Fermentation Equipment and its Application

  • Fermentation can be defined as a metabolic process in which raw materials such as sugar or carbohydrates are converted into acids, gases and alcohols. Micro-organisms such as yeast and bacteria play a central role in the fermentation process.
  • Fermentation process requires a fermentor, substrate, inoculum and optimum conditions.
  • The equipment used to carry out fermentation is called a fermentor.
  • A fermentor can be defined as an equipment or a vessel in which fermentation process is carried out.
  • A fermentor is a complex machine. It is used to create optimum conditions that are necessary for the fermentation process.

 Design of a fermentor

  1. A fermentor should be large enough to allow creation of fermentor bacteria.
  2. The material used in the manufacture of a fermentor should be strong enough to tolerate the pressures of fermentation media and gases produced.
  3. The material should be non-corrosive.
  4. It should not contribute toxic ions or substances to the fermentation media, because these toxic ions can harm the growth of microbial culture.
  5. It should have an inlet for oxygen and outlet for carbon dioxide in case aerobic microbes are used for fermentation.
  6. It should be completely sealed from outside environment to avoid contamination.
  7. It should be easily sterilize.
  8. It should provide a facility to add anti-foaming agents as well as a temperature controlling system.
  9. A pH detecting system is also important. An outlet for the withdrawal of media for determination of pH as well as inlet for addition of acids and alkalis should be provided. It is important to maintain pH at optimum levels for growth of culture.
  10. The most important thing is that a fermentor should provide aseptic means of withdrawal of fermented product and introduction of culture samples.
  11. The fermentor facilitate the stirring of media so that inoculum and fermentation media mix properly. It also ensures that the gases required for growth of microbial culture are available to all microbes.

Types of Fermentors and its Uses

1. Laboratory Scale fermentor

  • It is the smallest type of fermentor.
  • Capacity of this fermentor is minimum one to two litres and maximum up to twelve to fifteen litres.
  • It is used for research and development purposes.
  • Laboratory scale fermentor is used for determination of optimum condition of growth and biosynthesis of micro-organism.

2. Pilot scale fermentor

  • Pilot scale fermentor are of intermediate size.
  • It is used in large scale studies of fermentation process.
  • The minimum size range is of 25 to 100 gallons that is 94 litres to 378 litres.
  • The maximum size range is 2000 gallons that is 7570 litres.

3. Industrial scale fermentor

  • Industrial fermentor is a large scale fermentor. It is used in industries for fermentation of industrially important product.
  • The minimum size range is 5000 gallons to 10,000 gallons that is 18927.06 litres to 37854.12 litres.
  • The maximum size range 10,0000 gallons that is 378541.18.

This was the overview of fermentor and important aspects that are considered under fermentation process but there are important aspects that are carried out during fermentation process.

  1. Media formulation
  2. Inoculum preparation
  3. Sterilization of nutrient media and preparation of media.
  4. Fermentation process
  5. Recovery
  6. Purification
  7. Waste disposal

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Introduction to Industrial Microbiology

Industrial microbiology is a branch of science that deals with the study and uses of micro-organism which have industrial and economic applications. In industrial microbiology, microbes classified, characterized are isolated from their natural environment. Further, these micro-organism are produced on a large-scale by use of a fermentor.

Fermenting tanks with yeast being used to brew beer. Photo by Kafziel

Modern Fermenting Tanks
Photo by Kafziel

The most important applications of industrial microbiology are using microbes to convert inexpensive raw materials into economically important products. Further, these products go through detection, analysis, assay, purification and recovery to make them safe and effective. The end products are valuable, but the microbes creating these products are equally important.

During the process of industrial fermentation, microbial cells produce extra-cellular as well as intra-cellular enzymes. These enzymes play an important role in fermentation, degradation and utilization of media components. Enzymes can be used as a product by itself and have many end use applications. For example, enzymes like amylase and proteases are used in a variety of products.
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Growth of a Bacterial Cell

Growth of a Bacterial Cell

It can be defined as orderly increase in  quantity of all cell components of a cell.The cell increases in its size,shape and weight and further undergoes cell division.The bacterial cell undergoes different growth phases.This growth phases of bacterial cell can be studied by plotting a growth curve.

As we know studying growth pattern of an individual bacterial cell is impossible due to its small size so here we use population of a bacterial cell for determining the growth curve. The population of a bacterial cell is obtained by innoculating  a small number of innoculum from old culture to a suitable  fresh culture media and growth of a cell is monitored at regular interval of time.The growth curve is plotted by using logarithm of cell number against time.

A bacterial cell undergoes four different growth phases and they are as follows:-

  1. Lag phase
  2. Log phase
  3. Stationary phase
  4. Death phase

Graphical representation of a growth phases of bacterial cell.

Growth Phases of Bacterial population

Growth Phases of Bacterial population

Where,On X axis L is equal to logarithm of number of cell.

On Y Axis T is equal to time in hours

Growth phases of a bacterial cell

1.Lag phase

Initially when we innoculate a old bacterial cell to a new fresh medium the bacterial cell does not carry out immediate cell division  and the inoculated population remains steady.As the bacterial cell get exposed to a new fresh medium these cells try to adjust in the new environment like present nutrients,pH,temperature as well as on other hand this cell synthesize amino-acids,protein,RNA,enzyme and intermediate metabolic products which are further necessary for growth and cell division .

