BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 5. INDUSTRIAL BIOTECHNOLOGY

Stages of Biotechnological Production

Biotechnological production involves the following key stages:

- preparation of raw Materials (culture medium) and biological agents, namely a pure culture of the producer strain;

- Fermentation, leading to product formation;

- Product Recovery and purification;

- formulation of commercial product preparations.

Preparation of Raw Materials and Biological Agents

The First stage of biotechnological production involves preparing a nutrient medium primarily composed of carbon and nitrogen sources, along with macro- and microelements in the form of salts. Certain nutrients at excessively high concentrations can inhibit microbial Cell growth: glucose suppresses growth at concentrations > 50 g/L, ammonia > 3 g/L, iron > 1.15 g/L, magnesium > 8.7 g/L, phosphorus > 10 g/L, and zinc > 0.038 g/L.

A crucial requirement in preparing the culture medium is maintaining strict asepsis. To achieve this, equipment and medium components are sterilized. The most common method is thermal sterilization, which involves exposure to high temperatures (120-150°C). Radiation sterilization (γ-irradiation) and chemical sterilization (using bactericidal compounds) are also employed. Certain substrates do not require sterilization, such as methanol, ethanol, and concentrated acetic acid.

Industrial biotechnology utilizes industrial microbial strains as biological agents. These strains are developed through two main approaches:

1) by introducing additional genes into microorganisms or increasing their copy number or activity;

2) through the Selection of producer microorganisms.

Industrial microbial strains must meet the following requirements:

- ability to grow on inexpensive nutrient media;

- high growth rate and rapid end-product formation;

- minimal formation of by-products;

- Stability of the producer strain regarding its production characteristics;

- safety for humans and animals;

- resistance to contamination/infection.

Unlike wild-type strains, industrial microorganisms are characterized by high productivity. For instance, the productivity of an industrial Penicillium strain (50 g of penicillin per 1 L of nutrient medium) is 10,000 times higher than that of the wild-type strain.

Fermentation

The central phase of industrial biotechnological manufacturing is fermentation, which encompasses all operations from the inoculation of the producer strain to the completion of growth, Biosynthesis, or biotransformation processes.

Fermentation is carried out in specialized vessels known as fermenters or bioreactors. The structural layout of a bioreactor is shown in Fig. 5.1.

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Fig. 5.1. Schematic diagram of a bioreactor design

The following parameters must be controlled in bioreactors: optimal Temperature, pH (5.5 - 8.5), oxygen concentration, culture agitation intensity, and uniform delivery of nutrients. As microorganisms consume nutrients, they generate heat. To prevent overheating, bioreactor walls are cooled with Water. The optimal temperature for microbial cultivation typically ranges from 20 to 50°C. Consequently, bioreactors provide optimal conditions for vital microbial activity.

The design of each bioreactor is tailored to obtain a specific product. For example, the volume of a bioreactor used for Yeast biomass production reaches 1500 m³ (the cylindrical reservoir for growing yeast on petroleum Hydrocarbons is 40 m high and 20 m in diameter). In penicillin production, bioreactor volume does not exceed 100 m³. Research and development work generally utilizes bioreactors with a capacity of no more than a few liters.

Industrial fermentation is a multi-stage process involving the cultivation of a starting culture (5 - 10 ml), flask incubation (200 - 1000 ml), seed fermenter growth (10 - 100 l), and scaled-up production in an industrial fermenter (1000 - 100,000 l). The fermentation process flow is illustrated in Fig. 5.2.

Fig. 5.2. Fermentation process flow diagram

There are three main Types of fermentation processes:

- Batch fermentation — microorganisms are cultivated without adding fresh nutrient medium during the process;

- Fed-batch fermentation — fresh nutrient medium is periodically added during the process, increasing product yield by 25 - 100% compared to standard batch fermentation;

- Continuous fermentation — fresh nutrient medium is continuously supplied to the bioreactor while an equivalent volume of cell suspension is simultaneously removed.

Each fermentation system has its own Advantages and disadvantages. Batch fermentation is the most common, whereas continuous fermentation is the most promising and economically advantageous.

During fermentation, sterile air is sparged through the nutrient medium to supply microorganisms with oxygen. Because oxygen has low solubility in water, its reserves in the medium are rapidly depleted under conditions of intensive Microbial growth. Under such conditions, microbial Cells synthesize proteinases capable of degrading Proteins, which are the desired products of microbial biosynthesis. There are two primary approaches to solving the aeration problem in culture media:

- utilizing microbial strains deficient in Proteolytic Enzymes;

- introducing genes from the aerobic bacterium Vitreoscilla into the microbial genome, which encode an oxygen-binding protein (bacterial Hemoglobin).

Introducing the Vitreoscilla Gene into E. coli cells leads to enhanced synthesis of cellular and recombinant proteins, as well as activated ATP synthesis under low oxygen concentrations in the nutrient medium.

Product Recovery and Purification

Products of microbial biosynthesis may be localized either within the microbial cells or in the nutrient medium. Filtration or centrifugation is employed to separate microbial cells from the culture medium. If the product is intracellular, cells are disrupted using physical Methods (ultrasonication, osmotic Shock) or chemical methods (Cell wall lysis via butanol, toluene, or enzymes).

Techniques such as extraction, ultracentrifugation, crystallization, and Chromatography are used to isolate valuable products from the nutrient medium. Spent culture media must be tested for viable microorganisms to eliminate any risk of environmental release.

Formulation of Commercial Products

Depending on the production technology, biological preparations are classified into three main groups:

1) biopreparations containing viable microorganisms (plant protection agents, bacterial fertilizers, silage inoculants, biodegradants);

2) biopreparations containing inactivated cell biomass and its Processing derivatives (fodder yeast, fungal mycelium);

3) biopreparations based on purified microbial metabolites (Vitamins, Amino Acids, Antibiotics, Lipids, Polysaccharides).