What is biotechnology?

Biotechnology is a field that deals with obtaining of products, substances and processes necessary for humans, employing microorganisms. The prime source of biotechnology is connected with wine and cheese production already in olden times. Certainly, this terminology had not been used yet at that time. Today, it is not so easy to understand, where are the limits of the biotechnology's applicability, as it is combined with other scientific and technical fields such as molecular biology, genetics and gene engineering, biochemistry, chemical technology, etc.

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

1. For food industry requirements - citric acid, amino acid and other food additives;
2. For agriculture - plant protection agents, modified food;
3. For medicine - antibiotics, interferon, vitamins, vaccines and other preparations;
4. For environment protection - contamination degrading substances and processes;
5. In energetics - biogas, ethanol and other energy sources;
6. For the chemical industry - ethylene, acetone, buthanol and other substances.

And these are not all applications. There are studies and developments which make it possible to augment the ever more complicated live cells. Biotechnology continues to develop, and nobody will take the risk of predicting its development potentialities. Already now, these development rates have reached such a level that makes the society to control its directionality.

To reach the necessary goal, the biotechnological process has usually 3 major stages:

1. Preparation of nutrient media for the cultivated microorganism and the cultivation process;
2. The course of the microorganism reproduction process in bioreactors (called also fermenters) or in other equipment;
3. Obtaining of the final product or substance from the cultivated medium. This stage includes operations such as separation, purification and other technologies, which are connected with obtaining the commodity form.

What is the bioreactor necessary for?

Bioreactor is the equipment that ensures the conditions for microorganisms' reproduction. The microorganisms' sizes can be different depending on their type. However, in any case, we are dealing with several ?m. In miroorganisms' reproduction, their number can reach 1 million cells/ml. To visualise microorganisms, it is possible to look through the electromicroscopic enterprises' collection:

Electromicroscopic enterprises' collection

Microorganisms grow in a nutrient medium, from which the energy required for growth is taken. In addition, oxygen that is supplied with the help of compressed air can be used as the nutrient element. To proceed successfully the microorganisms' growth, it is necessary to observe precisely the environmental conditions, as microorganisms are much more sensitive than humans in this respect. Therefore, also in this microorganisms' earthly home, the bioreactor, a much higher comfort level should be ensured. The temperature that is well regulated in the shower cannot be changed by more than 1°C to one or the other side. The environment should be so acidic as is more pleasant for the given microorganisms, and there should be no deviations to other tastes. The more microorganisms have grown, the more oxygen should be given to them. So, don't leave them without the nutrition solution and don't forget to supply oxygen! It should not be also forgotten whether all the necessary nutrient components are retained. Otherwise, these are so sensitive and can either die or fail to do the work they had been entitled to do, i.e. something different starts being synthesised. It should not be also exaggerated - if too much oxygen is given, they can "choke"! And it is difficult to predict the sequences. In addition, it should be kept in mind that this universe - bioreactor should be well sealed. Otherwise, the "savages" (i.e. wild yeast) will break in there and will take away our microorganisms' nutrition. Thus, in some time, having looked at what is occurring in the bioreactor, we will see that our fine microorganisms are not there any more, but it is full with wild yeast. Therefore, we should see to it that everything is hermetically sealed. If samples are to be taken, it should be done so that the "bad" microorganisms would not get into the bioreactor vessel. Microorganisms are demanding - they will not reproduce all of a sudden. They acknowledge only stainless steel, good glass, teflon and, perhaps, some inert material. There should not be simple zincified screws in the inner part of the reactor, and sensors with brass parts should not be used! And before they start living in this home, it should be well cleaned and washed, and the survivals of "bad" microorganisms should be annihilated. Annihilating sterilisation is the only way, how it could be done.

