What is Biotechnology?

Biotechnology involves using microorganisms to create essential products and processes for humans. Its origins trace back to ancient wine and cheese production, though the term “biotechnology” was not used then. Today, its boundaries are hard to define as it integrates with fields like molecular biology, genetics, gene engineering, biochemistry, and chemical technology.

Biotechnology has pushed into our everyday life more than we usually imagine. It has offered applications for different fields:

  1. For food industry requirements – citric acid, amino acids, etc.;

  2. For agriculture – plant protection agents, modified food;

  3. For medicine – antibiotics, interferon, vitamins, vaccines, etc.;

  4. For environment protection – contamination degrading substances and

  5. In energetics – biogas, ethanol, and other energy sources;

  6. For the chemical industry – ethylene, acetone, butanol, etc.

These are not the only applications. Studies and developments continue to enhance complex live cells. Biotechnology’s rapid progress makes it difficult to predict its future, necessitating societal control. Typically, biotechnological processes involve three major stages to achieve their goals:

  1. Preparation of nutrient media and cultivation of microorganisms;

  2. Microorganism reproduction in bioreactors or other cultivation equipment;

  3. Obtaining the final product, including separation, purification, and other related processes.

What is the bioreactor necessary for?

A bioreactor is a vessel that provides the necessary conditions for microorganism reproduction. Microorganisms, typically a few micrometers in size, can reproduce to reach 1 million cells/ml

Microorganisms grow in a nutrient medium, requiring precise conditions. Oxygen is supplied via compressed air. Adequate oxygen and nutrient levels are crucial to prevent microorganism death or undesirable byproducts. The bioreactor must be sealed to prevent unwanted contamination, for example, by wild yeast. All components must be thoroughly cleaned and sterilized before use to eliminate any harmful microorganisms.

Bioreactor's preparation for operation

In order to realize the cultivation of microorganisms it is necessary:

  1. To prepare the bioreactor for cultivation;

  2. To sterilize the bioreactor;

  3. To prepare the inoculum.

Preparing the bioreactor involves cleaning the vessel, hoses, and tubes, installing sensors, and connecting necessary devices like pumps and flasks. Sterilization of the vessel and its components is essential to prevent contamination. Depending on the bioreactor’s design and size, different sterilization methods are used in the laboratory:

  • Autoclaving – installation of the bioreactor vessel, its hoses and tubes (which are disconnected) in the autoclave.

Variant 1 is commonly applied for glass or steel/glass reactors up to 10 liters, as the installation of a larger reactor in the autoclave is difficult.

Variant 2 is mainly used in pilot-scale bioreactors for larger-scale production where in situ sterilization ensures aseptic conditions

During the sterilization, the bioreactor and the corresponding gear should be hermetically sealed to avoid the penetration of the microorganisms wandering around in the environment.

The nutrient medium can be sterilized separately and then under sterile conditions fed up (for example, with the help of a peristaltic pump) in the bioreactor. The nutrient also can be first fed up in the bioreactor and then be sterilized along with the bioreactor.

Growth of the Microorganisms

The microorganism cultivation process, fermentation, starts with the moment when the preliminarily prepared inoculum (in a flask or other smaller bioreactor) is fed up in the bioreactor under sterile conditions.

The reproduction of the microorganism culture is characterized by 3 phases in time cycles:

  1. To prepare the bioreactor for cultivation;

  2. To sterilize the bioreactor;

  3. To prepare the inoculum.

During fermentation, phases progress from enzyme synthesis in the lag phase to rapid growth in the exponential phase until nutrients are depleted or toxins accumulate. The stationary phase follows, where metabolism continues for secondary metabolite production. Extended stationary phase may lead to decreased cell activity in the death phase. Batch fermentation durations vary (8 hours to 5 days), depending on microorganism strain, substrate, and fermentation goals.

In terms of the realization principle, there are three main types of the cultivation process:

  1. Batch;

  2. Fedbatch;

  3. Continuous.

  • In batch fermentation, fresh nutrient and inoculum are added to the bioreactor initially, followed by separation and sterilization after fermentation for the next cycle.
  • In fed-batch fermentation, nutrients are continuously or intermittently added based on growth or biosynthesis rates, with periodic partial or complete discharge when the bioreactor reaches capacity.
  • Continuous processes, like chemostats, involve ongoing discharge of cultivated solution, catering to long-term production needs based on technical and production requirements.
  • Fed-batch fermentation, essential in biotechnology, optimizes microbial growth and product yield by controlling nutrient addition, achieving higher biomass concentration
  • Continuous processes, like chemostats, are ideal for large-scale production and products with constant demand. They offer economic benefits and can run for a month without CIP or SIP, but need detailed fermentation kinetics and technical adjustments

History of biotechnology

1. The empirical period

It lasted for approximately 8000 years. Ancient people intuitively used methods to obtain bread and beer even though they did not know about microorganisms. A few thousand years ago acetic acid was first obtained. The first distillation in wine production was done in the 12th century. Vodka from bread grain was the first time obtained in the 16th century, but champagne – 18th century.

2. Etiological period (1856 – 1933)

This name is associated with the Greek word – aitia. During this period great French scientist L. Paster (1822 – 1856) did important research in microbiology. He formulated fermentations microbiological nature, proved living cell ability to reproduce in an oxygen-free environment, made a scientific base of vaccine prophylaxes. In 1859 L. Paster prepared liquid medium. In 1881 R. Kohs demonstrated the cultivation of bacterias – their reproduction in a liquid medium – which showed opportunity of microorganism reproduction with an aim – specific process realization (fermentation, acidification, etc.). In this period also began production of compressed yeast.

3. Biotechnical period

To a large extent, this period was based on research published in 1933 by A. Kluever and A. Perkin “Researching methods of mycelia funguses metabolism”, which describes methods of microorganism submerged cultivation. A breakthrough in the development of industrial biotechnology was the beginning of large-scale antibiotics production when WW II started because it was necessary to provide the wounded man with antimicrobial preparation.

The most significant achievements during this period are:

  1. Principle of bioreactor design;

  2. Theory of electrophoresis;

  3. Decryption of DNA structure.

4. Genetic techniques period

It started in 1972 when P. Berg created the first recombinant DNA molecule thus showing the possibility of purposeful manipulation with the genetic material of bacteria. Other significant achievements during this period are:

  1. Begins monoclonal antibody use in diagnostics;

  2. Begins production of human insulin;

  3. Cloning.