Kombucha in learning

Fungi are unbelievably important in biotechnology, because they can be used for fermentation. Kombucha is an example of a fermentation process which can easily be carried out at home and which, at the same time, draws on the extensive professional scientific knowledge that lies behind it. In the following section, you can read about fermentation, performing tasks and carrying out a kombucha brewing project.

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What is fermentation?

A fermentation process can be compared with an industrial plant. During the process, products are manufactured by the plant’s workers, who are much like a collection of microorganisms. A clear definition of fermentation goes as follows:

Fermentation is a process whereby a mass culture of microorganisms is used to make a product.


During fermentation, all possible processes are taking place inside the cells. They are not always easy to control. Getting an overview of what is happening can therefore become a disorganized and chaotic process.

Using the so-called black box model can be a tangible way of understanding a fermentation process. With this model, we view the cell as a factory that takes up materials from the surroundings and converts them to products, which are then discharged.

The materials that are coming in are called substrates. Substrates represent all those substances that the cells use to grow. These are either directly incorporated and used in the cells or are shunted through a biochemical pathway. The large collection of pathways is referred to as the cell’s metabolism. The cell’s primary objective is to reproduce, and therefore the substrates are particularly used to make new cells. The mass of cells is called biomass. At the same time, as a by-product of this, the cells secrete metabolic products which they have synthesized.

The chemical compounds formed in this metabolic process are termed metabolites. There is a difference between primary and secondary metabolites.

Those metabolites that are vital for cells are referred to as primary metabolites. They are chemical compounds that are essential for cells to continue living. An example of primary metabolites is amino acids, which are used to make proteins. Other examples are ethanol and lactic acid, which are formed as part of the process whereby cells charge up their “energy batteries” to carry out energy-dependent reactions.

Secondary metabolites are chemical compounds, produced by the cell, which are not directly essential. The secondary metabolites are produced by the microorganisms because they help the organisms survive, and so their production has been selected through evolution. One example of a secondary metabolite is penicillin, which is produced by molds. Penicillin is an antibiotic, which kills bacteria and can be used advantageously by molds to outcompete bacteria. Antibiotics are a classic example of a secondary metabolite, but there are many secondary metabolites whose function is not known.

Nowadays, many different small molecules and enzymes are produced by large-scale fermentation. Modern biotechnology has also opened up the possibility of genetically modifying microorganisms so that they make products that they would not normally be able to make. An example of this is the production of insulin by yeasts. Insulin is a hormone produced in the pancreas, which induces cells in the body to take up sugar from the blood. Diabetics have problems producing insulin, and so being able to produce the hormone in a fermentation vessel is a fantastic achievement.

Biomass can also be a desirable product in itself. Danish yeast plants produce around 25,000 tons of baker’s yeast each year, which is sold in Danish supermarkets and can be used in baking.
Yeasts and molds are examples of fungi-based fermentation, but there are now many different types of microorganisms being used as cellular factories:

Bacteria Yeasts
E.g.: Escherichia coli
Prokaryotes
Anaerobic anaerob
Very rapid growth
E.g.: Saccharomyces cerevisiae
Eukaryotes
Anaerobic
Very rapid growth
Molds Mammalian cells
E.g.: Aspergillus niger
Eukaryotes
Rapid growth
Strictly aerobic
E.g.: HeLa (cancer cell from humans)
Eukaryotes
Slow growth
Strictly aerobic

Each microorganism has its advantages and disadvantages. Some microorganisms are easy to use in fermentation but cannot always make the products you are interested in. Mammalian cells are especially difficult to ferment with as they have slow growth and can only grow under precisely the right conditions. On the other hand, they can produce valuable proteins, which cannot be made in other cellular factories.

