Submerged fermentation is a type of fermentation in which the microorganisms are suspended in a liquid medium. The liquid medium also contains various other nutrients and growth factors in the necessary proportions in a dissolved or a particulate solids form.
The main application of submerged fermentation technique is in the extraction of metabolites (secondary metabolites) which are needed to be in liquid form for use.
APPLICATIONS OF SUBMERGED FERMENTATION
The primary application of submerged fermentation is in the extraction process of metabolites (mostly secondary metabolites) that find applications in their liquid form.
Citric acid is one of the most important metabolites as the production volume of it is high, for the production of antibiotics like penicillin.
Submerged liquid fermentations are traditionally used for the production of microbially derived enzymes like cellulolytic enzymes.
CITRIC ACID PRODUCTION
Citric acid is widely distributed in plant and animal tissues.
It is an intermediate of the Krebb’s cycle, by which carbohydrate gets converted to CO2, in nature.
Citric acid can be produced on the industrial scale by employing submerged state fermentation as the fermentation method.
Type of bioreactor used for submerged fermentation:
Stirred tank bioreactor
Selection of strain and storage:
Various criteria should be checked for the selection of production strains such as:
-High citric acid yield.
-Stability of the strain.
-Adequate amount of sporulation, etc.
Microorganisms used for the production of citric acid:
-Species of PenicilliumandAspergillus.
–Aspergillus niger is used as the principal fungus for citric acid production as it can produce large quantities of citric acid while growing on a carbohydrate medium.
Maintenance of the culture of the selected strain is the next important step in citric acid production and is done so by the storage of spores.
Steps used to carry out fermentation to ensure abundant production:
-High sugar concentration.
-Limited nitrogen/phosphorus concentration.
-Very low concentration of heavy metals like iron and manganese.
Submerged fermentation process:
-The strain used for the submerged fermentation of citric acid is Aspergillus japonicus.
-The organism shows sub-surface growth.
-Citric acid is produced within the culture solution.
-Using submerged fermentation for the production of citric acid is economical as compared to other fermentation methods.
Uses of citric acid:
It Is extensively used in the production of carbonated drinks.
It is used in plasticizers.
It is used as a chelating and sequestering agent.
Used in the pharmaceutical and food industries as an acidulant.
The advantages of submerged fermentation include:
The duration of the process is short, therefore saves time.
The overall cost of the process is low and the yield of products is high, making it a very economical process.
The process of purification and processing of the products is far simpler compared to other processes.
The cost of handling is low and the handling of the fermenter is easy therefore it reduces the labour involved.
The overall volumetric productivity of this process is low.
The effluent that is generated during the process is high in quantity.
The equipment that is used is expensive and complex.
The products that are obtained by using this process may be of low concentration.
-Submerged fermentation is a type of fermentation in which the microorganisms are suspended in a liquid medium. The liquid medium also contains various other nutrients and growth factors in the necessary proportions in a dissolved or a particulate solids form.
-Submerged fermentation is a technique in which the overall moisture content of the process is high. Therefore, it is better suited for bacteria or other microorganisms that require high moisture contents for growth.
-It is a very widely used technique for many reasons, one prominent one being that the overall purification step is much easier compared to other techniques.
-The main application of submerged fermentation technique is in the extraction of metabolites (secondary metabolites) which are needed to be in liquid form for use.
Figure 1 Overview of the process of submerged fermentation
PRINCIPLE OF SUBMERGED FERMENTATION:
-In submerged fermentation, the growth/development of the desired microorganisms occurs in the liquid environment.
-The primary substrates that are used in this technique are molasses and broth.
-The composition of the broth used is such that the proportion of the broth and the nutrients is such that the production of antibiotics, industrial enzymes etc. is optimum.
-In submerged fermentation, the rate of utilisation of the substrates is high. Therefore, the rate of depletion of them is high. For this reason, the nutrients need to be constantly replenished.
-A specific microorganism is used as the starter culture for this process. This starter organism may be fungi, bacteria or any other suitable organism. A nutrient rich broth is taken in a flask and this starter culture is then inoculated in it to begin the process.
-This technique demands high oxygen levels as the enzymes and other products are produced when microorganisms responsible for production react sufficiently with the broth and the nutrients and break them down to produce the desired products. This process requires oxygen and it is therefore an important aspect of the process.
-In the process, the compounds that are bioactive need to be secreted into the reactant broth/medium.
