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.




Chromatography is a separation and purification technique which is used for the separation of solutes in a mixture, biomolecules etc. on the basis of distribution of the sample to be separated between stationary phase (phase which is not mobile and is usually mounted against a support like a chromatographic column) and the mobile phase (which is continuous/is poured or passed over the stationary phase)

Ion Exchange Chromatography is a method used for the separation as well as purification of ionically charged biomolecules like proteins, polynucleotides, nucleic acids etc. 

This technique finds a wide array of applications in the scientific world because of its simplicity and high resolution. 


The process of ion exchange can be defined as the reversible exchange of the ions present in a solution, with the ions electrostatically bound to the inert support medium.

The main factor which governs the process of ion exchange chromatography is the electrostatic force of attraction present between the ions. This electrostatic force of the ions depends on their relative charge, radius of the hydrated ions and the degree of non-bonding


Usually, ion exchange separations are carried out in columns packed with an ion-exchanger. The ion-exchanger is a support medium which is inert and insoluble. The medium may be capable of covalently binding to positive (anion exchanger) or negative (cation exchanger) functional groups. The ions which do bind to the exchanger electrostatically are called counterions. 

The conditions of separation can be manipulated in such a way that some compounds are electrostatically bound to the ion exchanger while some are not, therefore, helping in the separation of the desired compound.

The sample which contains the sample to be separated is allowed to percolate through the exchanger for a certain amount of time that will be sufficient for the equilibrium of the ions to be achieved. 

E–   Y  E–   X+ + Y+

In the equation mentioned above, 

  • E–  : Charged cation exchanger.
  • Y+ : Counterion of the opposite charge associated with the exchanger matrix.
  • X+ : Charged molecule bearing a charge similar to the counterion to be separated. This molecule is capable of exchanging sites with the counterion as shown above.

Once the exchange of the counterion with the sample has been achieved, the rest of the uncharged and like charged species is washed out of the column.

The ions that did bind can then be eluted out by either percolating the medium with increasing concentration of Y+ (works by increasing the possibility that the Y+ will replace the X+ in the above-mentioned equation due to it being present in a higher concentration). The elution can also be carried out by increasing the pH of the solvent and hence converting X+ to an uncharged species.

Simply put, once the sample containing the specific ions to be separated it passed through the ion exchanger column, the sample ions (which act as counterions to the ions of the exchanger column) bind with the ions on the exchanger column and form associations. However, the ions of the same charge as the exchanger column, present in the sample solution, repel each other and, therefore, do not bind and pass through the column.

The principles which have been mentioned above also apply to other macromolecules such as proteins and nucleic acids which are capable of showing the presence of both positive and negative ions. The type of molecules can bind to both anionic and cationic exchangers. 


The two main types of materials used to prepare ion exchange resins are:

  1. Polystyrene, and
  2. Cellulose

Polystyrene resins are prepared by the polymerization reaction of styrene and divinyl benzene. These resins are very useful for separating compounds with a small molecular weight. 

Cellulose based resins have a much greater permeability to macromolecular polyelectrolytes as compared to polystyrene resins and they also possess a much lowers charge density. 

Based on the type of charge carried by these ion exchangers and their strength, the ion exchange resins can broadly be classified into four types:

Strong cationic exchange resins:Weak cationic exchange resins:
Sulphonated polystyreneSulphopropyl cellulose Condensed acrylic acidCarboxymethylcellulose
Strong anionic exchange resins:Weak anionic exchange resins:
Polystyrene with -CH2NMe3ClDiethyl (2 hydroxypropyl)quaternary amino celluloseDiethylaminoethyl celluloseDiethylaminoethyl agarose

The buffer is that component of the chromatographic column that helps in the maintenance of the pH. The choice of these buffers is usually dictated by the compounds to be separated and whether the ion exchange is anionic or cationic.

  • Anion exchange chromatography should be carried out with cationic buffers.
  • Similarly, cation exchange chromatography should mostly be carried out with only anionic buffers for satisfactory separation and results. 

      Examples of some buffers used in this technique are:

  • Ammonium acetate
  • Ammonium formate
  • Pyridinium formate
  • Ammonium carbonate etc.
  1. The most significant application of ion exchange chromatography is in amino acid analysis.
  2. This technique is used to determine the base composition of nucleic acids.
  3. Ion exchange chromatography is used as a method of purification of water. Water is completely deionized using this technique.
  4. This technique is used for the ultra-purification of metal ion free reagents.
  5. It can also be used for the separation of a varied number of vitamins, biological amines, organic acids as well as bases. 


Biophysical Chemistry principles and techniques – Upadhyay and Nath


BY: Reddy Sailaja M (MSIWM030)

Microorganisms are minute creatures from time immemorial and omnipresent in all kinds of ecosystems on earth. Microorganisms are of different types: Bacteria, Fungi, Protozoa, Algae and Virus. These are not visible through naked eye and require microscope for their structural and functional evaluation.

Industrial microbiology deals with the application of various microorganisms for industrial processes that are beneficial to mankind.

Characteristics of industrially important microorganisms include:

  • Non pathogenic and non-contagious
  • Easy and rapid growth on industrial scale
  • Need of inexpensive medium for culture and growth
  • Production of spores
  • Easy inoculation
  • Desired product should be produced rapidly on large scale
  • capability to genetic manipulation, if required

Major industrial products produced by microorganisms:

  • Pharmaceutical drugs
  • Vaccines
  • Organic acids and solvents
  • Steroids
  • Dairy products
  • Enzymes
  • Beverages
  • Antibiotics
  • Amino acids
  • Vitamins

Figure 1: Major applications of microorganisms


Yeast species was widely used from thousands of years to produce beverages like wine, brandy, whiskey etc. Yeasts fall under kingdom Fungi and are eukaryotic, single celled organisms. Yeasts species, Saccharomyces cerevisiae  was allowed to grow on malted cereals and fruit juices to produce ethyl alcohol.

Beyond yeast, bacteria – Acetobacter, Lactobacsp. B. bucheri, etc and fungi – Pichia fermentans, Cyberlindnera mrakii etc are usually used in wine production


Discovery of Penicillin, an antibiotic by Alexander Fleming in 1928 is a significant step in medical microbiology history in the 20th century. Antibiotic is a bioactive substance produced by a microorganism that has the ability to either inhibit the growth or kill the other microorganism. Fungi are the main source of antibiotics.

Fungi of Actinomycetes group produce antibiotics like tetracyclin, streptomycin, actinomycin D etc. While, antibiotics like Penicillin, Cephalosporin etc are produced by filamentous fungi.

