CHAPTER IV: Industrial fermentations

Industrial manufacturing of microbial products usually relies on fermentation. Industrial fermentation is the cultivation of large quantities of microbes or other isolated cells to produce a substance of commercial interest. This operation takes place in bioreactors (fermenters).

IV.1. Fermentation

It is a natural phenomenon which consists of a transformation of a food by a microorganism in the absence of oxygen. Man used it for 3 reasons: to make it more digestible, to keep it longer and to produce a substance of interest.

According to the AFNOR NFX 42-000 standard of December 1986: it is defined as “biofermentations which use microorganisms”, fermentation is defined by the “degradation of carbohydrate substrates without the use of oxygen.

Today, fermentation is characterized as “the oxidation of organic compounds without the use of oxygen, through characteristic enzymatic systems, with various organic compounds as electron acceptors”.

Several definitions can be attributed to fermentation:

• It is all the deterioration of food by microorganisms.

• Any process that results in acidic alcoholic beverages or dairy products.

• Any large-scale microbial process that occurs in the presence or absence of air.

• Any metabolic process that releases energy and occurs only under anaerobic conditions.

• Any metabolic process that releases energy contained in a sugar or another organic molecule that does not require oxygen (O2) or an electron transport chain.

IV.2. Fermentation in a liquid medium (submerged) :

It was fermentation in a stirred liquid medium which was used first, it was traditionally used for the industrial production of enzymes due to the ease of controlling different parameters such as pH, temperature, aeration and humidity. This type of fermentation is carried out in stirred vessels (Bioreactors) and requires strict aseptic conditions.

IV.2.1. Bioreactors or fermenters :

The main function of a bioreactor is to ensure a controlled environment allowing optimal growth and production of microorganisms or cells in large quantities. A fermenter is generally built on the model of a bioreactor without, however, an aeration system. In the field of biotechnology, the term fermenter is sometimes used without any distinction from that of bioreactor. It makes it possible to differentiate the type of culture (bacteria, yeast for fermenter and animal cells for bioreactor).

                                                                     Diagram of a bioreactor

A bioreactor includes:

• A tank or enclosure made of glass (for laboratory models) or stainless steel.

• A cap if necessary to prevent air from the interior environment and that from the exterior environment.

• A syringe with catheter to inject a solution.

• An agitation system comprising one or more turbines depending on their size.

• Sensors for measuring temperature (thermometer), pH (pH meter), dissolved oxygen concentration (oximeter probe), level, etc.

• A computer-managed control system allowing all operating parameters to be recorded and controlled.

The bioreactor thus has four essential roles: container, sterilizer, aerator, and above all agitator.

 Container role :

The tank must be made of stainless steel and must resist the heat of sterilization and corrosion. It must also be impervious to external contamination and withstand the addition of acids, bases, anti-foams and additives which are made during the fermentation process.

 Role of sterile enclosure :

Before inoculation, the culture medium must be sterilized under pressure at 120°-125°, as quickly as possible. The bioreactor must be designed to ensure aseptic operations excluding any contaminating organisms.

The air must also be sterilized through a sterilizable absolute filter.

 Role of aerator :

The reactor must have a sterile air injection system which must be dissolved and dispersed throughout the medium.

 Role of homogenizer :

In many cases, the reactor must ensure homogenization of nutrients, air, temperature and dissolution of the injected air.

 Control role :

Ideally, the reactors must allow direct control of important parameters: pH, T°, dissolved oxygen, etc. It is essential to eliminate the heat produced by circulation of cold water and possibly add the anti-foam, acids, bases or nutrients.

IV.2.2. Liquid phase systems :

Bioreactors with liquid media allow the use of batch, feed-batch, and continuous processes.

      IV.2.2.1. Batch (discontinuous) process :

This technique consists of cultivating microorganisms in a closed system, the same volume of medium is used to carry out the growth, production and accumulation phases of the product. All medium is then removed and processed, and the final products are harvested only at the end of the culture.

The latency and acceleration phases correspond to a period of metabolic adaptation of the microorganism to the environment. The lag phase will be reduced as much as possible in industrial fermentation. The exponential growth phase is the phase during which the specific growth rate Q x expo is maximum. The amount of nutrients is in excess and therefore the biomass increases the fastest. The products formed during this phase are the primary metabolites.

In this mode of operation, all of the nutrients necessary for biological growth are introduced when the reaction starts. No addition or withdrawal is made subsequently and the reaction takes place at constant volume. The only possible operator actions concern only environmental variables (pH, temperature, stirring speed, aeration, etc.).

IV.2.2.2. Feed-batch process (fed batch) :

The principle is the same as the first process, but during fermentation, certain components of the medium or certain precursors are added in a controlled manner.

Fermentation begins in a small volume of culture medium (called stock), seeded with the inoculum. If the concentration of the inoculated inoculum is high, fermentation starts more quickly. In the exponential phase of bacterial growth, the sterile culture medium is introduced into the bioreactor without withdrawing product, leading to an increase in volume in the tank over time.

IV.2.3. Continuous systems :

Continuous and constant growth is ensured by a regular addition of nutrients and a parallel elimination of part of the cells, waste and formed products.

During the exponential phase, the nutrient medium becomes depleted of nutrients, while the products of microbial metabolism accumulate. If we renew the medium in the fermenter by adding new medium and eliminating the medium which contains the cells formed and the metabolites produced, the culture remains in exponential phase. This is the case for continuous cultures.

