Continuous Culture

Exponential growth in batch culture may be prolonged by the addition of fresh medium to the vessel Provided that the medium has been designed such that growth is substrate limited (i.e. by some component of the medium), and not toxin limited, exponential growth will proceed until the additional substrate is exhausted This exercise may be repeated until the vessel is full However, if an overflow device were fitted to the fermenter such that the added medium displaced an equal volume of culture from the vessel then continuous production of cells could be achieved. If medium is fed continuously to such a culture at a suitable ra steady state is achieved eventually, that is, formation ol new biomass by the culture is balanced by the loss ol cells from the vessel. The flow of medium into the vessel is related to the volume of the vessel by the term dilution rate, D, defined as:

where F is the flow rate (dm3 h-1)

I V is the volume (dm3). '¡iu,s D is expressed in the units h riie net change in cell concentration over a time riod may be expressed as:

I'ihIci steady-state conditions the cell concentration remain- constant, thus dx/dt = 0 and:

Thus, under steady-state conditions the specific growth rate is controlled by the dilution rate, which is an experimental variable. It will be recalled that under batch culture conditions an organism will grow at its maximum specific growth rate and, therefore, it is obvious that a continuous culture may be operated only at dilution rates below the maximum specific growth rate. Thus, within certain limits, the dilution rate may be used to control the growth rate of the culture.

The growth of the cells in a continuous culture of this type is controlled by the availability of the growth limiting chemical component of the medium and, thus, the system is described as a chemostat. The mechanism underlying the controlling effect of the dilution rate is essentially the relationship expressed in equation (2.5), demonstrated by Monod in 1942:

where s is the steady-state concentration of substrate in the chemostat, and s = K,D/{pm„-D). (2.13)

Equation (2.13) predicts that the substrate concentration is determined by the dilution rate. In effect, this occurs by growth of the cells depleting the substrate to a concentration that supports the growth rate equal to the dilution rate. If substrate is depleted below the level that supports the growth rate dictated by the dilution rate, the following sequence of events takes place:

0) The growth rate of the cells will be less than the dilution rate and they will be washed out of the vessel at a rate greater than they are being produced, resulting in a decrease in biomass concentration. GO The substrate concentration in the vessel will rise because fewer cells are left in the vessel to consume it.

(iii) The increased substrate concentration in the vessel will result in the cells growing at a rate greater than the dilution rate and biomass concentration will increase.

(iv) The steady state will be re-established.

Thus, a chemostat is a nutrient-limited self-balancing culture system which may be maintained in a steady state over a wide range of sub-maximum specific growth rates.

The concentration of cells in the chemostat at steady state is described by the equation:

where x is the steady-state cell concentration in the chemostat.

By combining equations (2.13) and (2.14), then:

Thus, the biomass concentration at steady state is determined by the operational variables, SR and D. If SR is increased, x will increase but ,v, the residual substrate concentration in the chemostat, will remain the same. If D is increased, fx will increase ( /x = D) and the residual substrate at the new steady state would have increased to support the elevated growth rate; thus, less substrate will be available to be converted into biomass, resulting in a lower steady state value.

An alternative type of continuous culture to the chemostat is the turbidostat, where the concentration of cells in the culture is kept constant by controlling the flow of medium such that the turbidity of the culture is kept within certain, narrow limits. This may be achieved by monitoring the biomass with a photoelectric cell and feeding the signal to a pump supplying medium to the culture such that the pump is switched on if the biomass exceeds the set point and is switched off if the biomass falls below the set point. Systems other than turbidity may be used to monitor the biomass concentration, such as COz concentration or pH in which case it would be more correct to term the culture a biostat. The chemostat is the more commonly used system because it has the advantage over the biostat of not requiring complex control systems to maintain a steady state. However, the biostat may be advantageous in continuous enrichment culture in avoiding the total washout of the culture in its early stages and this aspect is discussed in Chapter 3.

The kinetic characteristics of an organism (and, therefore, its behaviour in a chemostat) are described by the numerical values of the constants Y, /xmax and Ks. The value of Y affects the steady-state biomass concentration; the value of /xlmix affects the maximum dilution rate that may be employed and the value of Ks affects the residual substrate concentration (and, hence, the biomass concentration) and also the maximum dilution rate that may be used. Figure 2.4 illustrates the continuous culture behaviour of a hypothetical bacterium with a low Ks value for the limiting substrate, compared with the initial limiting substrate concentration. With increasing dilution rate, the residual substrate concentration increases only slightly until D approaches /xmax when s increases significantly. The dilution rate at which x equals zero (that is, the cells have been washed out of the system) is termed the critical dilution rate (Dait) and is given by the equation:

Thus, Dait is affected by the constants, /xmax and Ks, and the variable, SR; the larger SR the closer is Dait to Mmax- However, /xm.lx cannot be achieved in a simple steady state chemostat because substrate limited conditions must always prevail.