METHODS OF CARRYING OUT SUBMERGED FERMENTATION:
The primary two types of techniques that are used in submerged fermentation are:
Fed Batch fermentation, and
These are discussed below:
FED BATCH FERMENATION:
In batch-fed fermentation sterilized growth nutrients are added to the culture. Fed batch fermentation is widely used in bio-industries as it helps in the increase of cell densities in the bioreactors. In these processes, the broth is usually highly concentrated to prevent or stop dilution from occurring. To maintain the culture growth rates, the nutrients are added as and when needed. Doing so, promotes the reduction of the risk of overflow metabolism.
Parameters of fed-batch fermenters:
Size- small lab scale fermenters: 1-2 L to 15 L
pilot scale fermenters: 25-100 G to2000 G
large fermenters: 5000 G to 5,00,000 G
Working volume – less than total volume as head space is left to allow to allow aeration, splashing, foaming.
Ph control – This is done by the addition of acid /alkali.
Temperature control – Heating/cooling coils are used for the temperature control inside the bioreactor. In these devices, a ‘heat transfer fluid’ is passed through the coils or the jackets of the devices which help maintain the heat equilibrium.
Agitation: – Impellor: The agitator is mounted on a central drive shaft. Impeller blades are mounted on the shaft. The blades that are used usually cover two thirds of the total diameter of the vessel.
Most batch reactors also use baffles. Baffles are immobile blades. These work by breaking up/promoting the dissipation of the flow with the help of a agitator that rotates. They are usually fixed on the inside wall of the vessel.
Aeration – Aeration is done with the help of a sparger.
–Principal modes of injecting air:
Impeller air injection—air is fed to impeller by hollow drive shaft and then injected into the medium through holes in impeller.
Two phase injection— mixture of air and nutrient medium fed in foam or suspension form
Sparger air injection– air fed by sparger orifices
Advantages: -Initial capital expenditure is lower
-It is simple and feasible to remove contamination, if any occurs during the process,
Disadvantages: – It is less effective for the production of biomass and primary (growth-associated) metabolic products.
-Batch-to-batch variability of the product
-Increased non-productive down-time, involving cleaning, sterilizing, refilling and post sterilization cooling.
-The probes and the instruments may tend to get damaged due to repeated, periodic sterilization processes.
Continuous fermentation: An open system is constructed for continuous fermentation. In continuous fermentation, the rate of utilization of the nutrients by the microorganisms is equal to the rate of input of the externally supplied nutrients and growth factors. Due to this continuous process, a steady-rate of production is achieved.
Working mechanism: -Continuous addition of fresh fermentation medium occurs with constant stirring and agitation.
-Constant volume is maintained by incorporating an airflow weir.
-The rate of removal of broth or the spent fermentation broth is equal to the rate of addition of the fresh medium during the utilization of broth via the microorganisms present.
-There comes a stage then, where the rate at which the microbial cells grow is equal or proportionately equal to the rate at which the cells are displaced.
-The primary variables that need to be maintained to ensure the optimal production of substances using this technique include temperature, pH and gas levels (like oxygen and carbon dioxide).
The examples of the substrates used in submerged fermentation are:
Fermentation is a process of metabolism that produces chemical changes within organic substrates with the assistance of enzyme action. Microorganisms like lactic acid bacteria or yeasts are important for many kinds of fermentation as they produce the necessary enzymes required for the process. (These enzymes include proteinase, amylase, cellulase, etc.)
In the fermentation process, these microorganisms convert carbohydrates in the food like sugars and starches to acids and alcohols, which enhance the flavour and texture of foods, and also acts as natural preservatives. Many different day to day foods and drinks that we consume are fermented products. This includes beer, wine, cheese, amongst others. Fermentation can also be done at home for various foods like kimchi, curd, yogurt, kombucha, etc.
Benefits of Fermentation
Fermentation is and was one of the best ways to preserve fermentable foods, before pasteurization and refrigeration was discovered. It is a simple, easy, convenient method which extends the life of produce and dairy products, without needing added stabilizers and preservatives.
A range of complex flavours can be unlocked by fermenting foods. Bland and undesirable food can transform into salty, sour, tangy or sweet. Many of the largest contributors to the flavour of the food industry are fermented products including vinegar, cheese and wine.
We have a natural flora of microorganisms in our gut which consists of “good bacteria” which contributes to maintaining a good balance in our digestive and immune systems. It also protects us from preventing growth of other harmful microorganisms. This may deteriorate by excess use of antibiotics or consumption of processed foods. Fermented foods are rich in good bacteria and help maintain healthy, balanced gut flora, thus strengthening the immune system.
TYPES OF FERMENTS
Different types of ferments are categorized on the basis of types of starter culture and microorganisms used.
Generally, bacterial strains are of beneficial bacteria and they are also present in the desired food to be fermented.
Yogurt: Lactobacillus bulgaria and Streptococcus thermophilus are used in the fermentation of yogurt. It consumes lactose, milk sugar, in milk to form lactic acid.
