Fermentation Technology

PETER F. STANBURY

INTRODUCTION

Microorganisms are capable of growing on a wide range of substrates and can produce a remarkable spectrum of products. The relatively recent advent of in vitro genetic manipulation has extended the range of products that may be produced by microorganisms and has provided new methods for increasing the yields of existing ones. The commercial exploitation of the biochemical diversity of microorganisms has resulted in the development of the fermentation industry and the techniques of genetic manipulation have given this well-established industry the opportunity to develop new processes and to improve existing ones. The term fermentation is derived from the Latin verb fervere, to boil, which describes the appearance of the action of yeast on extracts of fruit or malted grain during the production of alcoholic beverages. However, fermentation is interpreted differently by microbiologists and biochemists. To a microbiologist the word means any process for the production of a product by the mass culture of microorganisms. To a biochemist, however, the word means an energy-generating process in which organic compounds act as both electron donors and acceptors, that is, an anaerobic process where energy is produced without the participation of oxygen or other inorganic electron acceptors. In this chapter fermentation is used in its broader, microbiological context.

MICROBIAL GROWTH

The growth of a microorganism may result in the production of a range of metabolites but to produce a particular metabolite the desired organism must be grown under precise cultural conditions at a particular growth rate. If a microorganism is introduced into a nutrient medium that supports its growth, the inoculated culture will pass through a number of stages and the system is termed batch culture. Initially, growth does not occur and this period is referred to as the lag phase and may be considered a period of adaptation. Following an interval during which the growth rate of the cells gradually increases, the cells grow at a constant, maximum rate and this period is referred to as the log or exponential phase, which may be described by the equation

dx/dt = µx (1)

where x is the cell concentration (mg ml-1), t is the time of incubation (h), and µ the specific growth rate (h). On integration equation (1) gives

xt = x0eµt (2)

where x0 is the cell concentration at time zero and xt is the cell concentration after a time interval, t h.

Thus, a plot of the natural logarithm of the cell concentration against time gives a straight line, the slope of which equals the specific growth rate. The specific growth rate during the exponential phase is the maximum for the prevailing conditions and is described as the maximum specific growth rate, or µmax. Equations (1) and (2) ignore the facts that growth results in the depletion of nutrients and the accumulation of toxic by-products and thus predict that growth continues indefinitely. However, in reality, as substrate (nutrient) is exhausted and toxic products accumulate, the growth rate of the cells deviates from the maximum and eventually growth ceases and the culture enters the stationary phase. After a further period of time, the culture enters the death phase and the number of viable cells declines. This classic representation of microbial growth is illustrated in Figure 1. It should be remembered that this description refers to the behaviour of both unicellular and mycelial (filamentous) organisms in batch culture, the growth of the latter resulting in the exponential addition of viable biomass to the mycelial body rather than the production of separate, discrete unicells.

As already stated, the cessation of growth in a batch culture may be due to the exhaustion of a nutrient component or the accumulation of a toxic product. However, provided that the growth medium is designed such that growth is limited by the availability of a medium component, growth may be extended by addition of an aliquot of fresh medium to the vessel. If the fresh medium is added continuously, at an appropriate rate, and the culture vessel is fitted with an overflow device, such that culture is displaced by the incoming fresh medium, a continuous culture may be established. 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. In this system a steady-state is eventually achieved and the loss of biomass via the overflow is replaced by cell growth. The flow of medium through the system is described by the term dilution rate, D, which is equal to the rate of addition of medium divided by the working volume of the culture vessel. The balance between growth of cells and their loss from the system may be described as

dt/dt = growth - output

or

dx/dt = µx - Dx

Under steady-state conditions,

dx/dt = 0

and, therefore, µx = Dx and µ = D.

Hence, the growth rate of the organisms 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 a chemostat culture.

The mechanism underlying the controlling effect of the dilution rate is essentially the relationship between µ specific growth rate, and s, the limiting substrate concentration in the chemostat, demonstrated by Monod in 1942:

µ = µmaxS/(Ks + s) (3)

where Ks is the utilization or...