Fermentation : Defination, Principle and Batch Fermentation Method

 The process of fermentation involves biochemical activity of organisms during their growth, development, reproduction, senescence, and death. Fermentation technology employs organisms to produce food, pharmaceuticals, and alcoholic beverages in industries on a large scale.

Principle of Fermentation

The principle involved in industrial fermentation technology is that organisms are grown under optimum conditions and are provided with raw materials and other necessary requirements like carbon, nitrogen, salts, trace elements, and vitamins. The end products formed due to their metabolism during their life span are released into the media. These end products are extracted by human beings as they are commercially valuable.

Some major end products of fermentation produced on a large scale industrial basis are wine, beer, cider, vinegar, ethanol, cheese, hormones, antibiotics, complete proteins, enzymes, and other beneficial products.

Batch Fermentation

In batch fermentation process, the microorganisms are inoculated in a fixed volume of batch culture medium. The organisms during their growth consume the nutrients, and the growth products (i.e., biomass and metabolites) start accumulating. Since the nutrient environment within the Fermenter is continuously changed, the rate of cell metabolism also changes, and ultimately, cell multiplication stops due to limitation of nutrients and accumulation of toxic excreted waste products.

Growth Characteristics in a Batch Culture of a Microorganism. 1) Lag Phase, 2) Transient Acceleration, 3) Exponential Phase, 4) Deceleration Phase, 5) Stationary Phase, 6) Death Phase

There is the complex nature of batch growth of microorganisms. In the initial lag phase, no apparent growth is observed; however, biochemical analyses show metabolic turnover signifying that the cells are acclimatising to the environmental conditions and will start growing. Then comes the transient acceleration phase when the inoculum begins to grow. This phase is quickly followed by the exponential phase where the organisms are growing at fastest rate as the nutrients are in excess, environmental conditions are optimum and growth inhibitors are absent.

In the batch fermentation process, the exponential growth occurs for a limited period. With the change in nutrient conditions, the growth rate decreases and begins deceleration phase followed by the stationary phase at which the growth sto completely because of nutrient exhaustion. The death phase when the grow rate has come to an end is the final phase of the cycle. Mostly t biotechnological batch processes are stopped before this stage because decreasing metabolism and cell lysis.

Advantages of Batch Fermentation

1. It Requires less space.
2. It Can be easily handled, and
3. There is Less chances of contamination.

Disadvantages of Batch Fermentation

1. It is time consuming method.
2. It requires more time for cleaning, sterilisation, and cooling.
3. Product yield is low.

Read More

Applications of rDNA Technology and Genetic Engineering in Medicine

 Genetic engineering has an important role to play in the production of medicines. Microorganisms and plant-based substances are used for producing various drugs, vaccines, enzymes, and hormones at low costs. Genetic engineering involves the study of inheritance pattern of diseases in man and collection of human genes that provide a complete map for inheritance of healthy individuals.

Vaccines

  Recombinant DNA technology is used for producing vaccines against diseases by isolating antigen or protein present on the surface of viral particles. Vaccines contain a form of an infectious organism that does not cause disease but allow the body immune system to form antibodies against the infective organism.

   When an individual receives vaccination against any viral disease, the antigens produce antibodies to act against and inactivate the viral profeins. The scientists with the help of recombinant DNA technology have transferred the genes for some viral sheath proteins to vecinia virus which was used against small pox. Vaccines produced by gene cloning are non-contaminated and safe as they contain only coat proteins against which antibodies are produced. Vaccines against viral hepatitis influenza, herpes simplex virus, virus-induced foot and mouth disease in animals are being produced by gene cloning.

Hormones

  Insulin was commercially produced in 1982 through biogenetic or recombinant DNA technology. Its medicinal use was approved by Food and Drug Administration (FDA) of the USA in the same year. The human insulin gene has been cloned in large quantities in E. coli bacterium that can be used for synthesising insulin. Humilin is the commercially available genetically engineered insulin.

