Types and Stages of Fermentation Process – Explained!

This article throws light upon the three types of fermentation process. The fermentation process consists of four stages. The four stages are: (1) Inoculum Preservation (2) Inoculum Build-up (3) Pre-Fermenter Culture and (4) Production Fermentation.

A classification, based on the product formation in relation to energy metabolism is briefly discussed below (Fig. 19.15).

Type I Fermentation:

When the product is formed directly from the primary metabolism used for energy production, it is referred to as type I and may be represented as.

Substrate A → Product

Substrate A → B → C → D → Product

Growth, energy metabolism and product formation almost run in a parallel manner (Fig. 19.15A). In this type, trophophase and iodophase are not separated from each other e.g. production of ethanol, gluconic acid and single-cell protein.

Type II fermentation:

In type II category, the product is also formed from the substrate used for primary energy metabolism. However, the product is produced in the secondary pathway, as illustrated below.

Substrate A → B → C → D ….Primary metabolism

→ E → F G → Product

At the beginning, the growth of the microorganisms is accompanied by high substrate utilization with little or no product formation. Now the growth is slowed down but the substrate consumption is high, and this is coupled with product formation. As is evident from Fig. 19.15B, in type II fermentation, the trophophase and idiophase are separate. Production of some amino acids, citric acid and itaconic acid are good examples of type II fermentation.

Type III fermentation:

There is a clear distinction between the primary metabolism and product formation in type III fermentation (Fig. 19.15C) as they occur at separate times. Substrate consumption and rapid growth occur in the first phase and the product formation occurs in the second phase. The product is formed from amphibolic metabolic pathways and not from primary metabolism e.g. production of vitamins and antibiotics.

Overlap of different types of fermentations:

Types I, II and III fermentations, originally categorized by Garden (in 1959) are not very rigid. There are intermediate forms based on the composition of the nutrient culture medium, strain of the microorganism used and product formation. For instance, industrial production of lactic acid falls between type I and II, while production of the antibiotic amyloglycoside is intermediate between types II and III.

It is sometimes difficult to categorize the industrial fermentations under any one of these types (I, II, III) due to complex nature of the process e.g. mycelium producing microorganisms in relation to antibiotic production.

The Fermentation Process:

The fermentation process basically consists of inoculum preservation, inoculum build-up, pre-fermenter culture and finally production fermentation. A brief account of the four stages of fermentation is given below.

Inoculum preservation (culture maintenance):

The preservation of high-yielding strains of microorganisms for fermentation is very important for product formation in substantial amounts. The ultimate purpose of preservation is to maintain the strains, as long as possible, without cell division. There are different methods of preservation.

Storage at low (2-6°C) temperature:

In this method, the microorganisms can be stored in a refrigerator in liquid culture or as stab culture. Although this is the easiest method of preservation, there is a high risk of contamination.

Storage by freezing:

The microbial cultures can be frozen and preserved for several years. In the freezers, the preservation can be done at -18°C or, at -80°C. For preservation at -196°C, liquid nitrogen must be used. It is very important that the freezing (and later thawing when required) is done slowly (usually with a change of 1°C/min) to prevent damage and killing of the microorganisms. If proper care is not taken, as many as 95% of the cells may be killed by freezing and thawing.

Storage by lyophilization:

Preservation of microorganisms by lyophilization (i.e., freeze drying) is the best method, although, it requires special equipment. In fact, lyophilization is the method of choice by many fermentation biotechnologists.

The storage of microorganisms can be done by any one of the three techniques described above. However, for each method, optimal conditions for preservation must be worked out for each strain separately. In general, the preserved master strains are cultivated once in two years for checking of their activity. When needed for use, the working strains can be obtained from the master strains.

Inoculum builds up:

The preserved cultures have to be revived for their industrial use. This can be done by growing the cultures in liquid or on solid media. The actual process and the conditions used for inoculum build-up largely depend on the preservation technique used. There are wide variations in the growth times which depend on the type of preservation and the organisms used as given below.

Refrigerated cultures (2-6°C):

Bacteria 6-24 hours

Actinomycetes 1-3 days

Fungi 1-5 days

Frozen cultures (18°C, -80°C, -196°C):

Bacteria 6^48 hours

Actinomycetes 1-5 days

Fungi 1-7 days

Lyophilized cultures:

For all organisms 4-10 days

For proper growth, and to obtain sufficient quantity of inoculum, a series of cultures are prepared. For good fermentation yield, the number of cells and spores, nutrient medium, temperature and age of the inoculum are important.

The inoculum build-up is suspended in a surface-active agent such as Tween 80 and transferred to the bioreactor for fermentation.

Pre-fermenter culture:

Fermenter pre-culture or pre-fermenter culture is often required for inoculating large sized bioreactors. Inadequate quantity of inoculum will not only delay the product formation, but also reduce the yield drastically. By culturing the microorganisms (the inoculum build-up) in small fermenters, the size of the inoculum can be increased for large-scale industrial use.

Biotechnologists have worked out the requisite inoculum concentrations for optimal fermentation e.g., for bacterial fermentation, the inoculum concentration should be between 0.2 to 3.0%; for fungal fermentation, it is in the range of 5-10%.

Production fermentation:

The general features and the different types of bioreactors are already described (See p. 239-244). The size of the fermenter used mainly depends on the product. For example, a small bioreactor (1-20 litre size) can be used for producing diagnostic enzymes and substances for molecular biology by recombinant microorganisms, while large bio­reactors (≥450 litres) are employed for producing single-cell protein and amino acids.

A diagrammatic representation of a generalized fermentation process is depicted in Fig. 19.16.

For appropriate production by fermentation, several parameters need to be carefully considered and optimized. These include composition of nutrient medium, carbon and nitrogen sources, batch to batch variations, effect of sterilization on nutrients and on pH, and alterations in temperature and aeration. The parameters—temperature, pressure, aeration and stirring are briefly described.

Temperature:

The temperature must be so maintained that there occurs maximal growth of microorganisms with optimal product formation, although this is not always possible. In general, there are two temperature ranges to run the fermentations a mesophile range (20-45°C) and a thermophile range (> 45°C).

Sometimes, two different temperatures are used for the same fermentation process—a higher temperature is employed for good growth (in trophophase), and then the temperature is decreased for optimizing product formation (in idiophase).

Pressure:

Appropriate maintenance of hydrostatic pressure, particularly in large sized bioreactors is very important. This is because pressure influences the solubility of O₂ and CO₂ in the culture medium. An overpressure in the range 0.2-0.5 bar is generally used.

Aeration:

A bioreactor gets aerated by the supply of O₂ and therefore, adjustment must be made to furnish required amount of O₂ to the microorganisms. Usually, the aeration rate is in the range of 0.25-1.25 vvm (volume of air/volume of liquid/minute).

Stirring:

The type and the speed of impellers determine the stirring rate in a fermenter. In general, the impeller speed decreases as the size of the fermenter increases. Thus, for a small bioreactor (size 1-20 litres), the impeller speed is in the range of 250-350 rpm, while for a large bioreactor (size around 450 litres, the impeller speed is 60-120 rpm.