1. Continuous cultivation by E. Börje Lindström

2. Theory Two types of apparatus are usually used; the chemostat and the turbidistat. aeration Reactor (S) Medium tank (S 0 ) Recovery tank Pump In both types there is a flow of fresh medium into the growth vessel (reactor)and the same amount of volume is leaving the growth vessel. The cell concentration is regulated in different ways: - In a chemostat the level of the bacterial population is dependent on a limiting factor (S 0 ) in the medium tank and the flow rate of the medium to the reactor is kept constant. - In a turbidistat the cell concentration in the reactor is registered continuously and regulated by changing the flow rate of medium into the reactor with no limiting factor in the medium. Stämmer verkligen detta? Något ämne i mediet är väl alltid begränsande förr eller senare?

3. Chemostat Cultivating an aerobic bacterium in a continuous way demands effective aeration and stirring. These prerequisites can be provided in stirred tank reactors. With a good stirring the composition of the medium and the air supply in the reactor will be homogeneous throughout the reactor and the bacteria will grow optimally. However, the process is always started batch-wise with no addition of new medium. The inoculated bacteria will then grow as fast as possible (  max ) until the concentration of the limiting substrate, (S), will decrease and hence the growth will slow down.

4. Chemostat, cont. Then the pump is started and new amount of nutrients will be available for the bacteria. The added limiting substrate, S, will now be consumed rapidly by the bacterial population in the vessel

5. Chemostat, cont. The growth of the bacteria in a continuous process is therefore dependent on the flow rate of new medium into the reactor and the limiting substrate, S 0, in the medium. At this steady state the growth of bacteria is equal to the removal of bacteria to the recovery vessel. At each fixed flow rate of medium a ”steady-state” will be obtained which means that: The cell population will be constant and kept at this level until the flow rate is changed.

6. Calculations For bacteria in a continuous one-reactor process using nutrients which all are in solution the following exponential equation is used: N t = N 0 x e  t (1) Where:  = the specific growth rate constant N t = number of cells at time t N 0 = the number of cells at time = 0. According to Monod et al. (ref.??) the following equation is valid:

7. Calculations, cont. K s is a concentration constant for the limiting factor which gives  = 0.5  max.  max is maximal  for the bacterium and the medium used Where: (S) is the concentration of the limiting factor in the reactor  =  max x (S)/K s + (S) (2)  (S)  max KsKs  max /2

8. Calculations, cont. Using the following nomenclature: V = the reactor volume (liter, L) F = the flow rate of the medium into the reactor (L/hr) D = dilution = F/V (tim -1 ) (X) = the cell concentration (bacteria/L) Y =  (X)/  (S) = the recovery constant of cells per unit limiting factor The following can be obtained:

9. Changes of cell concentration At steady state we have: Growth of bacteria - outflow of bacteria to the recovery vessel = V x (X) x  - F x (X) = 0 This gives:  = F x (X)/ V x (X) = F/V = D This means that increasing the dilution can be done until the growth rate constant reaches  max Conclusion: - At a dilution higher than  max the culture will be washed out because the growth of bacteria can’t compensate for the loss of bacteria to the recovery vessel.

10. Changes in the limiting factor At steady state we have: Amount of factor entering the reactor – amount of factor leaving the reactor - consumption of the factor by the growing bacteria = This gives: F x (S 0 ) – F x (S) –V x  x (X)/ Y = 0 (X) = Y((S 0 ) –(S)) Conclusion: - This means that knowing Y, (S 0 ) and (S), the amount of bacteria in the reactor can be estimated.