Organic acids and solvents:

Organic acids and solvents are produced from the microorganisms mainly for the pharmaceutical and other industrial needs. These compounds can be produced either from glucose or in the form of end products from pyruvate or ethanol.

Microorganisms that produce organic acids include:

Aspergillus niger – Citric acid

Acetobacter aceti – Acetic acid

Lactobacillus – Lactic acid

Salmonella – Formic acid

Escherichia coli – Butric acid and malic acid

Acetobacter xylinum – Ascorbic acid


Enzymes, also called biological catalysts occur naturally in the living system and regulate biochemical reactions. As enzymes have wide applicability in both medical and non-medical fields, microorganisms are genetically modified to produce industrially important enzymes. Amylase was the first industrial enzyme to be produced in the year 1896. Amylase has the ability to regulate indigestion and other digestive system related disorders.

Enzymes are widely used in food preservation, food processing, leather and paper industries, detergents and scientific research and development sectors like molecular biology.

MicroorganismSubstrate for growthEnzyme producedApplications
Aspergillus niger, Penicillium sp.PectinPectinaseAlcohol production, clarification of fruit juices
Saccharomyces diastaticusStarchAmylaseAlcohol production, starch removal glucose syrups production
Bacillus sp.ProteinsProteaseBread, baked foods, waffles production
Aspergillus sp.LipidsLipaseFood and aroma enhancement, biofuel degradation
Cellulomonas sp.CelluloseCellulaseAlcohol and glucose production
Streptomyces sp.XylanXylanasePaper production, biofuel production
Actinomyces, Streptomyces sp.ChitinChitinaseFood additive, therapeutic agent, antifungal and antitumor

Table 1: Enzymatic application of microorganisms

Amino acids:

Amino acids are building blocks of proteins and have high demand as supplements in food and nutraceutical industries. These are also used as supplements in bakery and packed foods. Microorganisms utilize amino acids for their metabolism and growth. Microorganisms are stimulated to produce extra amino acids, such that the extra amino acids are excreted into the surrounding medium.

Lysin and glutamic acid are the prime amino acid supplements in the preparation of bread and other nutritional supplements. Glutamic acid in the form of Monosodium Glutamate is used as flavor enhancing compound in the packed foods.

Glutamic acid is being produced from Corynebacterium gluatmicum. Corynebacterium sp. also used in:

  • Steroid conversion – an important process in the development of pharmaceutical products.
  • Hydrocarbon degradation – breakdown of plastic and oils for environmental protection.

Other amino acid producing bacteria include:

L-alanine – Corynebacterium dismutans, Pseudomonas dacunhae, Escherichia coli

L-arginine – Serratia marcescens, Bacillus subtilis

L-aspartic acid – Escherichia coli


Vitamins are critical for vital functions and a healthy life. As the human body unable to synthesize these compounds, it is necessary to be supplied in the diet in small amounts. Vegetables and meat are sources of vitamins.

Microorganisms are grown in bulk quantities for the commercial production of vitamins like – thiamine, riboflavin, folic acid, vitamin B12, ascorbic acid, beta-carotene etc.

Some microorganisms that produce vitamins include:

Beta-carotene – Blakeslea trispora, Phycomyces blakesleeanus

Riboflavin – Mycocandida riboflavin, Candida flareri, Clostridium buytilicum

Vitamin B12 – Pseudomonas denitrificans, Bacillus megaterium, Propionibacterium freudenreichii.

Pharmaceutical drugs:

Trichoderma polysporum produces ‘Cyclosporin A’ which is used as immunosuppressive agent during organ transplantation. Moscus purpureus produces ‘Statins’ that has the ability to reduce blood cholesterol levels.

Single cell proteins:

Microorganisms that are rich in protein content can be used as protein supplements for human and domestic applications and are called single cell protein (SCP).

Algal species, Spirulina is the most popular SCP being produced commercially as a protein supplement.

Other SCP producing microorganisms include:

Bacteria – Pseudomonas fluorescens, Lactobacillus, B.megaterium

Algae – Chlorella pyronoidosa, Chondrus crispus

Fungi – Aspergillus fumigates, A. niger, Rhizopus cyclopium

Yeast – S.cerevisae, C.tropicalis, C.utilis

Apart from the above applications, microorganisms have major applications in vaccine production, biofuel production and in treatment of malnutrition.


BY- Ria Fazulbhoy (MSIWM031)


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.


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.

  1. Yogurt: Lactobacillus bulgaria and Streptococcus thermophilus are used in the fermentation of yogurt. It consumes lactose, milk sugar, in milk to form lactic acid.
  1. 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.

  1. 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.
  1. 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. 




Biochip development began with the first work of sensory technology. An American company called Affymetrix developed the first biochip, called Gene chip, which contains a large number of DNA sensors used to detect errors. Biochip is a small laboratory version, which uses more than hundreds of simultaneous chemical reactions. They are specially designed to work in the natural environment, especially within living organisms. It is not an electronic device. Biochips contain millions of biosensors, which act as a micro reactor used to detect certain analytics such as enzymes, proteins, biological molecules, and antibodies.

Working of the Biochip

Biochip has different processes such as DNA, RNA, protein fragments, etc., represented by a point on a chip. These probes bind the existing targets in the sample to be tested. Due to hybridization a link between the investigations and their purpose is made. Biochip scanners and microarray image analysis software and used for target identification and signal testing. The results are calculated at the mathematical level and interpreted into the biological context.

 Components in a Biochip:

Biochip has two components which includes a transponder and a student.


Biochips contain an inactive transponder which means that these transponders require a small amount of electricity to operate. The transponder contains the following four components.

  • Antenna Coil – It is the smallest base used to send and receive signals from a scanner.
  • Computer microchip –It maintains a unique identification number ranging from 10-15 digits.
  • Tuning capacitor –It is charged with a very small signal sent by the operator.
  • Glass capsule – It is made from materials that are incompatible with soda-lime glass. It is used to hold an antenna coil, capacitor and microchip.


It contains a coil called exciter used to create an electromagnetic field (emf) with the help of radio signals. It provides the power needed to start the chip. The receiving coil is there to receive the shipping code backed from the excited chip inserted.