Sauerkraut: This fermented cabbage uses Lactobacillus strain of bacteria.
Yeast ferments are very common and are used for various foods. Yeasts ferment on the naturally occurring sugars in the food to form alcohols.
Wine: This alcoholic drink is formed by using sweet sugary fruits. The most common is grapes. Yeasts digest the sugars and form alcohol. When air is allowed, wine can be converted to vinegar by the formation of acetic acid.
Beer: When malted grains like barley and wheat are fermented by yeasts, which consume the sugars present, to form alcohol, beer is formed. Ginger beer is made when a fermented starter is made from fresh ginger and wild yeast, which are naturally present in the ginger, in the presence of air.
Sometimes, bacterial strains and yeast strains are used together in symbiosis for the fermentation of particular foods. Most starters need to be maintained and shared. Examples include kefir, kombucha, sourdough bread, Tha bai, etc.
The science of fermentation is known as zymology. Microorganisms play an important role in the fermentation technology. Through their actions the fermentation process transforms and preserves food. Thousands of years ago, people have started fermenting food. Now we have other techniques to preserve food still the fermentation process persists. People are interested in fermented food due to its interesting flavours, their vitamins and enzymes, or organic acids and “good bacteria”.
BACKGROUND OF FERMENTATION TECHNOLOGY:
Fermentation comes under the umbrella of industrial microbiology (it’ the interdisciplinary study that uses microbes, to produce commercial products or carry out important chemical transformations usually grown on large scale). So fermentation can be carried out both in small scale and large scale production.
Now the microbial biotechnology joined with the industrial microbiology which leads to many fold increase of products and also to produce New products, by means of genetic manipulations of these microorganisms which are originally not produced by these microorganisms.
Fermentation refers to any large scale process in which the commercial products like vitamins, enzymes, vaccines, organic acids are produced from the raw materials by using different microorganisms either aerobically or anaerobically.
In microorganisms, fermentation is the aerobic degradation of organic nutrients to produce adenosine triphosphate (ATP) for their growth.
The product that is produced during fermentation(or microbial growth) was of two groups.
Primary metabolites :It’s the product which is formed during the active growth stage (log phase) of the microorganism.
Ex. Alcohol produced by yeast. By the action of primary metabolic pathway of yeast (EMP pathway), the alcohol is produced and will be accumulated during the active growth stage of yeast.
Secondary metabolites :These are the products which are formed either at the end of active growth stage or at the beginning of the stationary phase are referred to as secondary metabolites.
Example: Antibiotics are usually produced in the stationary phase. These are formed by the secondary metabolic pathways That is not essential for the growth of microorganisms. Few organisms show this characteristic.
TYPES OF FERMENTATION :
There are various types of fermentation process based on its characteristics.
Based on the state, the fermentation process can be classified into two.
Submerged (liquid) fermentation :Fermentation is carried out in liquid forms.
Ex. From sugarcane industrial waste alcohol can be produced- Molasses by yeast.
Solid fermentation :With about 30-50% moisture level the Fermentation is carried out in solid condition.
Example: Gibberellic acid produced from grains by fungus Fusarium moniliformi
Based on the microbial growth (culturing method):
Batch fermentation :It is a closed system of microbial fermentation in which the volume is already fixed.
Fed batch fermentation :As the fermentation process is continuing the substrate for fermentation (raw material) is added in increments.
Continuous fermentation :It is an opened system of microbial fermentation in which the input (nutrients solutions or media) is added continuously with the withdrawal of equal portion of converted or fermented solution.
LARGE -SCALE FERMENTATIONS:
Fermentation process is carried out in a closed vessel which is called as Fermentor. The volume of the Fermentor may varies from 5-10 lit to 500,000 lit. Similarly the Fermentor model will vary for both aerobic and anaerobic process.
DESIGN AND PARTS OF FERMENTOR :
There are various types of Fermentor. But the basic structure of Fermentor is as follows:
The components are:
Inlet :to add the the substrates for microorganisms etc
Outlet :to collect the products and check the samples
Stirrer :to mix the contents inside the Fermentor
Air sparger :by releasing small bubbles into the Fermentor to provide oxygen to the microorganisms
pH meter : to check the pH and maintaining by adding acid or alkali
Thermometer : to check the temperature
Outer jacket :for cooling process ( because during the Fermentation, lot of heat will be generated)
Sterilization unit :stem is supplied to sterilize the medium and container
Scale up : it refers to sequence wise step in any Fermentation to yield Maximum product
Scale up of fermentation
INDUSTRIAL APPLICATION OF FERMENTATION PROCESS :
Presently, “ Fermentation technology” is replaced by the word “Bioprocess technology” . The commercial products formed by the fermentation technology is very useful for the industrial purposes.
