There are both disadvantages and advantages to both surface and submerged processes.
Surface processes are used in small plants whilst submerged processes are preferred for large-scale production.
Fermentation and Scaling-up.
The development of fermentation processes and the isolation of suitable strains of microorganisms to obtain a good yield of product takes many years. At the same time it is also necessary to develop cost-effective media, to measure any additives and their effects, to design and test the reactor and to investigate aeration, stirring, pH and growth temperature. This could lead to a fermentation system that may work well in the laboratory but that doesn’t mean it will work well on a larger scale. When tones of microorganisms are involved they may not behave the same way as when there is a few grams as it is a living organism. Solving these problems and moving from a laboratory system to a full-scale production is the next challenge. This is normally done in a series of carefully monitored stages, each stage being a ten-fold increase on the last one. Each time the optimum growth conditions are determined. In this way the final fermenter size is reached.
In these established industrial fermentations it is still necessary to move through these stages to help to ensure that there are enough actively growing organisms to put into the final fermentation vessel. This is known as Scaling-up and without this process the time taken for the number of organisms to increase so that they effectively ferment the material in the final vessel would make the fermentation uneconomic.
Selecting a strain.
Enzymes are only produced by living cells and microorganisms are used to produce enzymes on an industrial scale. The scope for detecting new and useful organisms is huge. E.g. 10’s of millions of microorganisms of many different types exist in a gram of soil. Less than 1% of the world’s microorganisms have been researched so there is much more scope for research. Strain selection is necessary to find the microorganisms that will produce an enzyme that will do exactly what is required. Strains must therefore not make humans ill; some could be genetically altered. Organisms that are selected to produce the enzymes that are used in the food industry have to comply with very strict rules and regulations. The responsibility of the final product’s safety lies with the product manufacturer.
Intracellular and Extracellular enzymes.
The enzymes are always produced inside the cells but many pass out through the cell walls into the growth medium. These are known as extracellular enzymes. Enzymes that stay within the cell are known as intracellular enzymes. Most of the enzymes that are used in industrial applications are extracellular enzymes produced by microorganisms during fermentation; the process can be either using a submerged or a surface culture.
Extracellular enzymes have 3 main advantages over intracellular ones: -
- the enzyme is already outside the cell so there is no need to break open the cell - often a difficult and expensive process;
2. Only a limited number of enzymes are secreted into the growth medium so isolating the desired enzyme is much easier;
- The extracellular enzymes are more robust and are therefore less likely to be broken down by heat or chemicals than the intracellular ones.
Isolated enzymes are usually more efficient in biotechnology than whole cells because the enzyme concentration is higher and no unwanted enzymes are there.
Aseptic technique guidelines.
- Sterile gloves, masks and hats must be worn at all times.
- All staff must wear a freshly laundered lab coat.
- Always work in a draught-free area, preferably with the cultures inside a fume cupboard.
- Ensure that benches are thoroughly washed with antiseptic before starting any aseptic work.
- Always flame the tops of bottles and culture vessels before transferring any culture.
- Use only sterile pipettes or flamed nichrome loops for transferring cultures.
- Allow flamed loops to cool before placing them into liquid cultures or onto agar gels.
- Make sure that any culture medium and agar plates are sterile before use.
- Never inoculate an unlabelled plate or culture bottle.
- Incubate all cultures in a sealed incubator.
Cell growth and enzyme production.
The microbial cells are encouraged into exponential growth in the starter vessel and seed vessel. This means that there is a rapid increase in the number of cells, so there are enough cells to go into the main fermentation vessel. In the industrial production of a protease, the microorganisms in the starter and seed vessels are given a rich nutrient medium with a lot of protein so that they can easily get the amino acids they need to grow.
Not much protease is produced because as soon as the enzyme leaves the cell it is in the presence of protein, which it breaks down into amino acids that the bacterium can absorb. This inhibits further production of protease. In the fermentation vessel, the bacterial cells are allowed to grow for a further 1-8 days. The microorganisms commonly used for enzyme production require a source of organic carbon and nitrogen, as well as minerals, water and oxygen. The medium in the fermenter vessel is usually a relatively cheap feedstuff e.g. potato flour, cornsteep liquor, or soy bean meal with added sugars and salts. It is important that the medium meets all of the basic nutritional needs of the microorganism that is being used. The nutrient medium does not have much protein in it.
Each bacterium produces a lot of protease because when the enzyme leaves the cell it does not immediately come into contact with protein that it can break down into amino acids for the bacterium to absorb – so each bacterium is producing more enzyme in order to maximize the harvest of amino acids from the small amount of protein that is available. If the organisms are given too little nutrients then they will not survive at all. The biotechnologists provide a medium that contains just sufficient nutrient to ensure that the microorganisms in the main fermenter produce the maximum amount of enzyme and continue to grow without increasing the number of cells. This stage of population growth is called the exponential growth phase and it is the production phase of the fermentation. It is also important to control the other growth factors e.g. oxygen level, pH and temperature are also monitored and controlled. Maximum enzyme production is achieved when each of the growth conditions is maintained at its optimum.
Enzyme production is more efficient today than 100 years ago as: -
- Genetic manipulation of microorganisms has improved yields;
- Low-cost nutrients and advanced monitoring have improved fermentation technology;
- New methods extract a greater proportion of the enzymes from the cell;
- Down stream processing techniques are more efficient;
- Continuous production in special reactors is more efficient than batch processes.