Growing conditions

To carry out a successful fermentation in the best way possible, it is essential to make sure that the microorganisms are well suited to the process. This can be achieved by fulfilling their need for good growing conditions as much as is possible. The fermentation is carried out in a growth medium which contains the substrates that are necessary for the microorganisms’ survival. At the very least, a growth medium should contain the following five components:

  • Water is always the largest component of one’s growth medium. Few organisms can tolerate environments without water. Moreover, water is used in a medium to maintain homogeneity and to control temperature. In addition, mineral water contains minerals, which are one of the other essential components in a growth medium.
  • Energy is essential for the microorganisms to carry out their metabolic processes. In those few instances where you is working with so-called phototrophic organisms, such as plants, the energy can come from light. One is typically working with so-called chemoorganotrophic microorganisms, which use energy-rich organic compounds as their energy source. Therefore, energy is generally supplied in the form of a carbon source.
  • Carbon, of all raw materials, is the most commonly used building block in cells and it is contained in all organic compounds. Because of this, it should constitute a substantial part of the growth medium. The carbon source, as we have said, is also most often the source of energy. The most common carbon sources are sugars, e.g. glucose. Alternatively, one can also use oils, fatty acids and alkanes.
  • Nitrogen is another important building block in all microorganisms. Nitrogen forms part of amino acids, DNA and other important compounds in the cell. Nitrogen can come from both inorganic compounds such as ammonia and nitrates, or organic compounds such as simple amino acids or proteins.
  • Minerals is the group name for all the raw materials mentioned below, which microorganisms use in small concentrations. Examples of some of the many minerals are: phosphorus, potassium, iron, zinc and copper. Minerals can be found in small concentrations as impurities from other sources, such as mineral water.

In some fermentation processes, it may also be necessary to add vitamins, which are essential substances that the cell factory cannot synthesize, but which are indispensable for life. When, for example, one uses mammalian cells in a fermentation, a long list of vitamins is added to enable the cells to survive.

A growth medium is either complex or minimal:

Complex medium:
A medium in which the content of chemical compounds is undefined. This is made up of complex nutrient sources, such as yeast extract and corn-steep liquor, which contain many different substances.

Minimal medium:
A well-defined medium, the content of which has been precisely designed for a particular purpose. A minimal medium contains, for example, pure glucose and a pure salt. Demineralized water is used and minerals are added to this.

The advantage of a complex medium is that it is typically cheaper to use. In addition, the microorganisms often grow more quickly in a complex medium, as it contains many different substrates. So-called growth factors, which promote growth, may be present in a complex medium. A minimal medium, on the other hand, is used for scientific purposes as the composition of the growth medium is well-defined and one can therefore perform calculations and create models, which would not be possible with a complex medium. It is easier to reproduce a fermentation with a minimal medium since you have better control of what is happening.

The need for oxygen is different for anaerobic and aerobic microorganisms. If they are strictly aerobic, a supply of gaseous oxygen is required. This is the case, for example, with molds. In practice, supplying oxygen is one of the most difficult challenges, as it needs to be bubbled into the vessel in such a way that the oxygen dissolves in the medium and comes into contact with all the microorganisms in the vessel.

Optimum growing conditions can vary considerably from organism to organism. Some microorganisms prefer glucose as their carbon source rather than glycerol. One example of this is the yeast Saccharomyces cerevisiae, which through evolution has developed the ability to grow extraordinarily well on glucose. By contrast, it does not grow well on glycerol and other alternative carbon sources. It is therefore important to know the background of your microorganism.

The table below gives some examples of adjectives that can describe a microorganism.

Adjective Meaning
Aerobic
Anaerobic
Strictly anaerobic
Facultative anaerobe
Acidophile
Halophile
Xerophile
Barophile
Oligotroph
Autotroph
Heterotroph
Phototroph
Chemotroph
Psychrophile
Mesophile
Thermophile
Can grow with oxygen
Can grow without oxygen
Can only grow without oxygen
Can grow both with and without oxygen
Tolerates low pH
Tolerates high salt concentrations
Tolerates low humidity
Tolerates high pressure
Can grow at low nutrient concentrations
Atmospheric carbon dioxide as carbon source
Organic compounds as carbon source
Sunlight as energy source
Chemical compounds as energy source
Tolerates low temperatures
Tolerates moderate temperatures
Tolerates high temperatures

Types of fermentation reactors

Finally, we will look at how the fermentation process can best be carried out in practice. In industry, fermentation is carried out in large vessels. The precise way in which the growth medium is supplied can have a drastic effect on product yield. There are three ways in which these vessels can be operated.