Lymphokines

Lymphokines are proteins regulating the immune system in the human body. u. Interferon is an example of lymphokines that are used to fight viral diseases (such as hepatitis, herpes, and common cold) and cancer. Such drugs can be manufactured in large quantities in bacterial cells. Lymphokines are also helpful in AIDS. Interleukin-II is a commercially available genetically engineered substance that stimulates the multiplication of lymphocytes.

Somatostatin

  Somatostatin is used in some of the growth related abnormalities. This drug appears to be species specific and the polypeptide obtained from other mammals has no effect on humans, hence it is extracted from the hypothalamus of cadavers. Genetic engineering has helped in the chemical synthesis of gene which is joined to the PBR 322 plasmid DNA and cloned into a bacterium. This transformed bacterium is converted into a somatostatin synthesising factory.

Production of Blood Clotting Factors

  Blockage of coronary arteries by cholesterol or blood clots causes heart attack. Plasminogen is a substance found in blood clots. Genetically engineered tissue Plasminogen Activator (tPA) enzyme is used to dissolve these clots in individuals who have suffered heart attacks.

Cancer

  Antibodies cloned from a single source and targeted for a specific antigen (monoclonal antibodies) have proved useful in the treatment of cancer. Monoclonal antibodies have been targeted with radioactive elements of cytotoxins (e.g., Ricin from castor seed) to make them more deadly. Such antibodies seek cancer cells and kill them with their radioactivity or toxin.

Read More

Bergey's Manual of Systemic Bacteriology

 Bergey's manual is an accepted reference on the identification of bacteria. It has undergone gradual transformations and expansion since the time of its first publication. 

 The American Society of Microbiology published first edition of Bergey's Manual of Determinative Bacteriology in 1923. Professor David H. Bergey (Chair Person) and other four colleagues acted as the members of the editorial board. Afterwards, there was a sequel of eight editions, an abridged version and several supplements. At present, ninth edition (published in 1994) is available. It is used to classify bacteria based on their structural and functional attributes by arranging them into specific familial orders. However, this process has become more empirical in recent years.

Bergey's Manual of Systematic Bacteriology  Manual of Systematic Bacteriology

It is the main resource for determining the identity of prokaryotic organisms, emphasizing bacterial species, using every characterizing aspect. First edition of this manual consists of four volumes. It's first volume was published in 1984, the second in 1986 and final two volumes in 1989. This manual has much broader scope. It includes all information of earlier manuals. In addition it includes the information on taxonomy, ecology, cultivation, maintenance and the preservation of organisms. It includes many kinds of information such as...

1. Descriptions and photographs of species,
2. Test of distinguish to distinguish among genera and species,
3. DNA relatedness among organisms and
4. Various taxonomic studies.

There are four divisions of kingdom Procaryotae according to Bergey's Manual of Systematic Bacteriology.

Division I : Gracilicutes

Includes prokaryotes with thin cell walls e.g. Gram negative bacteria.

Division II : Firmicutes

Includes prokaryotes with thick cell wall e.g. Gram positive bacteria. 

Division III : Tenericutes

Includes the prokaryotes that lack cell wall.

Division IV : Mendosicutes

Includes the prokaryotes lacking peptidoglycan in their cell wall.

Second edition of Bergey's Manual of systematic Bacteriology consists of five volumes. Volume I was published in 2001, Volume II in 2005 and the other three volumes in 2007. In comparison to the first edition, second edition has certain changes. e.g .,

1. Volume - I includes The Archaea and The Deeply Branching And Phototrophic bacteria.
2. Volume II includes the Gram-negative Proteobacteria. It includes medically important genera are Escherichia, Neisseria, Pseudomonas, Rhizobium, Rickettsiae, Salmonella and Vibrio.
3. Volume - III includes the Gram- positive bacteria with low G + C content in their DNA. They are the members of phylum Firmicutes . It includes rods and cocci and also pleomorphic Mycoplasma. They may form endospores. Its classes are Clostridia, Mollicutes and Bacilli.
4. Volume - IV includes the Gram - positive bacteria with high G + C content in their DNA. They have more than 50- 50 % G + C content. 
5. Volume - V includes ten phyla. They are located here for convenience. Includes morphologically diverse gram - negative organisms. They may not be related. Organisms included are Plantomycetes, Chlamydia, Spirochaetes, Bacteroilds, Fusobacteria, Chlamydiae, Acidobacteria, Verrucomicrobia and Pictyoglomus.