 Three types of Biochips are available: –

DNA Microarray

It contains a large number of tiny DNA dots that are fixed on a solid surface. It is used to calculate speech levels with a large number of genes. Each DNA tag contains probes which is Pico moles of some kind. Typically, a probe-target hybridization is detected and calculated by the detection of a fluorophore which refers to the fluorescent chemical compound that can emit light over a luminous spectrum. It is set to determine the equal amount of nucleic acid series in a target. The new macromolecule arrangement was a macro range about nine X-12 inches and the first machine which is a based animation analysis was unveiled in 1981.

Microfluidic Chip

They are the site of a chemical laboratory. They are used for a wide range of responses such as DNA analysis, molecular biological processes and many other chemical reactions. These chips are quite complex because they contain thousands of substances. These parts are physically designed as a bottom-up full-custom Set, which can be very large staff.

Microarray Protein

These chips are used to track activity and protein synthesis, and to find their performance on a large scale. Its main advantage is that it can be used to track large amounts of protein in the same way. This protein chip has a support surface such as a microtiter plate or beads, nitrocellulose membrane, glass slide. This is automatic, fast, economical, highly sensitive, consumes a small number of samples. The first method of protein chips was introduced in antibody microarrays in scientific literature in 1983. The technology that supports this chip was very easy to develop DNA microarrays, which have become the most widely used microarrays.


Biochips have the following benefits –

  • They are very small in size and strong and fast.
  • It can make thousands of organisms in just seconds.
  • Biochip and help with various diseases.


Biochips have the following disorders –

  • They are expensive.
  • They can be repaired inside the human body or without their permission.
  • They can raise major issues of personal privacy.


  • The biochip can be used to track any person or animal anywhere in the world.
  • It can be used in various fields such as the BP sensor, the oxygen sensor in the medical field.
  • Biochips can be used to store his medical and financial information.



In Latin, the word beer, bibere: meaning to drink. The beer-making process is known as brewing. The ancient Egyptians practiced beer brewing from barley as far back as 4,000 years ago. However, evidence indicates that Egyptians learned the craft from the Tigris and Euphrates tribes, where man’s culture is said to have originated. However, hops’ use is much more recent and can be traced back to a couple of centuries ago. 

Types of Beers :

It is possible to classify barley beers into two broad groups: top-fermented beers and bottom-fermented beers. This distinction is dependent on whether, at the end of fermentation, the yeast stays at the top of the brew (top-fermented beers) or the bottom of the sediment (bottom-fermented beers). 

Bottom-fermented beers 

Bottom-fermented beers are often referred to as lager beers because they have been processed for clarification and maturation or ‘lagered’ in cold cellars after fermentation. The strains of Saccharomyces uvarum (formerly Saccharomyces carlsbergensis) are yeast used in bottom-fermented beers. Most of the world’s lager beers are of the Pilsener kind (70 percent-80 percent). 

a. Pilsener beer: This is a medium hop, pale beer. 3.0-3.8 percent by weight is the alcohol content. It is traditionally lagered for two to three months. However, modern breweries dramatically reduced the lagering period that has been reduced in many breweries across the globe to about two weeks. The water is soft for the Pilsener brew, producing relatively few ions of calcium and magnesium. 

b. Dortmunder beer: a pale beer, but with fewer hops (and therefore less bitter) than Pilsener. It has a thicker body and taste, in any case. The alcohol level is also 3.0-3.8 percent and is slightly longer in a classical lager: 3-4 months. Brewing water, which contains significant quantities of carbonates, sulfates, and chlorides, is difficult. 

c. Munchner: This is a mildly sweet beer with a dark, aromatic, and full-bodied flavor since it is just mildly hopped. The alcohol content, ranging from 2 to 5% alcohol, may be very high. The water used for brewing is high in carbonates but low in other ions. 

d. Weiss (Weizen): Weiss beer from Germany made from wheat and steam beer from California, USA, are both highly effervescent bottom-fermented beers. 

Top-fermented beers 

Top-fermented beers with Saccharomyces cerevisiae strains are brewed. 

a. Ale: While it can be said that lager beer is of German or continental European origin, ale (Pale ale) is an English beer of its own. English ale is a pale, heavily hopped beer with a 4.0 to 5.0 percent (w / v) alcohol content, often as high as 8.0 percent. During and sometimes after fermentation, hops are added. Therefore, its high ester content is very bitter and has a sharp acid taste and wine aroma. The mild ale is sweeter since it hops less vigorously than the traditional pale ale. 

b. Porter: This is a medium-brown, heavy-bodied, highly foamed beer made of medium malts. It contains fewer hops than ale and is sweeter as a result. It has an alcohol content of approximately 5.0%. 

c. Stout: Stout is a heavy-bodied, very dark, vigorously hopped beer with a heavy aroma of malt. It is made from dark or caramelized malt; caramel can be added occasionally. It has a moderately high alcohol content, 5.0-6.5 percent (w / v), and is usually kept for up to six months, with fermentation in the bottle. Some stouts are less hopped than usual, which is sweet.

Raw Brewing Materials 

The raw materials are barley, malt, yeast, hops, water, and adjuncts. 

Malt from barley 

As a cereal for brewing, barley has the following benefits. Its husks are thick, hard to crush, and stick to the kernel. After mashing, this makes malting and filtration much more straightforward than for other cereals such as wheat. The second benefit is that during storage, the thick husk is protection against fungal attack. Third, the temperature of gelatinization (i.e., the temperature at which the starch is transformed into a water-soluble gel) is 52-59 ° C, far lower than the optimal temperature of barley malt alpha-amylase (70 ° C) as well as beta-amylase (65 ° C). 

Adjuncts :

Adjuncts are starchy materials that were initially introduced because a malt with higher diastatic power ( i.e., amylases) was developed by the six-row barley varieties needed to hydrolyze the starch in the malt. The definition now encompasses products other than those which are amylase hydrolyzed. For example, the word now contains added sugars (e.g., sucrose) to improve the beer’s alcoholic content. Starchy adjuncts, typically containing little protein, lead to fermentable sugars after their hydrolysis, which raises the alcoholic content. 

Hops :

Hops (varieties include: H. lupuloides, H. cordifolius, H. neomexicanus) are the dried cone-shaped female flowers of the hop-plant Humulus lupulus. 

(a) Hops, particularly against beer sarcina (Pediococcus damnosus) and other beer spoiling bacteria, have some antimicrobial effects. 

(b) They contribute to colloidal stability and foam head retention of beer because of the bitter substances’ colloidal origin. 

(c) During the wort’s boiling, the hops’ tannins help to precipitate proteins; if not extracted, these proteins create a low-temperature haze in the beer. 