Batch

A batch is the simplest operational mode of fermentation. This involves adding growth medium and a culture of microorganisms to a closed system and leaving it to take care of itself. After a period of time, the vessel can be opened and the product extracted from the fermentation liquid. Batch fermentation proceeds as follows:

The growth of biomass starts with the lag phase in which the culture acclimatizes to its new environment. This is followed by the exponential growth phase: The microorganisms begin to divide and growth is exponential. At some point, this growth flattens out and the organisms enter a stationary phase in which there is no further growth. This can be due to many things but often results from the limited substrate source being used up. When there are no more sugars left, the culture also stops growing. After some time in the stationary phase, the living cells die in the so-called death phase.

In batch fermentation, production depends on what metabolites are being produced and on whether they are primary or secondary metabolites. If it is a primary metabolite, it is produced at the same time as normal growth during the exponential growth phase. Secondary metabolites are often first produced in the stationary phase. This is due to the microorganisms becoming exposed to a stress factor, which inhibits normal growth. As a response, they therefore begin to produce secondary metabolites, which can possibly help them cope with the stress factor.
One explanation of this phenomenon is that a secondary metabolite can become involved in carbon catabolite repression. When there is a high concentration of a certain carbon source, a complex regulatory system comes into play. When there is no longer a high concentration, a number of genes become subject to regulation, such as upregulation of a secondary metabolite gene expression.

In a batch, growth can stop for many different reasons. These can typically be factors which do not conform with the microorganism’s required growth conditions; for example, the development of low pH, high temperature, or oxygen deficiency, to name a few.

One of the major challenges of the batch process in industry is that high concentrations of metabolic products can become toxic to the cells. For example, high ethanol concentrations can kill the yeast S. cerevisiae. Although this yeast is known to tolerate high concentrations of ethanol, there is nevertheless an upper limit. Therefore, a strong spirit can only be made using a distillation process.

Chemostat

A chemostat is an extended version of a batch in which the vessel is fitted with inward-flowing and outward-flowing hose. A problem with batch fermentation is that it stops as soon as the substrate runs out in the medium. A chemostat attempts to solve this problem by emptying the contents of the vessel at a certain point and refilling it with fresh growth medium. The inward and outward flows are the same and therefore the volume of the vessel contents remains unchanged. A chemostat is also referred to as a continual fermentation as there is a constant flow through the vessel.

The aim of the chemostat is to achieve a so-called steady state, which denotes an environment in which all concentrations of substrate, product and biomass are kept constant. This means that there is no accumulation of substrate, product or biomass in the vessel. At steady state, a chemostat can theoretically continue operating indefinitely.

Steady state is used in industry, but also has many applications in research into determining some of the different parameters, e.g., how much substrate is converted to product.

There are advantages and disadvantages in using a chemostat. One of the advantages is that one can maintain a constant low substrate concentration, which is often preferable. Moreover, a chemostat can continue operating indefinitely, yet it also means an increased risk of contamination. A chemostat is not a closed system, and therefore one needs to ensure that no undesirable microorganisms enter the process. It is also often the case that the microorganisms’ productivity falls off after a while as a result of spontaneous mutations.

Fed-batch

The third and final mode of operation in fermentation is a fed-batch. Fed-batch is often the solution which gives the largest product yield. As with a chemostat, a fed-batch attempts to solve the same problem with a batch, namely that at some point in time, the nutrients run out. Fresh medium is therefore introduced but, in contrast to a chemostat, there is no outflow of medium. Flow into the vessel rises exponentially in line with the exponential cell growth, and at the same time, it increases the volume in the vessel. The crucial factor is therefore the size of the vessel. When the vessel is full, one can either empty it completely or draw off just a certain amount to avoid having to start the process again