Read More

General Nature and Basic Structure of Enzyme

 A living cell is capable of performing a multitude of  biochemical reactions in order to survive grow and multiply. This involves

• Degradation of complex nutrients into simpler forms so that they can be absorbed by the cell.
• Uptake of these simple nutrients.
• Chemical transformation of these simple nutrient molecules into various precursor metabolites, so that they are available for biosynthesis.
• Generation of ATP and other bio reactive molecules, which co-operate in cellular biochemical reactions.
• Biosynthesis of cellular molecules and structural components of the cell etc.

All these diverse types of chemical reactions can occur with a high degree of specificity and rate, under normal growth conditions for the organism. These chemical changes are brought about by the living cell through the role and participation of a special class of molecules known as enzymes . Enzymes function in the cell by acting as a biocatalyst.

Discovery of Enzymes

The term enzyme was coined by Kuhne in 1878, suggesting in yeast (en = in, zyme = yeast). It was examined that cell free extract from yeasts is capable of causing conversion of sugar into alcohol. This lead to the understanding that the molecules present inside the cells of yeasts are responsible for these chemical transformations. Kuhne called them as enzymes.

Later on, it was established that all biochemical activities of a living cell are attributed to these magic molecules called enzymes. The first enzyme to be discovered was 'amylase'. Its presence in malt extract was detected in 1833 by two French chemists Pain and Persuses.

General Nature of Enzymes

Enzymes are regarded as organic biocatalysts which are capable of functioning both extra cellular and intra cellular. Following are the major characteristics of enzymes. 

1] With exception of a few catalytic RNA molecules or ribozymes, all enzymes are protein in nature. Over 90 % enzymes are globular proteins. Many of them are conjugated proteins, having non protein component. 

Enzymes may be monomeric or multimeric proteins. As they are proteins, they share all major characteristic of proteins .

• They are macromolecules with high MW.
• They are non dialyzable and are unable to pass through semi permeable membrane.
• They are amphoteric in nature. i.e. they possess both types of lonizable groups : -NH₂ and -COOH. which on ionization . yield positively charged ammonium (NH4+) ion and negatively charged carboxyl (COO-) ion.
• As they are amphoteric, they possess specific isoelectric pH. At this pH, both -NH₂ and -COOH groups get equally ionized such that their net electrical charge becomes minimum.
• They show electrophoretic mobility. If the enzyme solution is at pH, above isoelectric value, they acquire negative charge and hence move towards anode and vice versa.
• They are colloidal in nature.
• They can be salted out by salts like ammonium sulfate
• They can be precipitated out by protein denaturing solvents like acetone and alcohol.
• They can absorb maximum UV light at 280 um wavelength.

2]. Enzymes are mostly thermo labile and get denatured at high temperature, usually above 60°C. However, some enzymes are found thermo stable and can withstand high temperature up to 70°C - 80°C or even more.

3]. Enzymes are highly specific in action. They can act on a specific substrate molecule to bring about specific biochemical reaction .

Catalytic RNA - Ribozyme

  Apart from proteins, a few RNA have also been recognized to have catalytic activity. They are called ribozymes or catalytic RNA. They were first recognized by Thomas Cech in 1982. The ribozymes have two types of common roles :

1. RNA processing, where the RNA is involved in RNA splicing, RNA ligation and RNA replication.  
2. Peptide bond formation during protein synthesis. In ribosomes, they function as a part of rRNA and participate in peptide bond formation.

Basic structure of Enzymes

In general, enzyme molecule consists of two components.