Aquatic Water 

Water is so critical that the natural water available in the world’s great brewing centers has given beers unique to these centers a special character. As is the case, water with a vital calcium and bicarbonate ion content is ideal for developing darker, sweeter beers. 

(a) With the addition of calcium sulfate (gypsum), the water can be ‘brutalized.’ Gypsum addition neutralizes the carbonates’ alkalinity. 

(b) Acids: lactic acid, phosphoric acid, sulfuric acid, or hydrochloric acid can be added. CO2 is emitted, but there is an unwelcome risk that the resulting salt may remain. By gas stripping, the CO2 released is extracted. 

(c) Water may be decarbonated by the addition of lime calcium hydroxide or by boiling. 

(d) Water can be enhanced by ions’ exchange, eliminating all the ions if desired. 

One or more of the methods described above may be used simultaneously.

Brewer’s Yeasts :

Yeasts typically produce sugar alcohol under anaerobic conditions, although not all yeasts are inherently suitable for brewing purposes. In addition to alcohol production, brewing yeasts can generate a balanced proportion of wort sugars and proteins that generate esters, acids, higher alcohols, and ketones. The distinctive taste of beer contributes to these compounds. Several features differentiate the two kinds of brewers’ yeasts (i.e., the top and bottom-fermenting yeasts). 

(a) S. Uvarum typically occurs alone or in pairs (formerly S. carlsbergensis). S. Cerevisiae typically produces chains and even cross-chains sometimes. 

(b) S. Cerevisiae sporulates with greater ease than S. uvarum. 

(c) S. Cerevisiae can only ferment the fructose moiety; in other words, it lacks the enzyme system necessary to ferment the galactose and glucose-formed melibiose. 

(d) S. Cerevisiae strains have a more significant respiratory system than S. Uvarum, which is mirrored in the two groups’ various cytochrome spectra. 

After fermentation, yeasts have reused a variety of times, depending on the individual brewery tradition. In this practice, mutation and pollution are two risks.

Brewing Process: The brewing process involves the sequential events of malting, cleaning, mashing, operation, wort boiling treatment, fermentation, storage, and packaging.


Malting is intended to produce amylases and proteases in the grain. The germinated barley generates these enzymes to allow it to break down the carbohydrates and proteins in the grain to nourish the germinated seedling before its photosynthetic systems are sufficiently developed to support the plant. 

1. For brewing, not all barley strains are suitable. Barley grains are cleaned during malting; broken barley grains and foreign seeds, sand, metal bits are removed. 

2. At 10-15 ° C, the grains are then soaked in water. The grain absorbs water and ultimately increases in volume by about 4 percent. Embryo respiration begins as soon as the water is absorbed. 

3. Microorganisms develop in steep water, and the steep water is altered at 12-hour intervals until the grain’s moisture content is about 45 percent to allow grain deterioration. Steeping takes between two and three days. 

4. The grains are then drained from the moisture and moved for germination to a malting floor or a rotating drum. 

5. The heat produced by the sprouts hastens germination. Sometimes, moist, warm air is blown about 30 cm deep through the beds of germinating seedlings. Water on them may also be sprinkled. 

6. The starch granules in the endosperm are located inside the cells. Hemicellulose, which is broken down by hemicellulases before amylases can invade the starch, is composed of these cell walls. The grain also synthesizes alpha-amylase. Beta-amylase is already present but is bound to proteins and released by proteolytic enzymes and is not synthesized. Enzyme modification or development is completed in 4-5 days of seedling growth. 

Klining, which includes heating the green malt in an oven at a relatively mild temperature, prevents further reactions in the grain before the moisture content is reduced by around 40 percent to 6 percent. The heating temperature depends on the form of beer made. 

Klining takes 20-40 hours at 80-900C for the form of Pilsener. 

For Munich beers, drying at 100-1100CC takes up to 48 hours. 

Some alterations in the gross composition of the barley grain occur at the end of malting. As cattle feed, the rootlets are removed and used. At each point of malting, weight loss known as malting loss occurs, and the accumulated loss can be as high as 15 percent. Barley malt resembles swollen grains of unthreshed rice with its rich enzyme content and can be processed for significant periods before being used.

Malt cleaning and milling operations: 

1. The barley is carried to the upper part of the brewing tower. Subsequent processes take place on increasingly lower floors throughout the brewery process. 2. On the ground level floor, laggering and bottling usually are performed. 

3. Gravity is used in this way to transport the goods, and the pumping cost is removed. 

4. The barley malt is cleaned of dirt at the top of the brewing tower and passed over a magnet to remove pieces of metal, particularly iron, and then milled to expose malt particles to malt enzymes’ hydrolytic effects during the mixing process. 

The smaller the particles, the larger the malt extract would be. However, very fine particles hinder and unduly prolong filtration. Therefore, the brewer has to find a compromise particle size that will offer maximum extraction but allow a relatively quick filtration rate. The crushing is done regardless of the particle size, preserving the husks that contribute to filtration while reducing the endosperm to fine grits. 

Mashing : 

1. Mashing determines the nature of the wort. 

2. The mashing object is to extract as much as possible of the soluble portion of the malt and hydrolyze insoluble portions of the malt and adjuncts enzymatically. 

3. Mashing consists of combining the ground malt and adjuncts at temperatures suitable for the malt-derived amylases and proteases. 

4. Wort is known as the aqueous solution resulting from mashing. 

Starch (55%) and protein (10-12%) are the two largest grain’s dry weight components. The controlled breakdown of these two components affects the character of beer enormously. 

The degradation of starch during mashing : 

Around 55 percent of the dry weight of barley malt is produced by starch. 20-25 percent of the malt starch is composed of amylose. The alpha- and beta-amylases are the main enzymes in malt starch breakdown. 

Breakdown of proteins during mashing 

During malting, the breakdown of malt proteins, albumins, globulins, hordeins, and glutenins begins and continues during mashing through proteases that break down proteins into polypeptides and polypeptidases by peptones that break down polypeptides into amino acids. There is no pronounced optimum temperature of protein breakdown, but it occurs uniformly up to 60 ° C during mashing, above which temperature proteases and polypeptidases are greatly retarded. 

General environmental circumstances that affect mashing : 

A combination of temperature, pH, time, and concentration of the wort affects mashing progress. When the temperature is sustained for long periods at 60-65 ° C, a maltose-rich wort occurs because the beta-amylase activity is at its peak, and this enzyme mainly produces maltose. 