Bioreactor's preparation for operation

To realise the microorganisms' cultivation, it is necessary:

1. To prepare the bioreactor for cultivation;
2. To sterilise the bioreactor;
3. To prepare the inoculum.

To prepare the bioreactor for cultivation means that the bioreactor vessel and the required leads are cleaned and washed, all sensors are installed in the bioreactor, and other devices (pumps, titrated flasks, etc.) required for the process are connected. The bioreactor vessel and its leads should be sterilised to avoid possible infectious (non-sterilised) disease breeders. In the laboratory bioreactor, one of the following sterilisation methods is used, depending on the bioreactor's construction and volume:

1. Autoclaving - installation of the bioreactor vessel and the leads (which are disconnected) in the autoclave.
2. Sterilisation in the place - steam supply to the bioreactor's jacket. In this case, the bioreactor is not moved somewhere else, and the bioreactor jacket is usually connected by a 3-way valve to the steam line pipes.
3. Sterilisation in situ - in this case, the heating of the inner part of the bioreactor vessel is ensured by the heaters present inside the bioreactor. In this case of sterilisation, the glass part of the bioreactor vessel should be protected with a metal jacket.

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

Variant 2 is commonly applied for steel and steel/glass reactors with a steel jacket.

Variant 3 is applied mainly for bench-top type reactors with the built in powerful enough heaters, a thermostat or a steam generator. This variant is commonly applied in the corresponding reactors up to 20 litres.

During the sterilisation, the bioreactor and the corresponding leads should be hermetically sealed to avoid the penetration of the microorganisms wandering in the environment. More detailed information on the bioreactor's sterilisation is available in the following references:

The nutrient can be sterilised separately, and then it is sterilely fed up (for example, with the help of a peristaltic pump) in the bioreactor. The nutrient can be also fed up in the bioreactor and then be sterilised jointly with the bioreactor.

Growth of Microorganisms

The microorganism cultivation process, fermentation, starts with the moment when the preliminary prepared inoculum (in a retort or other smaller bioreactor) is sterilely fed up in the bioreactor.

The reproduction of the microorganism culture is characterised by 4 phases in time cycles:

1. Lag phase;
2. Exponential phase;
3. Stationary phase;
4. Death phase.

During the lag phase, the cell metabolism is aimed at synthesising enzymes in a definite reproduction nutrient. The lag phase length can vary also for the same microorganism culture in a definite nutrient, since it depends on different factors, e.g. on how many non-growing cells are present in the inoculum.

The exponential phase is the growing period in which the cell division with the logarithmic increase in the population number occurs. Such a dramatic increase in the growth rate is a limited period of time in a fixed amount of the nutrient. The nutrient resources are exhausted, or also the process is inhibited by the separation of the toxic metabolite.

The growth stops, and the so-called stationary phase sets in, although the cell metabolism goes on acting, and the process of separating the secondary metabolite can begin. In many cases, the aim of fermentation is the obtaining of the secondary metabolite rather than biomass, since the former can be used as the raw material for obtaining valuable products and preparations. In this case, the fermentation is purposefully retained at the stationary phase.

If the fermentation is continued for some time in the stationary phase, then a gradual decrease in the cell activity, i.e. the death phase can begin.

The batch fermentation process duration can varied from about 8 h till 5 days. Its mainly depends from microorganisms strain, substrate art and fermentation task.

In terms of the feeding character, the cultivation process can have 3 types:

1. Batch;
2. Fedbatch;
3. Continuous.

In the batch process, the bioreactor is supplied with a fresh nutrient, and thereby the inoculum is fed up there. At the end on the fermentation process, the content is passed to the separation stage, the reactor is cleaned and sterilised to be ready for the next process.

In the fedbatch process, a fresh nutrient (the feeding up intensity is commonly connected with the growth or biosynthesis rate) is supplied in the bioreactor continuously or in portions. When the bioreactor is full, it is partially or completely discharged. The process is finished or resumed.

In the continuous process or chemostate, the solution cultivated in the bioreactor is continuously discharged. The continuous process can proceed for a very long time, and its duration is commonly determined by the production requirements and technical factors.

Most widespread is the fermentation with fedbatch, and the latter is commonly applied for biological products. In this case, the drawbacks of the batch process are prevented with minor technical changes.

Continuous processes or chemostates are applied most frequently for large-scale production of biochemicals. From the viewpoint of production, such processes are more economic, although essential technical modifications as well as a deeper insight in the given fermentation kinetics are required.

More about principles of biotechnology and bioreactors