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 saturation constant, which is numerically equal to the substrate concentration when µ is half µmax. At steady-state, µ = D, and, therefore,

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]

Where s is the steady-state concentration of substrate in the chemostat, and

s = KsD/(µmax - D) (4)

Equation (4) 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 that 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:

(i) 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.

(ii) 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 that may be maintained in a steady-state over a wide range of sub-maximum specific growth rates.

Fed-batch culture is a system that may be considered to be intermediate between batch and continuous processes. The term fed-batch is used to describe batch cultures that are fed continuously, or sequentially, with fresh medium without the removal of culture fluid. Thus, the volume of a fed-batch culture increases with time. Pirt described the kinetics of such a system as follows. If the growth of an organism were limited by the concentration of one substrate in the medium the biomass at stationary phase, xmax, would be described by the equation:

xmax = YSR

where Y is the yield factor and is equal to the mass of cells produced per gram of substrate consumed and SR is the initial concentration of the growth limiting substrate. If fresh medium were to be added to the vessel at a dilution rate less than µmax then virtually all the substrate would be consumed as it entered the system:

FSR = µ(X/Y)

where F is the flow rate and X is the total biomass in the vessel, i.e. the cell concentration multiplied by the culture volume.

Although the total biomass (X) in the vessel increases with time the concentration of cells, x, remains virtually constant; thus dx/dt = 0 and µ = D. Such a system is then described as quasi-steady-state. As time progresses and the volume of culture increases, the dilution rate decreases. Thus, the value of D is given by the expression

D = F/(Vo + Ft)

where F is the flow rate, V0 is the initial culture volume, and t is time. Monod kinetics predict that as D falls residual substrate concentration should also decrease, resulting in an increase in biomass. However, over the range of growth rates operating the increase in biomass should be insignificant. The major difference between the steady-state of the chemostat and the quasi-steady-state of a fed-batch culture is that in a chemostat D (hence, µ) is constant whereas in a fed-batch system D (hence, µ) decreases with time. The dilution rate in a fed-batch system may be kept constant by increasing, exponentially, the flow rate using a computer-control system.

3 APPLICATIONS OF FERMENTATION

Microbial fermentations may be classified into the following major groups:

(i) Those that produce microbial cells (biomass) as the product.

(ii) Those that produce microbial metabolites.

(iii) Those that produce microbial enzymes.

(iv) Those that modify a compound which is added to the fermentation – the transformation processes.

(v) Those that produce recombinant products.

3.1 Microbial Biomass

Microbial biomass is produced commercially as single cell protein (SCP) for human food or animal feed and as viable yeast cells to be used in the baking industry. The industrial production of bakers' yeast started in the early 1900s and yeast biomass was used as human food in Germany during the First World War. However, the development of large-scale processes for the production of microbial biomass as a source of commercial protein began in earnest in the late 1960s. Several of the processes investigated did not come to fruition owing to political and economic problems but the establishment of the ICI Pruteen process for the production of bacterial SCP for animal feed was a milestone in the development of the fermentation industry. This process utilized continuous culture on an enormous scale (1500 m3) and is an excellent example of the application of good engineering to the design of a microbiological process. However, the economics of the production of SCP as animal feed were marginal, which eventually led to the discontinuation of the Pruteen process. The technical expertise gained from the Pruteen process assisted ICI in collaborating with Rank Hovis MacDougall on a process for the production of fungal biomass to be used as human food. A continuous fermentation process for the production of Fusarium graminearum biomass (marketed as Quorn®) was developed utilizing a 40 m3 air-lift fermenter. This process was based on sound economics and has proved to be a major economic success.

3.2 Microbial Metabolites

The kinetic description of batch culture may be rather misleading when considering the product-forming capacity of the culture during the various phases, for, although the metabolism of stationary phase cells is considerably different from that of logarithmic ones, it is by no means stationary. Bu'Lock et al. proposed a descriptive terminology of the behaviour of microbial cells which considered the type of metabolism rather than the kinetics of growth. The term 'trophophase' was suggested to describe the log or exponential phase of a culture during which the sole products of metabolism are either essential to growth, such as amino acids, nucleotides, proteins, nucleic acids, lipids, carbohydrates, etc. or are the by-products of energy-yielding metabolism such as ethanol, acetone and butanol. The metabolites produced during the trophophase are referred to as primary metabolites. Some examples of primary metabolites of commercial importance are listed in Table 1.

Bu'Lock et al. suggested the term 'idiophase' to describe the phase of a culture during which products other than primary metabolites are synthesized, products which do not have an obvious role in cell metabolism. The metabolites produced during the idiophase are referred to as the secondary metabolites. The interrelationships between primary and secondary metabolism are illustrated in Figure 2, from which it may be seen that secondary metabolites tend to be synthesized from the intermediates and end-products of primary metabolism. Although the primary metabolic routes shown in Figure 2 are common to the vast majority of microorganisms, each secondary metabolite would be synthesized by very few microbial taxa. Also, not all microbial taxa undergo secondary metabolism; it is a common feature of the filamentous fungi and bacteria and the sporing bacteria but it is not, for example, a feature of the Enterobacteriaceae. Thus, although the taxonomic distribution of secondary metabolism is far more limited than that of primary metabolism, the range of secondary products produced is enormous. The classification of microbial products into secondary and primary metabolites should be considered as a convenient, but in some cases, artificial system. To quote Bushell, the classification should not be allowed to act as a conceptual straitjacket, forcing the reader to consider all products as either primary or secondary metabolites. It is sometimes difficult to categorize a product as primary or secondary, and the kinetics of production of certain compounds may change, depending on the growth conditions employed.

At first sight it may seem anomalous that microorganisms produce compounds which do not appear to have any metabolic function and are certainly not by-products of catabolism as are, for example, ethanol and acetone. However, many secondary metabolites exhibit antimicrobial properties and, therefore, may be involved in competition in the natural environment; others have, since their discovery in idiophase cultures, been demonstrated to be produced during the trophophase where, it has been claimed, they act in some form of metabolic control. Although the physiological role of secondary metabolism continues to be the subject of considerable debate its relevance to the fermentation industry is the commercial significance of the secondary metabolites. Table 2 summarizes some of the industrially important groups of secondary metabolites.

The production of microbial metabolites may be achieved in continuous, as well as batch, systems. The chronological separation of trophophase and idiophase in batch culture may be studied in continuous culture in terms of dilution rate. Secondary metabolism will occur at relatively low dilution rates (growth rates) and, therefore, it should be remembered that secondary metabolism is a property of slow-growing, as well as stationary, cells. The fact that secondary metabolites are produced by slow-growing organisms in continuous culture indicates that primary metabolism is continuing in idiophase-type cells. Thus, secondary metabolism is not switched on to remove an accumulation of metabolites synthesized entirely in a different phase; synthesis of the primary metabolic precursors continues through the period of secondary biosynthesis.