EnzymeOptimum TemperatureTemperature for destruction Optimum pH
Alpha- amylase70° C80° C5.8
Beta- amylase60-65° C75° C5.4

As shown in Table, the optimal pH for beta-amylase activity is approximately the same as that of proteolysis. The mash concentration is essential: the thinner the mash, the higher the maltose content and the extract.

Methods for mashing 

There are three main techniques for mashing: 

(a) Methods of decoction, where part of the mash is moved from mash tun to the mash kettle where it is boiled. 

(b) Methods of infusion, where the mash is never boiled, but the temperature rises steadily. 

(c) Form of double mash, where starchy adjuncts are boiled and added to the malt. 

Mash Separation : 

Husks and other insoluble materials are removed from the wort in two stages at the end of mashing. The wort is separated from the solids first. Second, by washing or sparring with hot water, the solids themselves are liberated from additional extractable content. 

1. The traditional way of separating the husks and other solids from the mash is to strain the mash in a lauter tun that is a vessel about 10 mm above the real bottom with a perforated false bottom, which the husks themselves form a bed in which filtration takes place. 

2. In recent times, the Nooter strain master has come into use in large breweries, particularly in the United States. 

3. Filtration, like the Lauter tun, is through a bed shaped by the husks, but straining is through a series of triangular perforated pipes positioned at various bed heights instead of a false bottom. 

4. Whereas the Lauter tub is cylindrical, the strain master itself is rectangular with a conical rim. Among others, its advantage is that it can accommodate more significant amounts than the Lauter pool. 

5. Cloth filters located in plate filters and scanning centrifuges are also used, and the Lauter tun and the strainmaster. 

The sparging (or hot water washing) of the mash’s solids is performed at about 80 ° C with water and continues until the extraction is complete. The material that is left is known as spent grain after sparging and is used as animal feed. The liquid is often extracted by centrifuging from the spent grain; the extract is used for cooking the adjuncts. 

Boiling Wort : 

The wort is boiled in a brew kettle used to be made of copper for 1–1.5 hours. It is applied when corn syrup or sucrose is used as an adjunct at the beginning of boiling. It also adds hops, some before and some at the end of the boiling process. The boiling objective is as follows: 

(a) Wort concentrate: 5-8 percent of the volume is lost during the boiling by evaporation. 

(b) To sterilize the wort before its entry into the fermenter to reduce its microbial load. 

(c) To inactivate some enzymes so that the composition of the wort does not alter. 

(d) To extract from hops soluble materials which not only aid in the removal of proteins but also help in the removal of proteins; 

The bitterness of hops was also added. 

(e) To precipitate proteins which, due to heat denaturation and complexing, form large flocks with tannins extracted from hops and malt husks. In beer, unprecipitated proteins form hazes, but too little protein contributes to the foam’s head’s inadequate formation. 

(f) To produce the beer color: some of the beer colors come from malting, but the bulk forms during the wort’s boiling. Color is produced by various chemical reactions, including sugar caramelization, phenolic compound oxidation, and amino acid and sugar reduction reactions. 

(g) Removal of volatile compounds: removal of volatile compounds such as fatty acids that may contribute to beer’s rancidity. 

The amount of precipitation and flock forming can be increased during the boiling, agitation, and circulation of the wort. 

Pre-fermentation treatment of wort: 

The hot wort is not sent to the fermentation tanks directly. If dried hops are used in a hop strainer, then they are typically removed. The proteins and tannins are precipitated during boiling while the liquid is still warm. When the wort has cooled to around 50 ° C, some more precipitation occurs. The warm precipitate is referred to as “trub” and consists of 50-60% protein, 16-20% hop resins, 20-30 percent polyphenols, and around 3% ash. The wort is oxygenated at approximately 8 mg/liter of wort during the fermenter transition to provide the yeast with the required oxygen for initial growth.

Fermentation :

The cooled wort is pumped into fermentation tanks or allowed to flow by gravity, and yeast generally collected from a previous brew is inoculated or ‘pitched in’ at a rate of 7-15 x 106 yeast cells/ml. 

Top Fermentation :

For the yeasts’ initial growth, the wort is applied via a fishtail mist so that it is aerated to the tune of 5-10 mg/liter of oxygen. At a temperature of 15–16 ° C, yeast is pitched in at the rate of 0.15 to 0.30 kg/hl. For about three days, the temperature is allowed to increase to 20 ° C steadily. At this stage, it is cooled to a constant temperature. It takes approximately six days for the whole primary fermentation. During this time, Yeasts float to the surface; they are scooped off and used for future pitching. The yeast transforms into a hard leathery coating in the last three days of fermentation, which is also skimmed off. Occasionally, the wort is moved after the first 24-36 hours to another vessel in the so-called dropping method. The switch assists in aerating the system and also allows the cold-break sediments to be discarded. It is also possible to achieve aeration by paddle circulation and using pumps. Nowadays, the conventional open tanks are being replaced by cylindrical vertical, closed tanks. 

Bottom Fermentation :

Wort is inoculated per ml of wort to the tune of 7-15 x 106 yeast cells. Over three to four days, the yeasts then expand four to five times in number. At 6-10 ° C, yeast is pitched in and is allowed to increase to 10-12 ° C, which takes about three to four days. At the end of fermentation, it is cooled to around 5 ° C. CO2 is released, and this produces a head called Krausen that starts to collapse as the yeasts begin to settle after four to five days. The total duration of fermentation can last for 7-12 days. 

Beer Components :

Anaerobic conditions predominate during wort fermentation in the top and bottom fermentation; the original oxygen is only necessary for cell growth. Fermentable sugars are converted by cooling to alcohol, CO2, and heat that must be extracted. There is no fermentation of Dextrins and Maltotetraoses. Amino acids produce higher alcohols (sometimes known as fusel oils), including propanol and isobutanol. Using the tricarboxylic acid cycle, organic acids such as acetic, lactic, pyruvic, citric, and malic are also derived from carbohydrates. 

Lagging : 

(a) Lagering: The beer, known as the ‘green’ beer, is harsh and bitter at the end of the primary fermentation above. It has a yeasty flavor because of higher alcohols and aldehydes, perhaps. It is stored at low temperatures (around 0 ° C) in closed vats for times that are used for as long as six months before maturing in some cases. 

There is secondary fermentation during lagering. Yeasts are often added, using some sugars in the green beer. Secondary fermentation saturates the beer with CO2, and the development of secondary fermentation is accompanied by the exhaust rate of CO2 from the safety valve. Active fermentation of wort or Krausen can be applied occasionally. CO2 may be chemically applied to the beer at some times. Evaporation during secondary fermentation reduces materials that can undesirably influence taste and are found in green beer, e.g., diacetyl, hydrogen sulfide, mercaptans, and acetaldehyde. There is an improvement in the desired beer ingredients, such as esters. During the lagering phase, any tannins, proteins, and hop resins that are still left are precipitated. 

