# A

Medium

Medium

Reactor

Diverted product stream

Continuous f centrifuge

Concentrated biomass return

Feed pump

Medium in-flow

Stirred reactor

Product out-flow

Fig. 5.23 Continuous fermentation systems, (a) Plug flow reactor (shown with biomass feed-back), (b) Continuous fermentation systems - Chemostat.

that the culture volume within the reactor remains constant. Unlike the plug flow reactor, the chemostat vessel is inoculated once only and when batch growth has commenced the in-flow of fresh medium is initiated. It is assumed that mixing is perfect so that in-flowing medium is instantaneously and homogeneously distributed throughout the reactor.

The conditions within a chemostat are governed by two opposing effects: growth on the nutrients in the in-flowing fresh medium and dilution due to loss of culture through the exit tube and its replacement by fresh medium. Mathematically this may be explained as follows:

Growth in any culture is characterised by the general growth equation:

where Nt is the population at time t; N is the population at t = 0; n is the specific growth rate.

The specific growth rate (ji) is defined as the rate of growth per unit amount of biomass and has the units of reciprocal time. The specific growth rate of a microorganism in a culture is controlled by environmental factors such as nutrient supply and temperature. However, under ideal conditions, where growth is not limited by any external factor, each organism has a maximum specific growth rate (|vax) that is determined by the genotype.

Returning to the chemostat; from the general growth equation, the instantaneous growth rate of the population is given by:

The rate of loss of cells being removed from the growth vessel is:

D is the dilution rate and is equal to f/V, where f is the rate of addition of medium and V is the volume of culture in the vessel. It also has the unit of reciprocal time. The net change in population is therefore:

If the dilution rate (D) is greater than (imax the above expression will have a negative value and the population in the chemostat will tend, with time, towards zero, termed 'wash-out'. If the dilution rate is less than (imax the value of the expression is positive and the population size will increase. The increase in population density increases the rate of depletion of nutrients in the in-flowing medium and eventually the concentration of an essential nutrient will fall to a growth limiting value. In consequence, the specific growth rate falls until it is equal to the dilution rate. At this point dN/dt = 0 and a steady state is achieved, the population density (and composition of the spent medium) being constant. Different steady states can be achieved by varying the dilution rate between zero and some fraction of |vax. At each steady state the concentration of the growth limiting nutrient within the reactor will be zero. It follows, therefore, that apart from varying the dilution rate to regulate growth rate within the chemostat, it is also possible to exert control by varying the composition of the growth medium. The productivity of a micro-organism growing in a chemostat is always greater than that achievable using the same combination of organism and medium in a batch culture of equal volume. This is because, in the former, growth rate remains constant throughout, as there is no lag or stationary phase.

Some embellishments to the basic design and operation of chemostats are possible. One approach is the reverse of the conventional chemostat. Thus, a steady state may be established by automatic self-regulation of the dilution rate in response to an input signal from a sensor which measures biomass concentration or a growth-related parameter. Examples include the turbidostat where biomass concentration is determined using a light scattering spectrophotometric approach. Growth related parameters could be rate of carbon dioxide evolution, rate of oxygen consumption, exothermy or the concentration of any other growth related metabolite for which a suitable sensor exists. The advantage of the turbidostat, or related approach, is that it is possible to establish steady states where all essential nutrients are present in excess. This may provide a means of exerting greater control over the composition of the product stream than is possible with the simple chemostat.

It is possible to use a biomass concentration loop similar to that shown for the plug flow reactor shown in Fig. 5.23(a). In this way, the rate of formation of product and biomass can be increased to higher levels than are achievable with a simple chemostat. Another alternative is to use multiple chemostat vessels linked in series. In this arrangement, the outlet stream issuing from the first vessel may be augmented with additional nutrients as it is fed into the second vessel. Multi- stage chemostats allow the establishment of distinct steady states in each growth vessel. Like the single stage chemostat, the mutli-stage variety may also be fitted with a biomass concentration and feed-back loop system.

The types of continuous systems that may be used for brewing, have been classified by Portno (1970). Based on an earlier classification of Herbert (1961) these may also, somewhat confusingly, be differentiated into open or closed types. In this case, open types are those in which no attempt is made to retain yeast cells and the process flow issuing from the fermenter is of the same composition as that inside. Conversely, closed types have some method which separates the issuing beer from the yeast, the latter being restrained within the fermenter. Portno (1970) extended this classification to include another category, partially closed, defined as where the concentration of cells escaping is less than the concentration retained in the fermenter but greater than zero. Partially closed systems can be quantified by the retention (or closure) index (R %), defined as the restriction over escape of cells from systems in steady state:

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