The following are the commercial products obtained through the fermentation process by different microorganisms.
Most of the antibiotics can be produced by microbial fermentations. Sometimes, the antibiotic needs to be modified through some chemical processes, referred as semi- synthetic antibiotics. Here are some antibiotics and the organisms involved in its production.
Vitamin B12 and Riboflavin are vitamins which is commercially produced by pseudomonas denitrificans and Ashbya gossipii respectively.
L-glutamate, L- aspartate, glycine, cysteine, tryptophan etc are some common amino acids commercially produced by microbes.
Presently, these amino Acids can be produced by genetically modified bacteria.
For conversion of one compound to another product the microorganisms act as bio-catalyst in bio conversion process.
Ex. Most of the steroids are produced by this process
Enzymes can also be produced by fermentation process which is mostly used in food industry and molecular biology.
The following table shows the enzymes and the involved organisms
Bacillus licheniformis, Aspergillus
Acetic acid, citric acid, acetone, butanol etc are produced by microorganisms.
Single cell protein
By using fermentation technology protein is also produced which can be used as food referred to as Single cell proteins (SCP)
Example: The yeast, chlorella, spirulina etc .
Alcohols and alcoholic beverages
The yeast play a major role in the alcohol industry. As we know it is a facultative aerobe it can be grown aerobically and anaerobically. When, yeast grows aerobically its biomass increased with low alcohol, used for the production of baker yeast while if it grows anaerobically, the alcohol production will be Maximum.
Example: Whiskey, rum, otka etc are the commercial preparations of alcohol.
The microorganisms itself can be used as food. Following are some of the foods obtained from microbes.
Metabolism is the collection of chemical reactions takes place to sustain life of an organism.
The main purposes of metabolism is to convert food to energy to run cellular activity; to convert food to building blocks for nucleic acids, lipids, protein and carbohydrates; and to remove metabolic waste.
The metabolic reactions which are enzyme catalysed are responsible for the growth and reproduction of organism, to maintain their structure and interact with environment.
Metabolic reaction is of two types one is catabolic reaction, means the breaking down of compounds and another is anabolic reaction- the building up of the compounds. The catabolic reaction liberates energy and the anabolic reaction uses energy.
Metabolic pathways include the steps through which one chemical is transformed into another and each step is facilitating by an enzyme.
Enzymes are the key component of the metabolic reaction; they act as catalyst- allows the reaction to proceed more rapidly.
Metabolic reaction is of two types:
In catabolism the compound through the set of chemical reaction is broken down into simpler compound or molecules.
This is achieved by breaking down and oxidizing food molecules.
Catabolism is responsible to provide energy for working of the cell and component needed for the anabolic processes which build molecules.
The nature of these catabolic reactions based on the source of energy and carbon which is differ from organism to organism.
The chief metabolic processes in a cell are:
Tricarboxylic acid cycle
Anabolism is the set of constructive reactions which used energy released by the catabolic pathway to synthesize complex molecules.
The complex molecule construct cellular structure step by step, make up from small and simple precursor.
The biomolecules are necessary for the growth and reproduction, some biomolecule serve as the central metabolic intermediates.
Some organisms can synthesis all the necessary organic compound like autotrophs. They can be grown on simple media. On the other hand, the organisms which cannot synthesize organic compounds from atmosphere are known as fastidious organisms.
Following anabolic process takes place in organism:
Synthesis of glucose, lipids, amino acid and protein, nucleic acids
Synthesis of other growth factors like vitamins, hormones etc.
In the glycolysis process glucose and other sugar are partially oxidized to the smaller molecule i.e. pyruvate
Embden-Myerhof pathway, pentose phosphate pathway and Entner-Doudroff pathway are the three routes for the conversion of sugar into pyruvate.
It is anaerobic process in which organism obtain energy in the absence of oxygen, also called anerobic fermentation.
Tricarboxylic acid pathway:
Given by H. A. kerbs in 1973
Also known as citric acid cycle. Because citric acid is the first product of the kerb cycle which is as known as TCA cycle as the citric acid has three carboxylic group.
It is anaplerotic reaction which means one product of a cycle is taken up by the other cycle
Oxaloacetate is taken from TCA cycle and used for carbon source from the amino acid synthesis.
Pentose phosphate pathway:
It is an alternative pathway for the sugar degradation.
Its main function is to generate power in the form of NADH in extramitochondrial cytoplasm and the second function is to convert hexoses into pentose for the synthesis of the nucleic acids. The third function is complete degradation of pentose.