Lagering gives the beer its final organoleptic characteristics that are attractive, but it is hazy due to protein-tannin complexes and yeast cells. To remove these, the beer is filtered through kieselguhr or membrane filters. 

(b) Beer Treatment: no extensive lagering of bottom-fermented beers occurs. In different ways, they are handled in casks or bottles. In specific procedures, at the end of fermentation, the beer is moved to casks with a load of 0.2–4.00 million yeast cells/ml. It is ‘primed’ by adding a small amount of sugar combined with caramel to enhance its taste and appearance. The yeast grows in the sugar, and the beer is carbonated. Hops at this point are also often added. At around 15 ° C, it is kept for seven days or less. The beer is ‘fined’ by the addition of Isinglass after ‘priming.’ Yeast cells, tannins, and protein-tannin complexes are precipitated by Isinglass, a gelatinous substance from the swimming bladder of fish. Following that, the beer is filtered or pasteurized and distributed. 

Packaging : 

Beer is moved to pressure tanks and then distributed to cans, bottles, and other containers. Beer cannot contact oxygen during the transfer; CO2 loss or contamination with microorganisms is also not permitted. Alcohol is applied to tanks under CO2 atmosphere, distilled under a CO2 counter-pressure, and all the equipment is routinely washed and disinfected to achieve these objectives. 

Before being filled, bottles are thoroughly washed with hot water and sodium hydroxide. A pasteurizer passes through the filled and crowned bottles, which heats the bottles for half an hour at 60 ° C. It takes about half an hour for the bottles to hit the pasteurizing temperature, stay in the pasteurizer for half an hour, and cool down for another half an hour. This pasteurization method often creates hazards that lead some larger breweries to carry out bulk pasteurization today and aseptically fill containers.

Schematic representation of industrial process of production of malt beverages.




Fermenter is a system which provide controlled environmental conditions for the growth of microbes to obtain a desirable metabolite by preventing the entry of contaminants. Sterility is maintained by steam. Steam is maintained at 121 degree for a period of time and all the unwanted microbes are killed.

In designing a fermenter, following points must be considered

  1. The fermenter vessel must be able to work aseptically for a number of days.
  2. Evaporation loss should be less
  3. It must allow nutrient and reagent feed.
  4. Proper aeration and agitation should be provided.
  5. Temperature and pH control should be provided.
  6. Power consumption must be low.
  7. It should facilitate the growth wide range of microbes.


  1. Impellers: Mixes the nutrient media so that each and every cell gets equal amount of nutrients, It also prevents the settling down of nutrients. This impeller is connected to the motor.
  2. Temperature Sensor and pH sensor: Microbes grow only at a particular temperature and pH.Hence a sensor is provided at the top side of fermenter to keep a track on it.
  3. Water jacket: Helps in cooling down of the fermenter when the temperature rises. Water jacket provides the circulation of water which controls water. The reactors are covered with water jacket.
  4. Sparger: It is hollow plate which consist of two plates in which the top one contains holes on it. Oxygen passes to the lower plate which moves to the upper plate from where it comes out in the form of bubble .This bubble is broken by impeller releasing oxygen.
  5. Baffles: It helps in preventing vortex formation. Vortex formation prevents efficient mixing of nutrients hence it’s important to have baffles.


  1. Air lift Fermenter: Volume of the culture will be divided into two sections with the help of baffle. Only of the two section will be sparged with air or any other gas and this region where it is sparged is called riser. The other section is known as down-corner. Successive cycles of low and high pressure are created during growth medium circulation. Hence it is also called pressure cycle fermenter. It can be used for continuous process. It doesn’t require agitation and it’s cost effective.
  • Continuous Stirred Tank Bioreactor: It consists of a cylindrical vessel which is occupied with a motor driven central shaft which supports one or more impellers. Shaft is present at the bottom of the bioreactor. The air provided to the culture medium is through the spranger.Advantages of stirred tank reactors are that they provide efficient gas transfer for the growing cells.
  • Bubble Column Fermenter: It is simple to construct and operate. It consist of a cylindrical vessel with a ratio of 4:6 (height to diameter).The upper section of the fermenter is often widened to provide proper gas separation. The gas is spranger into liquid by the means of spranger and hence, adequate level of mixing is obtained. The liquid phase can be delivered by batch or continuous mode, which can either be countercurrent or concurrent.  They have very low maintenance cost and very little space for maintenance. It is widely used for waste water treatment, production of enzymes, proteins and antibiotics.
  • Fluidized Bed Fermenter: This type of reactor is used to carry out multiphase reactions. The fluid is passed through a solid material at high velocities to suspend the solids. It provides excellent mixing, increased mass transfer and enlarged surface area which increases the efficiency. Removal of heat and exchange if gases are easy. It requires more pumping power and also chances for erosion of internal components are more.
  • Hollow Fiber Membrane Fermenter: This fermenters are commonly used for suspension cell. Acyclic polymer or polysulphone fibers are used. Nutrients and oxygen from the intercapilary stream moves across the membrane into the extra capillary space. The product accumulates on the extra capillary site and thus can be harvested at considerably high consist of a cylindrical vessel which consist of individual fibers which are held together in a shell and tube heat exchange allows simultaneous separation of cell from extra cellular product no wash out required because cells are trapped. High productivity per unit of volume is obtained due to high cell this type of reactors diffusion may cause limitation in growth and accumulation of toxic products in fiber are observed.
  • Fixed Bed Fermenter: It is generally used for connecting an immobilized bio catalyst cell for enzyme with the substrate solution. The vessel is packed with a bed of immobilized enzyme particles. The substrate solutions is added from one side and the product is recovered from the other end. It is difficult to maintain pH and gaseous reactant.
  • Tray Fermenter: Generally its made up of wooden or plastic metal tray which are widely used for traditional solid state fermentation the substrate in trays area arranged one above the other so that steam sterilization of the substrate allow aeration by air, moistures applied to the substrate by double flow nozzle with water and air and forced aerator by a blower and can be carried out automatically.
  • Dialysis Unit Fermenter: It allows toxic waste metabolite or end product to diffuse away from the microbial culture and permits new substrate to diffuse through the membrane towards the culture.
  • Deep Jet Fermenter (High Pressure Consuming Bioreactor): The fermenter medium is circulated by pumps gas dissolution and the liquid movement. The gas mixed with high power jet of liquid is injected into the fermenter. Better gas dissolution is obtained in this design but it also leaves involvers more power consumption.




