Shake-flask batch culture provides a simple and convenient method of growing small amounts of microorganisms. However, if grams of biomass are required for protein purification or liters of medium are needed for product recovery then the limitations of the shake flask quickly become apparent. The most significant problem is getting enough oxygen to the culture: to grow a 5 liter culture in a single flask would require a 50 liter glass Erlenmeyer flask if the ratio of one-tenth volume of culture to the total volume of container is maintained. Furthermore, the shake flask is a highly dynamic system. As the organism grows, it excretes primary and secondary metabolites into the medium. Some of these metabolites might actually prevent efficient use of the substrate, but more significantly there may be a change in pH by many units. If a batch culture of 1 liter or more is to be grown efficiently, more controlled conditions are required.
▶The simple stirred tank fermenter
A culture will grow in a reproducible form if it is supplied with the same medium under the same conditions of temperature, pH, and oxygen concentration. A shake flask will provide constant aeration if the medium does not exceed 500 ml, but above this volume insufficient oxygen can be entrained at the surface of the culture. The stirred tank fermenter overcomes this primary obstacle to microbial growth in larger volumes by providing agitation via an impeller, rotating at the bottom of a circular vessel. Additional mixing of the culture may be provided by one or more baffles on the sides of the vessel. Oxygen, normally as sterile air, enters the fermenter underneath the impeller via a sparger. The sparger breaks up the flow of air into bubbles, which are broken into yet smaller bubbles when hitting the impeller. The combination of an impeller running at > 250 rpm, baffles, and sparger enable a highly efficient oxygen transfer to the culture. If the speed of the impeller and the rate of flow of oxygen to the fermenter are linked via a processor to an oxygen probe in the culture, the oxygen saturation of the culture can be monitored and regulated continuously.
At high biomass loads, a vessel is subject to some cooling to the atmosphere, but may even be warmed by the metabolic energy of the culture itself. Shake-flask temperature is regulated by the air temperature of an incubator, but a fermenter is often too large both spatially and in terms of heat capacity to be regulated in this way. Instead, a jacket surrounds the fermenter vessel linked to a thermostatically controlled water supply. This can be supplemented with warming/cooling coils in the culture itself. Finally the pH of the fermenter is kept constant with a probe linked to alkali and acid pumps.
▶Other fermenter types
The stirred tank fermenter provides an efficient means for the growth of most bacteria. However, as culture volumes approach hundreds of liters, the power demands of turning the impeller fast enough to ensure sufficient oxygen transfer can become too high. The airlift reactor has no internal moving parts and stirs the culture via the passage of the air itself. This design of reactor is also useful for cells that are prone to lysis by mechanical shear.
When oxygenation is not so important (e.g. when using anaerobic or microaerophilic cells) or when immobilized enzymes are used, much simpler reactors can be employed, such as fluidized or fixed bed reactors.
A stirred tank reactor regulated for temperature, pH, and oxygen so that all conditions remain constant can be adapted to a continuous mode of action. If the continuous culture grows so that it is limited by one of the medium components, it is called a chemostat, but limitations on biomass (turbidostat) or electron potential (potentiostat) can be imposed. In an industrial context, continuous culture is useful as it provides a constant production of biomaterials for downstream applications. In the research laboratory, the kinetics of the chemostat provide additional insight into the physiology and biochemistry of pure and mixed cultures.
We assume that the action of the impeller means that the medium coming into the culture vessel is mixed instantaneously. This is known as the replacement time (tr, the time for one complete volume change of the vessel).
If V is the volume of the reactor and F is the flow rate:
tr = V/F
If the flow rate is too high, the organism cannot divide quickly enough to maintain growth in the vessel, and eventually is diluted away (washout). The flow rate can be adjusted so that the rate of washout of biomass equals the maximum growth rate (mm). At this point any change in substrate concentration will have a direct effect on biomass. The set of conditions under which biomass remains constant over several volume changes of the chemostat is known as a steady state.
The specific growth rate of a chemostat culture is equal to the dilution rate, D
D = F/V
This is always the case as the net increase in biomass equals growth minus output. Over an infinitely small period, this becomes:
V.dx = V.mx.dt – Fx.dt
Dividing throughout by V.dt
dx/dt = (m – D)x
So at the steady state when dx/dt = 0, then m = D.
We can also calculate the biomass and growth-limiting substrate concentrations by rewriting the balance for the growth-limiting substrate (net increase in biomass = growth- output) for the case over an infinitely small period: