Method and apparatus for the production of secondary metabolites by the maintenance-state cultivation of microorganisms

Claim 1

A method of growing and maintaining a culture of microorganisms in a state wherein greater than 90 percent of the energy source available to the culture is utilized for maintenance energy, comprising the steps of providing in a closed system comprising a vessel connected to a filtration means in a manner which allows the continuous withdrawal of filtrate free of cells from the system, an initial fluid medium seeded with cells of selected microorganisms, the medium containing nutrient material comprising a source of maintenance energy for the cells, and initially cultivating the cells causing the mass of said cells to increase and the concentration of said source to decrease, introducing additional medium containing said source at a volume rate F R into the vessel as said mass is increasing wherein the volume rate is controlled so that the product of the rate F R and the energy substrate concentration S R of said source is constant, and further cultivating the cells until the rate of increase of said mass is progressively reduced to a minimum and a concentration of cells, X m , is obtained which satisfies the following relationship ##EQU2## thereby achieving said state, wherein X m is the cell mass per unit fluid volume in said system, V F is the fluid volume in said system, S F is the concentration of said source in said system, S A is the amount of said source assimilated into the cells per unit fluid volume in said system, and M c is a coefficient relating the rate of utilization of the energy source for maintenance energy to the cell mass, and wherein S F is permitted to decrease substantially to zero upon the cells reaching said state, filtering a portion of the medium in the system and products of metabolism therein from the cells, said portion being free of cells, and transferring said portion at a rate F S to a receiver.

Claim 2

A method of growing and maintaining a culture of microorganisms in a state wherein greater than 90 percent of the energy source available to the culture is utilized for maintenance energy, comprising the steps of providing in a closed system comprising a vessel connected to a filtration means in a manner which allows the continuous withdrawal of filtrate free of cells from the system, an initial fluid medium seeded with cells of selected microorganisms, the medium containing nutrient material comprising a source of maintenance energy for the cells, and initially cultivating the cells causing the mass of said cells to increase and the concentration of said source to decrease, introducing additional medium containing said source at a volume rate F R into the vessel as said mass is increasing wherein the volume rate is controlled so that the product of the rate F R and the energy substrate concentration S R of said source is constant, and further cultivating the cells until the rate of increase of said mass is progressively reduced to a minimum and a concentration of cells, X m , is obtained which satisfies the following relationship ##EQU2## thereby achieving said state, wherein X m is the cell mass per unit fluid volume in said system, V F is the fluid volume in said system, S F is the concentration of said source in said system, S A is the amount of said source assimilated into the cells per unit fluid volume in said system, and M c is a coefficient relating the rate of utilization of the energy source for maintenance energy to the cell mass, and wherein S F is permitted to decrease substantially to zero upon the cells reaching said state, filtering a portion of the medium in the system and products of metabolism therein from the cells, said portion being free of cells, and transferring said portion at a rate F S to a receiver.

Claim 15

A method of growing and maintaining a culture of microorganisms in a state wherein greater than 90 percent of the energy source available to the culture is utilized for maintenance energy, comprising the steps of providing in a closed system comprising a vessel connected to a filtration means in a manner which allows the continuous withdrawal of filtrate free of cells from the system, an initial fluid medium seeded with cells of selected microorganisms, the medium containing nutrient material comprising a source of maintenance energy for the cells, and initially cultivating the cells causing the mass of said cells to increase and the concentration of said source to decrease, introducing an additional medium containing said source at a volume rate F R into the vessel as said mass is increasing, wherein the volume rate is controlled so that the product of the rate F R and the energy substrate concentration S R of said source is constant, and further cultivating the cells until the rate of increase of said mass is progressively reduced to a minimum and a concentration of cells, X m , is obtained which satisfies the following relationship ##EQU3## thereby achieving said state, wherein X m is the cell mass per unit fluid volume in said system, V F is the fluid volume in said system, S F is the concentration of said source in said system, S A is the amount of said source assimilated into the cells per unit fluid volume in said system, and M c is a coefficient relating the rate of utilization of the energy source for maintenance energy to the cell mass, and wherein S F is permitted to decrease substantially to zero upon the cells reaching said state, remaining to a collector a fraction of the cells and medium and reducing said product of the rate F R and the energy substrate concentration S R until the contents in said system reach a desired fraction of the quantity present before said drawing off, and terminating said removal when the desired level is reached and increasing said product until the contents of said system are increased to a F R S R desired value, and thereafter maintaining said product F R S R constant and permitting the cell mass to increase until the value of S F is again decreased substantially to zero and the cell mass again reaches said state.

Claim 16

A method of growing and maintaining a culture of microorganisms in a state wherein greater than 90 percent of the energy source available to the culture is utilized for maintenance energy, comprising the steps of providing in a closed system comprising a vessel connected to a filtration means in a manner which allows the continuous withdrawal of filtrate free of cells from the system, an initial fluid medium seeded with cells of selected microorganisms, the medium containing nutrient material comprising a source of maintenance energy for the cells, and initially cultivating the cells causing the mass of said cells to increase and the concentration of said source to decrease, introducing an additional medium containing said source at a volume rate F R into the vessel as said mass is increasing, wherein the volume rate is controlled so that the product of the rate F R and the energy substrate concentration S R of said source is constant, and further cultivating the cells until the rate of increase of said mass is progressively reduced to a minimum and a concentration of cells, X m , is obtained which satisfies the following relationship ##EQU3## thereby achieving said state, wherein X m is the cell mass per unit fluid volume in said system, V F is the fluid volume in said system, S F is the concentration of said source in said system, S A is the amount of said source assimilated into the cells per unit fluid volume in said system, and M c is a coefficient relating the rate of utilization of the energy source for maintenance energy to the cell mass, and wherein S F is permitted to decrease substantially to zero upon the cells reaching said state, remaining to a collector a fraction of the cells and medium and reducing said product of the rate F R and the energy substrate concentration S R until the contents in said system reach a desired fraction of the quantity present before said drawing off, and terminating said removal when the desired level is reached and increasing said product until the contents of said system are increased to a F R S R desired value, and thereafter maintaining said product F R S R constant and permitting the cell mass to increase until the value of S F is again decreased substantially to zero and the cell mass again reaches said state.