BY- Shaily Sharma (MSIWM041)
-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
- Continuous fermentation
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:
- 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:
- Liquid media
- Fruit and vegetable juices
- Sewage and wastewater
- Sugars/ molasses etc.