Citric acid production and applications of submerged fermentation

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.


  • 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 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: 

  1. Stirred tank bioreactor
  2. Airlift fermenter.

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 Penicillium and Aspergillus.

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.

Article by– Shaily Sharma (MSIWM041)




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


-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.


The primary two types of techniques that are used in submerged fermentation are:

  • Fed Batch fermentation, and
  • Continuous fermentation

These are discussed below:

  • 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.Image result for fed batch fermentation diagram
  • 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.C:\Users\Shaily\AppData\Local\Microsoft\Windows\INetCache\Content.MSO\5358C9E5.tmp

-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:
  1. Liquid media
  2. Fruit and vegetable juices
  3. Sewage and wastewater
  4. Sugars/ molasses etc.