         6 . The process of  claim 4  wherein the transfer of the first inoculum liquid to the closed vertical bioreactor is done with a diaphragm pump. 
     
     
         7 . The process of  claim 4  wherein the placement of step a) is done within a laminar fume hood. 
     
     
         8 . The process of  claim 7  wherein the target molecule is ethanol. 
     
     
         9 . The process of  claim 7  wherein the transfer of the first inoculum liquid to the closed vertical bioreactor is done with a diaphragm pump. 
     
     
         10 . A process for the creation of an inoculum for an organism capable of photosynthesis of a target compound within a closed bioreactor, comprising
 a) placing an organism capable of making the target compound via photosynthesis within an initial liquid culture volume of 0.1 liter to 1.0 liter of OD 1.0 to 2.0 into a first container of size approximately 10 times the volume of the initial liquid culture with the placement done within a laminar fume hood;   b) adding sterile liquid growth media into the container by transferring the sterile liquid growth media under pressure without exposure to atmosphere in an amount 5 to 9 times the volume of the initial liquid culture medium, and less than the container size to enable a growth to a final OD from 1.5 to 2.0 to form a first inoculum liquid;   c) transferring the first inoculum liquid under pressure without exposure to atmosphere to a second container, of volume 5 to 9 times the volume of the first inoculum liquid;   d) adding sterile liquid growth media into the container by transferring the sterile liquid growth media under pressure without exposure to atmosphere in an amount 5 to 9 times the volume of the first inoculum liquid, and less than the second container size to enable a growth to a final OD from 1.5 to 2.0 to form a second inoculum liquid;   e) repeating steps c) and d) as necessary to form an inoculum suitable for introduction into the closed bioreactor; and   f) forming the target compound within the closed bioreactor.   
     
     
         11 . The process of  claim 10 , wherein the target compound is ethanol.