Two main types of continuous culture systems are generally used : Chemostats and Turbidostats

 Chemostat : control is done by limiting the power supply. In a chemostat, growth is limited by a factor (carbon source or amino acid for example). Determined by the variation in growth rate in different concentrations of the nutrient in the fresh growing medium (figure below).

 Turbidostat : control is done according to cell multiplication, measured by turbidimetry, this system is equipped with a photoelectric cell to measure the absorbance or turbidity in the culture chamber. The flow rate of the medium through the tank is automatically adjusted to maintain a predetermined turbidity or cell density. Under these conditions the culture medium does not contain any limiting nutrients.

The turbidostat works better at high dilution rates, while the chemostat is more stable and more effective at lower dilution rates. Continuous fermentation generally used for the production of single-cell proteins, ethanol, acetic acid, etc. from inexpensive raw materials such as starch, molasses and cellulose. It is also used in wastewater purification to eliminate chemical waste through continuous digestion.

IV.3. Fermentation in a solid medium :

These are cultures in which the substrates are neither solubilized nor suspended in a large volume of water. The substrates are only moistened.

Fermentation in medium or solid phase (FMS) is a technological process which reproduces the natural living conditions of microorganisms, in particular those of filamentous fungi and higher fungi, by allowing their development (adhesion) to the surface of a support organic.

From a fundamental point of view, solid medium fermentation is defined as fermentation involving moist solid particles with little or no free water and differs from liquid fermentation, where the nutrient medium is completely solubilized in a large volume of water, and fermentation in a submerged environment where the nutrient medium is for example in the form of a suspension of fine particles in the liquid phase.

Only organisms tolerant of low water activity are capable of growing under these conditions. These are mainly filamentous fungi and yeasts.

IV.3.1. The support :

The support is one of the most important parameters in solid-state fermentation. It must be chosen carefully depending on several factors such as particle size, porosity, biochemical composition, its water retention capacity and/or its capacity to contain nutrients (source of carbon, nitrogen and of mineral salts), its availability and its cost.

They are classified into two categories, inert and organic supports, and come in three forms:

1) In the form of natural organic materials (starchy or lignocellulosic). These are generally sources of insoluble, complex and heterogeneous polymers (bagasse, beet pulp, straw, wood, wheat bran, cassava, etc.). They serve as both substrate (carbon source) and support;

2) In the form of synthetic materials (polyurethane foam). They only serve as a support and therefore require the provision of a nutrient medium;

3) In the form of mineral materials (clay granules, perlite, pozzolan). They only serve as a support and therefore require the provision of a nutrient medium.

IV.3.2. Environmental factors and cultivation parameters :

 Temperature :

Temperature is one of the most difficult factors to regulate in solid fermentation. The low thermal conductivity of the air (compared to that of water), of the supports and the absence of free water limits the transfer of heat and its elimination thus favoring a rise in temperature of up to 20 °C above incubation temperature. This temperature rise depends on the type of microorganism, porosity, particle size and depth of the support.

This phenomenon occurs especially during the growth of the strain and is directly proportional to its heat-generating metabolic activity, of the order of 100 to 300 kJ per kg of cell mass. Poor heat dissipation can lead to temperature gradients within the culture medium during fermentation and can cause metabolic deviations, drying of the medium, degradation of secreted products, a reduction in nutrient availability or even the cessation of the vegetative phase.

 Humidity relative to water activity :

The water content, or rather the quantity of water available, is really very important since low humidity would limit the hydrolysis of the substrate, the solubilization and diffusion of nutrients and/or the accumulation of inhibitory compounds in solid particles. , while high humidity would reduce porosity, gas volume and gas exchange, while promoting bacterial contamination.

 Ventilation

Aeration is an important (essential) factor in fermentation in a solid medium since it will allow: oxygenation (especially for aerobic organisms such as filamentous fungi), dissipation of metabolic heat (regulation of the temperature of the medium) and the elimination of metabolism products (CO2, water vapor, volatile compounds).

 The pH

The pH is very difficult to homogenize and control in FMS. Indeed, during the culture, the metabolic activity of the strains will modify the pH of the medium either by acidifying it, by the production of acids or by the absorption of ammonium ions, or by alkalizing it, by the release of ammonia from the breakdown of proteins, urea or other amines.

IV.3.3. The different types of industrial reactors :

The number of reactors designed for FMS is very limited and they are not optimized. The four main types of reactors commonly used are :

a. Tray bioreactors :

This type of bioreactor is made up of stepped trays (wood, metal or plastic), containing a thin layer of support (5 to 15 cm), and placed in a controlled atmosphere chamber (temperature, humidity). The cultivation takes place in principle statically and the aeration of it is generally done passively (without forced aeration), although aeration can be carried out by circulating air from the bottom to the top of bedroom.

Tray bioreactors

            b. This type of bioreactor is generally composed of a column (glass or plastic) containing the solid support. The enclosure can optionally be equipped with a double envelope allowing water circulation and therefore better temperature regulation. The culture is generally static and aeration can be applied from the bottom of the column.

            c. Rotating drum bioreactors.

This type of bioreactor is made up of a tank, rotating around an axis (like a concrete mixer). This system allows adequate aeration (forced or not) and homogenization of the environment through periodic or continuous rotation.

Rotating drum bioreactors

d. Fluidized bed bioreactors.

This type of bioreactor consists of placing particles suspended in the air inside a column, consisting of a mixture of solids and fluid. This system allows continuous agitation and aeration, thus avoiding particle aggregation and promoting the elimination of metabolic heat. However, the main disadvantage of this type of bioreactor lies in its (too) high cost.

IV.4. Advantages and disadvantages of fermentation in a solid medium :