The Dilution Rate (D) and the continuous fermentation process

 In Upstream Bioprocesing

When a continuous fermentation process is carried out, it is critical that the condition of steady-state must be reached. The main process parameters that can be used for the chemostat technique are the dilution rate, specific growth rate and the yield of the product on the substrate.


The dilution rate (D), usually in units per hour (h−1), describes the relationship between the flow of medium into the bioreactor (F), that can be expressed in L·h−1, and culture volume within the bioreactor (V) in L

Residence time (t) is the inverse of the dilution rate and is also related to the reactor volume and flow rate:


In general, the continuous culture must go at least through four or five residence times before it can be considered to be in a steady-state (McNeil and Harvey, 2008).


The net change in cell concentration over a time period can be calculated using the biomass equation for the continuous reactor, where the term with the dilution rate is included:


The biomass concentration is expressed as X and µ is the specific growth rate in hours (h-1), basically, the term µX refers to the growth and DX is the output, therefore, under steady-state conditions, the cell concentration remains constant when:

Therefore, the change in biomass concentration (X) over time (t) is zero, and change in substrate concentration (S) over time (t) is zero, that is, no net accumulation of biomass or substrate. In some systems will be very difficult to reach a perfect steady-state, would be a pseudo-steady-state (McNeil and Harvey, 2008).


To reach a culture at steady-state, the specific growth (µ) must be equal than the dilution rate (D)


This equilibrium can be achieved controlling the substrate concentration (S) in the fermentation medium by the media addition of fresh media. The specific growth rate was demonstrated by Monod and is determined by the rate of flow of nutrient solution to the culture according to the next equation:


The inlet flow of fresh media and the outlet flow of product must be regulated to maintain a constant concentration of substrate (S) in the culture medium in the bioreactor, where the right equipment and a proper control SW are a key factors for this purpose (figure 1 represents an example of this system of a BIONET F0-BABY bioreactor connected to a Continuous Process Module, CPM). To reach the steady-state, the substrate concentration (S) determined by the dilution rate (D) can be predicted by the next equation:



The expression to calculate the biomass concentration (X) according of the yield coefficient (Yx/s) on the S at steady-state will be:


Is very important to establish experimentally a proper dilution rate for each process, different dilution rates lead to different product yields and qualities (Collet et al., 2004).


An inappropriate dilution rates (D) rate can carry out next situations:


  • If the D is greater than the maximum specific growth rate (µmax), the result is washout, dX/dt becomes negative
  • The substrate concentration in the vessel can be accumulated
  • The accumulation of substrate in the bioreactor can turn the relation of biomass concentration and be increased without control.


(McNeil and Harvey, 2008; Stambury et al., 2003)


The schematic representation of a simple chemostat system at lab scale is illustrated in figure 1, where a BIONET F0-BABY bench-top bioreactor can operate by a continuous process with pumps integrated into a BIONET Continuous Process Module (CPM), controlled and monitored by the software ROSITA, allowing an easily and scalable system for the development of experiments for setting of a proper dilution rate.


Figure 1. Continuous fermentation process at lab scale by using a BIONET F0-BABY bioreactor with a Continuous Process Module (CPM).



The continuous fermentation processes are more difficult to carry out, compared with batch and fed-batch fermentation processes, to establish a steady-state is a challenge, the contamination risks are high and require additional and specific equipment. Nevertheless, if these drawbacks can be solved, the main advantages are a cost-effective fermentation process, reducing the number of stages (cleaning, starter cultures, propagations, sterilizations, etc…), saving energy and time, avoiding the accumulation of toxics substances, and increasing the productivity of the process.


More info of Bionet Continuous Process Module for bench-top bioreactors.




Collet, C., Adler, N., Schwitzguébel, J.-P., Péringer, P., (2004) Hydrogen production by Clostridium thermotactic during continuous fermentation of lactose. Int. J. Hydrog. Energy, 29, 1479–1485

McNeil, B and Harvey, L.M. (2008) Chapter 4: Modes of Fermenter Operation. Practical Fermentation Technology. John Wiley & Sons, Ltd. ISBN: 978-0-470-01434-9

Stambury P.F., A. Whitaker and J.J. Hall (2003) Chapter 2: Microbial Kinetics Growth. Principles of Fermentation Technology, 2 nd ed. Elsevier Science. USA.

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