The time of lag phase may vary from species to species due to various environmental factor.At the end of lag phase the bacterial cells starts to multiply and cells enter in logarithmic phase of growth.

2. Logarithmic phase

This log phase is also called as exponential growth phase.In this growth phase of bacterial all cell multiply and carry out cell division.Cell division in this growth phase is constant and maximum.Here the number of cells increases exponentially with time.In this growth phase all bacterial cells are small in size and biologically active as they are constantly dividing so in most of the biochemical and biological studies bacterial cells from this exponential growth phase are used.

If we plot a graph of logarithm of cell number against time on X axis it result in a straight line with positive slope.The multiplication rate and generation time of bacterial cell varies from species to species under different environmental condition.At the end of log phase the growth rate decrease that is generation time of cell increases .After the end of log phase the bacterial cell enters in stationary phase.

3.Stationary phase

As the cells enter in stationary phase the number of cells remain constant because generation time of cell is very low.It occurs due to various factors like depletion of nutrients, accumulation of toxic waste products,change in pH etc. Due to all this factors in this phase the growth rate is equal to death rate and the total number of cells till the end of stationary phase remains constant.

If we plot a graph of log of cell number against time a straight line parallel to X axis is formed with the end of this phase the death phase starts.

4. Death phase

In death phase of growth the bacterial cells starts to die.Here death rate is higher than the growth rate and the death rate increases to maximum at the end of death phase. There are various factors responsible for this phase like exhaustion of nutrients,accumulation of toxic waste products.drastic changes in pH etc.

If we plot a graph of log of number of cells against time on X axis a straight line with negative slope will be observed.Here with the death phase the bacterial growth curve end.

These are the growth phases of a bacterial cell.


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Nitrate Reduction Test

Nitrate is a poly-atomic inorganic molecule and its molecular formula is NO3–  

Nitrate is reduced by micro-organism in two ways.

1. Assimilatory Nitrate reduction.

Assimilatory nitrate reduction mainly occurs during aerobic condition in absence of nitrogen.In this reduction process nitrate and ammonia are used for synthesis of organic nitro-compounds and amino-acids.In assimilatory nitrate reduction the nitrate do not play any role in energy production.

2. Dissimilatory nitrate reduction.

Dissimilatory nitrate reduction is carried out under anaerobic condition where bacteria use nitrate as a electron acceptor.

These assimilatory and dissimilatory nitrate reduction process is carried out nitrate reductase enzyme.This nitrate reductase enzyme converts nitrate to nitrite .The reaction is as follows

NO3 + NADPH + H+       Nitrate reductase           NO2–  + NADP+ + H2O           

Further this nitrite is converted to ammonia by nitrite reductase and the ammonia formed is assimilated.The nitrate reductase test is performed by using two indicators and the reduction of nitrate to nitrite is detected by using two indicators and that are sulphanilic acid and α- napthylamine.

Aim 

Aim of the experiment is to carry out nitrate reduction test .

Requirement

  1. Peptone nitrate broth
  2. Test culture.
  3. Reagent A-Sulphanilic acid.
  4. Reagent B- α- napthylamine.
  5. Zinc dust.

Composition of Peptone nitrate broth

  • Peptone            -0.5 gm
  • KNO3                 -0.2 gm
  • Distill water    -100 ml
  • pH                       -7

Principle

In nature many micro-organism posses nitrate reductase enzyme  when this micro-organisms are exposed to nitrate as a sole source of energy have a ability to reduce nitrate to nitrite.Here in nitrate reductase test we expose these micro-organism to a media containing nitrate as a sole source of nitrogen and energy and further this nitrite is detected by using two reagents Sulphanilic acid and α- napthylamine.

Procedure

  1.  Take sterile peptone nitrate broth and  innoculated it with loopful of  test culture  and this innoculated broth is incubated for about 24 hours at about 37 ° C .
  2. After incubation 0.5 ml of reagent A that is  Sulphanilic acid is added and then reagent B α- napthylamine is added observe the test tube for development of red colour.
  3. Further if no red colour develops a pinch of zinc dust is added and mix properly and test tube is observed for development of red colour.

Flow chart of Nitrate reduction test procedure

 

Reagent A is Sulphanilic acid and Reagent B alpha napthylamine

Reagent A is Sulphanilic acid and Reagent B alpha napthylamine

 

Conclusion

  • If development of red colour is observed after addition of reagents then it indicates positive nitrate reduction test.
  • If after addition of both reagents there is no red colour formation then it may indicate negative nitrate reduction test or false result  because there is a possibility that the microflora present in the tube may reduce the nitrate to other byproducts like ammonia,molecular nitrogen,nitrous oxide,nitric oxide and as the test reagents detects only nitrates so it can give a false negative result so to clear this doubt a pinch of zinc dust is added and tube is observed for red colour development .Here zinc ions reduce nitrates to nitrites and we can observe development of red colour as zinc ions confirms presence of residual nitrates and this development of red colour indicates negative nitrate reduction test.

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