Figure 2.5 illustrates the continuous culture behaviour of a hypothetical bacterium with a high Ks for the limiting substrate compared with the initial limiting substrate concentration. With increasing dilution rate, the residual substrate concentration increases significantly to support the increased growth rate. Thus, there is a gradual increase in s and a decrease in x as D approaches Dull. Figure 2.6 illustrates the effect of increasing the initial limiting substrate concentration

Continuous Culture Dilution Rate

Dilution rate

Fig. 2.4. The effects of dilution rate on the steady-state biomass and residual substrate concentrations in a chemostat culture of a micro-organism with a low K„ value for the limiting substrate, compared with the initial substrate concentration.

- Steady-state biomass concentration.

Steady-state residual substrate concentration.

Dilution rate

Fig. 2.4. The effects of dilution rate on the steady-state biomass and residual substrate concentrations in a chemostat culture of a micro-organism with a low K„ value for the limiting substrate, compared with the initial substrate concentration.

- Steady-state biomass concentration.

Steady-state residual substrate concentration.

Continuous Culture Microorganisms

Dilution rate

Fig. 2.5. The effect of dilution rate on the steady-state biomass and residual substrate concentrations in a chemostat of a microorganism with a high Ks value for the limiting substrate, compared with the initial substrate concentration.

_ Steady-state biomass concentration.

---Steady-state residual substrate concentration.

Dilution rate

Fig. 2.5. The effect of dilution rate on the steady-state biomass and residual substrate concentrations in a chemostat of a microorganism with a high Ks value for the limiting substrate, compared with the initial substrate concentration.

_ Steady-state biomass concentration.

---Steady-state residual substrate concentration.

Effect Biomass Chemostat

Dilution rate

Fig. 2.6. The effect of the increased initial substrate concentration on the steady-state biomass and residual substrate concentrations in a chemostat.

_ Steady-state biomass concentrations.

---Steady-state residual substrate concentrations.

sri, sr2 and sr3 represent increasing concentrations of the limiting substrate in the feed medium.

Dilution rate

Fig. 2.6. The effect of the increased initial substrate concentration on the steady-state biomass and residual substrate concentrations in a chemostat.

_ Steady-state biomass concentrations.

---Steady-state residual substrate concentrations.

sri, sr2 and sr3 represent increasing concentrations of the limiting substrate in the feed medium.

on x and s. As SR is increased, so x increases, but the residual substrate concentration is unaffected. Also, Dcrit increases slightly with an increase in SR.

The results of chemostat experiments may differ from those predicted by the foregoing theory. The reasons for these deviations may be anomalies associated with the equipment or the theory not predicting the behaviour of the organism under certain circumstances. Practical anomalies include imperfect mixing and wall growth. Imperfect mixing would cause an in the degree of heterogeneity in the fer-i,W'?S\uih -ome organisms being subject to nutrient L vvtiiM others are under severe limitation. This efCS' o„ is particularly relevant to very low dilu-1 ' - svskms when the flow of medium is likely to l""1 mi'ciniittcnt. This problem may be overcome by iVVc ol Uvd-back systems, as discussed later in this n ■ ''ler' ^'■H growth is another commonly encoun-T ed practical difficulty in which the organism adheres n'lhe iiinei surfaces of the reactor resulting, again, in i iiKu-i-c in heterogeneity. The immobilized cells are ^¡,¡^-1 m removal from the vessel but will consume substrate resulting in the suspended biomass concen-nai.on lviiv< lower than predicted. Wall growth may be limited by coating the inner surfaces of the vessel with frequent observation in carbon and energy limited chemostats is that the biomass concentration at low dilution rates is lower than predicted. This is attributed to the phenomenon of micro-organisms utilizing a greater proportion of substrate for maintenance at low dilution rates. Effectively, the yield factor decreases at low dilution rates. Bull (1974) has reviewed the major causes of deviations from basic chemostat theory.

The basic chemostat may be modified in a number of ways, but the most common modifications are the addition of extra stages (vessels) and the feedback of biomass into the vessel.

Multistage systems

A multistage system is illustrated in Fig. 2.7. The advantage of a multistage chemostat is that different conditions prevail in the separate stages. This may be

Medium inlet

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Responses

  • Yohannes
    When substrate is depleted in continuous culture, growth rate decreased?
    8 years ago
  • Sebastian
    What is the use of turbidostat in continuous culture?
    7 years ago
  • Ruby
    What are the advantage of turbidostat in continuous culture?
    7 years ago
  • Oona
    What happens when substrate for micro organism is depleted?
    7 years ago

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