Principles of microbial growth

The growth of organisms may be seen as the increase of cell material expressed in terms of mass or cell number, and results from a highly complicated and coordinated series of enzymatically catalyzed biological steps. Growth will be dependent on the availability and transport of necessary nutrients to the cell and subsequent uptake, and on environmental parameters such as temperature, pH and aeration being optimally maintained.

The quantity of biomass or specifi c cellular component in a bioreactor can be determined gravimetrically (by dry weight, wet weight, DNA or protein) or numerically for unicellular systems (by number of cells).

It is possible to develop mathematical equations to describe the essential features of organism growth in bioreactors. The original mathematical equation described by Monod (1942) gives specifi c growth (µ) as a function of the concentration of a substrate in the medium (S):

S is in limiting concentration in comparison with other essential nutrients, µmax is the maximum specifi c growth rate of the organism while ks represents a saturation constant. Ks is the substrate concentration at which µ=µmax/2.

In normal practice, an organism will seldom have ideal conditions for unlimited growth; rather, growth will be dependent on a limiting factor, for example, an essential nutrient. As the concentration of this factor drops, so also the potential growth of the organism will decrease. Reactions can occur with static or agitated cultures, in the presence or absence of oxygen, and in liquid or low-moisture conditions (e. g. on solid substrates). The MO can be free or can be attached to surfaces by immobilization or by natural adherence.

In biotechnological processes, there are three main ways of growing MO in the bioreactor: batch, fed-batch or continuous.

In a batch culture the MO are inoculated into a fi xed volume of medium and as growth takes place nutrients are consumed and products of growth (biomass, metabolites) accumulated. The nutrient environment within the bioreactor is continuously changing and, in turn, enforcing changes to cell metabolism. Eventually, cell multiplication ceases because of exhaustion or limitation of nutrient(s) and accumulation of toxic excreted waste products.

Fed-batch culture is, in the broadest sense, defined as an operational technique in biotechnological processes where one or more nutrients (substrates) are fed (supplied) to the bioreactor during cultivation (to prevent nutrient depletion) and in which the product(s) remain in the bioreactor until the end of the run. The advantage of the fed-batch culture is that one can control concentration of fed-substrate in the culture liquid at arbitrarily desired levels (in many cases, at low levels).

In contrast to batch conditions the practice of continuous cultivation gives near balanced growth with little fl uctuation of nutrients, metabolites or cell numbers or biomass. Continuous methods of cultivation will permit organisms to grow under steady state (unchanging) conditions, in which growth occurs at a constant rate and in a constant environment. In a completely mixed continuous culture system, sterile medium is passed into the bioreactor at a steady fl ow rate and culture broth (medium, waste products and organisms) emerges from it at the same rate keeping the volume of the total culture in the bioreactor constant.

Factors such as pH and the concentrations of nutrients and metabolic products, which inevitably change during batch cultivation, can be held near constant in continuous cultivations. In industrial practice, continuously operated systems are of limited use.

The main steps in fermentation process are represented in Fig. 1.

Fig. 1. Schematic representation of fermentation process.

The first step in a common fermentation process (Fig. 1) is the preparation of the substrate in which the selected microorganism will be developed. The medium, prepared with adequate formulation will be sterilized before inoculation with the microorganisms. The pure culture is propagated by several successive stages to obtain enough amount of biomass for inoculating the fermenter. When the desired number of cells is developed, the inoculum is pumped to the production fermenter.

When fermentation is finished, culture fluid goes to cell separation. As a result, biomass is obtained and can be used again in the fermentation process and the cell free supernatant will be treated by extraction and purification.

The residues from extraction goes to effluent treatment. In next lectures, we will study the most common wastewater biotreatments.

It is important to minimize the fermentation time. When the time is excessively long, the productivity decreases and consequently the cost of the process increases.

Batch cultivation systems represent the dominant form of industrial usage. The complex nature of batch growth of MO is shown in Fig. 2. The initial lag phase is a time of no apparent growth, but actual biochemical analyses show metabolic turn over indicating that the cells in the process are adapting to the environmental conditions and new growth will eventually begin. Later, the inoculum begins to grow to be quickly followed by the exponential phase. In the exponential phase, microbial growth proceeds at the maximum possible rate, nutrients are in excess, ideal environmental parameters and growth inhibitors absent. However, in batch cultivations exponential growth is of limited duration and as nutrient conditions change, growth rate decreases entering the stationary phase when overall growth can no longer be obtained due to nutrient exhaustion. The fi nal phase of the cycle is decline / death phase when growth has ceased. Most biotechnological batch processes are stopped before this stage because of decreasing metabolism and cell lysis.

Fig. 2. Growth characteristics in a batch culture of a MO.