  • Biocontrol agents or Compounds used for managing agricultural pests by means of specific biological effects are termed as Biopesticide.
  • Natural bio-control agents such as plants, certain minerals, microorganisms can be used as biopesticides by modifying them.
  • Majority of crop loss is due to pests or insects. As biopesticides has less adverse effects on environment compared to traditional insecticides, hence can be used to control pests.


  • Some of the earliest agricultural biopesticides used were plant extracts such as Nicotine during 17th century as reported in records.
  • In the year 1835, demonstration experiments involving biological control agents against Lepidopteron pests were carried by Agostine Bassi in White muscardine disease caused by a fungus Beauveria bassiana.
  • In the 20th century, studies involving use of biopesticides emerged rapidly and more number of biopesticides was developed. Bacterium Bacillus thuringenesis was considered to be the initial biopesticide.

Key features of bio-control agents used as biopesticides:

  • It should be specific to the target host.
  • It should have high multiplication rate.
  • It should be environment friendly with very less adverse effects.

Types of Biopesticides:

Three main types of biopesticides are used commonly:


Microorganisms used for insect control are called as bioinsecticides. Viruses, bacteria, fungi, protozoa and mites can be used as bioinsceticides.

  1. Bacterial biopesticides:
  2. Several different bacterial pathogens specific to type of insect they control are being used as insecticides. Examples: Bacillus, Clostridium, Pseudomonas, Enterobacter, Proteus, etc can be used.

Bacillus Thuringenesis: spore forming, rod shaped, gram positive bacterium. They produce parasporal crystals during sporulation. These crystals can be called as Cry proteins or delta endotoxins. These cry proteins prove to be toxic to insects, specifically to lepidopteron and coleopterans.

Mode of action of Bt:

  • Parasporal crystals containing cry proteins are ingested by the insects.
  • Once ingested, these crystals get dissolved in the alkaline environment of the gut of the insect.
  • Delta toxin which is inactive gets cleaved and thus is activated.
  • Specific receptors in the midgut respond to the toxins and binds with it.
  • Pores develop in the epithelium layer of the insect as soon as toxin is inserted.
  • Thus, epithelium is disrupted leading to cell lysis and death of insect.

Symptoms: Common symptoms include; larvae become static and sluggish, stops feeding, water oozes out from larval bodies, and finally larvae dies and falls off the leaf. Bt cotton works particularly against cotton Bollworms, gypsy moths and cabbage worms.

  • Fungal biopesticides:
  • Generally, Entomopathogenic fungi are used as biopesticides. Common examples are Beauveria bassiana (causes White muscardine disease) and Metarhizium anisopliae(causes green muscardine disease).

Mode of action of Beauveria bassiana:

  • It is a filamentous fungus. It is also called as imperfect fungi and belongs to class Deuteromycetes. Generally, used against American boll worm, Codling moth and Potato beetle.
  • By using spores, fungus invades haemocoel of Insects.
  • Spores germinate once they attach to the cuticle. Hyphae penetrates insect cuticle.
  • Penetration appresorium and penetration Peg is formed which helps in the penetration process.
  • Fungal enzymes like chitinases, proteases, and lipases helps to dissolve the cuticle. Once the cuticle is dissolved, hyphae enter the haemolymph and proliferate and colonise the entire insect and release blastospores.
  • Due to depletion of nutrition in haemolymph, insect dies.
  • Viral biopesticides:
  • They attack arthropods and other insects. Commonly used viral biopesticide is Baculoviruses.  Viral biopesticides are generally used to control lepidopteron larvae. Baculoviruses are composed of double stranded DNA and are very small viruses. Three subgroups are present in these viruses; Nuclear Polyhedrosis virus (NPV), Granulosis virus (GV), and Non Occluded viruses. 

Mode of action of NPV:

  • When insect ingests the virus, it enters the mid-gut of insects and infects the gut cells by membrane fusion.
  • In the nucleus, NPV un-coats itself and passes through the intestinal epithelium.
  • Infects the haemocoel.

Symptoms: includes; discoloration of larvae (turns yellow or brown), larval decomposition, infected larvae hangs itself upside down on twigs and larvae becomes swollen because of accumulation of viral fluid inside them.


  • Many fungi are known from genera like Dactylella, Arthrobotrys which can act as nematicide.

Fungus damage nematode in different ways:

  • Haustoria: fungi use these haustoria to penetrate into the body of the nematode and then it digests cell contents and uptakes nutrients of the nematode.
  • Catching by loop: loops are formed by fungal mycelium. When nematode passes through the loop, it constricts and gets trapped.
  • Production of adhesive hyphae: adhesive branches are produced by fungal mycelium which may stick with the nematodes body.
  • Hyphal mesh formation: mesh like cobweb is formed to tap the nematode.
  • The other groups which can acts as nematicide are soil fungi like Verticillium Chlamydosporium, Dactylella oviparisitica.


  • They are primarily used to control the weeds and include use of phytotoxins, pathogens and microbes.
  • Living microorganism is the active ingredient in bio-herbicide.
  • Most commonly fungi are used, though bacteria can also be used as bio-herbicides.
  • High degree of target specifity against weed can be obtained using bio-herbicides.
  • Bio-herbicides have no effect on non-target and beneficial plants and hence are used frequently.

Examples: fungi like Phragmidium violacerum, Phytopthora palmivora (targets milk-weeds) are used commonly.

Advantages of Biopesticides:

  • Less toxic compared to conventional pesticides.
  • The main advantage of using biopesticide is that they only affect target pest.
  • It provides correct identification of pest as biopesticides are highly specific.
  • Relatively cheaper.
  • The risk of pests developing resistance to biopesticides is low as mostly the agents used have multiple mode of action.

Few limitations of biopesticides:

  • When compared to conventional pesticides, they have a slower rate of control and lower efficacy and shorter persistence.
  • Biopesticides have much greater susceptibility to environmental conditions. This can be avoided by modifying the organism used but the process will be time consuming and costly.
  • Grower must require greater knowledge to use biopesticides effectively as they are not as robust as conventional pesticides.




  • Any fluid consumed for drinking purpose can be called as a beverage.
  • Beverages consist of diverse range of food products, mostly liquids like water, soft drinks, fruit beverages, etc.
  • Beverage like water is most essential for human body as it helps in excretion of food and digestion assimilation. Roughly, 60% of human body is made up of water.

History and origin of Beverage Industry:

  • The origin of beverage industry is linked with the antique civilization period.
  • Soft drink got its name in the year 1798, and is the combination of word ‘Soda Water’.
  • Major developments in Beverage and Food industry began in 19th century after development of Canning process by Nicholas Appert and process of pasteurization (process of heating at lower temperatures) by Louis Pasteur.
  • Mead which is a fermented beverage usually made from honey and water was the first ever beverage made as per reports. It is an alcoholic beverage. (Origin-7000 BC, china).

Classification of Beverages:

  1. Natural and Synthetic. (Contains artificial sweeteners).
  2. Carbonated and Non-carbonated.(example- club soda)
  3. Alcoholic and non-alcoholic.
  4. Stimulating and non-stimulating (beverages raising physiological activity) examples- coffee, tea, water.

Soft Drinks Processing/Production steps:

  1. Concentrate (removing all the water) preparation is the very first step in the preparation of any carbonated soft drink.
  2. Sugar syrup clarification: mixture containing sugars, essence, flavoring agents and water forms syrup. It is done to retain particles and crystals from syrup.
  3. Water and microbial stabilization: largest portion of the beverage is accounted by water.  Pre-filtration step is crucial as it ensures good economics of filter train, protects final filter and reduces initial bioburden. Final filtration step removes micro-organisms and makes water contaminant free.
  4. Carbonation: carbon dioxide is added to the beverage. Carbon dioxide injected should be microbe free.
  5. Bottle blower and bottle washer: using PET bottles bottle blowing can be done in any beverage. Air used to turn pre-forms to final PET bottle must be contaminant free. Quality of container must be maintained and it should be microbe free in order to produce good quality beverage.
  6. Bottle filler: for filling process, the filler bowl is pressurized and the gas used has to be microbiologically stable.

Production of Fruit Juices:

  • Variety of fruits can be used for making fruit juices. Fruits like orange, citrus, apples, grapes, cranberries, mangoes and so forth are used.
  • First, the fruits are washed properly and then graded to remove damaged ones. Then, according to the size fruits are separated and transferred to juice extractors.
  • In the juice extractor, oils are extracted from peels and the fruits are squashed to extract the juice.
  • Juice is then screened to remove seeds and pulp (pulp contains all the fiber, essential for controlling blood sugar levels).
  • In the next step, Juice is then sent to evaporators to remove most of the water by heat and vacuum.
  • The juice is then chilled and used as frozen concentrate. Chilling process generally removes oils and essence which are added back before adding to juice packager.
  • Filtered water is used to dilute the concentrate and then it is pasteurized and packaged under sterile conditions.

Production of stimulatory beverage: TEA:

  • Tea leaves are blended and dried to produce tea bags.
  • After the blending process, the tea is sent to tea packaging machines, where it can be packaged as individual packs or in bulk.
  • For powdered tea, tea leaves are blended and brewed using hot water. The liquid tea concentrate is spray dried and stored in drums.
  • Tea powder is packaged in jars and blended with sugars or sugar substitutes.
  • Flavoring agents can also be added to enhance taste and to elevate fragrance. Example- lemon.

Production of coffee process:

  • Coffee beans are extracted and stored in large containers.
  • Removing the endocarp layer is important from wet processed coffee and hence Hulling machineries are used for this process.
  • Then, grading and sorting according to the size is done.
  • Coffee beans are then selected and blended in a large blender system.
  • Roasting of coffee beans. At 205 degree Celsius beans pop up and they expand in size. This is called as light roast (first crack). To reach dark roast levels, coffee beans are heated at 240 degree Celsius.
  • After the roasting process, the roasted beans pass through transporting screw into an elevator. Through elevators, beans are passed into large grinder machines where grinding of beans takes place.
  • Next step is packaging.
  • Coffee beans can also be exported, as in most of the western countries; beans are used and are added directly into instant coffee maker machines, where they can be blended.
  • Coffee can also be made in powdered form and packaged.

Production of Distilled Spirits:

  • Based on preparation mode, alcohol beverages are fermented beverages (beer) and distilled beverages (Whiskey and brandy).
  • Materials used in  preparation of distilled beverages includes, fermented cereal mash, fermented fruits, molasses, juices, honey and so forth.
  • The phases in distilled spirit production are: receiving of grain, milling, cooking, fermentation, distillation, storage, blending and bottling.
  • Grains are received by grain elevator and the grains are weighed in the elevator.
  • Grains necessary for mash bill are grinded in the milling process.
  • Meal from the mill is received by cookers along with slurries with backslop, water and ammonia. Solubilization of starch takes place using steam-jet cooking.
  • Resulting mash is cooled at fermentation temperature.
  • In the fermentation process sugar is converted to alcohol by using yeast (Saccharomyces Cerevisae). Fermenters are used for the process of fermentation.
  • For cognac and scotches production, pot still distillation is used.
  • By-product is recovered (centrifugation, evaporation, drying and mixing).
  • Whiskies, brandies need to be stored for longer periods for better taste. Charred oak barrels are used for storage. After storage and product maturation(few years), they are

Blended, filtered and packaged as finished products.

Production of Beer (fermented beverage):

Few steps are involved in beer production:

  1. Malting: barley grain is used for beer preparation and is made ready for brewing. In malting process, malt goes through a very high temperature drying in a kiln, called Kilning process.The process is continued with gradual increase in temperature for few hours. After kilning, grains are called malt and are crushed to expose cotyledons, rich in carbohydrates.
  2. Mashing: starch conversion to sugars takes place in mashing process. Milled grains are mixed large vessel called mash tun. Sugar rich liquid called ‘Wort’ is generated in mashing process.
  3. Lautering: process of separating the wort (liquid with sugar obtained during mashing) from barley grains.
  4. Boiling: large tanks like kettle are used to boil the wort with herbs or sugars. In this step, flavor, color, aroma of the beer is decided. Hops are used which add flavors to beer.
  5. Whirlpool: whirlpool vessel is used to separate out solid particles in the hopped wort.
  6. Wort cooling: cooled at 20-26 degree Celsius and yeast is also added during this process.
  7. Fermentation: cooled wort is added to large fermentation tanks where upon action of added yeast fermentation process is carried out.
  8. Conditioning (beer aging/maturation): after fermentation process, beer is transferred to conditioning tank. In this process beer ages, smoothens.
  9. Conditioning process is done for several weeks and after that beer is filtered and force carbonated for Bottling (packaging of beer into bottles).