1. Apo enzyme and
2. Prosthetic group or cofactor

Apo enzyme is protein part of enzyme.
Prosthetic group or cofactors are non protein part of enzymes. They consist of vitamin or their derivatives or metal lons. (If these non protein components are bound loosely to protein, they are called cofactors) and if they are bound firmly (covalently), are called prosthetic groups. Apo enzyme and prosthetic group form complex to form active form of enzyme, known as Holoenzyme.

Classification of Enzymes

Read More

pH Effect on Microbial growth

 The self-ionization of water is a continuous process when it is in pure form. In this process, when there is collision of two H₂O molecules they dissociate into H⁺ and OH^-  ions. There is no free hydrogen ions in the water. Because the free hydrogen ions have tendency to attach to the H₂O molecule to form hydronium ion. This means, you will always find  H₃O⁺ or hydronium ions in water, instead of free H+ ions.

What is pH ?

pH stands for potential of hydrogen, or power of hydrogen. pH can be defined as measurement of hydrogen ion concentration in a solution, or mathematically it can be defined as negative logarithm of hydrogen ion concentration.

Formula of pH : -log [H+]

pH is applied only to aqueous solutions. That means where there is water, there is pH. pH was first described by Sorensen in 1909. pH reveals the acidity or basicity of water.
  The pH scale ranges from 0 to 14 where the value 7 indicates the neutral pH. pH less than 7 indicate acidic conditions, and greater than seven indicate basic or alkaline. In other word pH is about calculating the free hydronium ions and free hydroxyl ions in a given solution.

A solution with more H+ ions is acidic and gives a pH value less than 7 Similarly, a solution with more OH- ions, is basic and gives a value greater than seven.

Importance of pH

Certain reactions in our body require certain value of pH. Anything higher or lower value may cause damage. For example, if too much of hydrochloric acid is produced in the stomach, the patient will be advised to take antacids like magnesium hydroxide in order to neutralize the excess pH.
  In the same way plants cannot grow if there is continuous rising and falling of pH of soil. Animals in the river cannot survive when acid rains fall on the river.
  Similarly, pH plays an important role on microbial growth.

There were cases where microbes did not show growth in the absence of right pH conditions, even they were supplied with all the required nutrients.
  The pH value where a microbe can grow its best, is called the optimum pH.
Most bacteria grow best at a pH value near to 7, which means, most bacteria are neutrophils.
Some bacteria can grow at a pH range between 3 and 4. These are called acedophiles.
Alkaliphiles are the bacteria that can tolerate pH between 8 and 11.

Impact of pH on Microbial growth

The bacterial cell consists of several protein molecules, lipids, and nucleic acids. And the cell hosts several biochemical reactions. All these activities are regulated when there is optimum pH.
In lower pH conditions, the increased hydrogen ions break the weak hydrogen bonds of protein side chains and finally change the shape of the protein.
  When a protein is not in its original shape, it cannot perform its routine function, and ultimately the bacteria cannot survive.

There are two methods to measure pH in a microbiology lab. The first one is using pH paper. The pH paper changes its color when it is dipped into a solution. This color change is based on acidity and basicity of the sample solution.
Later, the color of the paper will be compared with the color chart provided along with the paper. These papers are coated with a pigment called Flavin, which is extracted from red cabbage. Flavin has ability to change color when it comes in contact with an acid or base. This method will not provide the exact pH value. However, this will provide a value which is closer to the actual pH value.
The other method to measure pH that gives an accurate value is using a pH meter.

Read More

INTRODUCTION TO BIOGECHEMICAL TRANSFORMATIONS IN SOIL : MINERALIZATION AND IMMOBILIZATION OF ELEMENTS

 Erth is a closed system, where the over all quantity of matter remains constant. Microorganisms need electron, energy and nutrients to grow. They are responsible for cyclic transformation of compounds, and therefore they are called biogeochemical agents. They carryout transformation of carbon, nitrogen, sulphur, phosphorus, iron etc. This cycling of elements is called biogeochemical cycling. Both biological and chemical process are involved in biogeochemical cycling.

The oxidation reduction reactions are mainly responsible for biogeochemical cycling of compounds. This changes the chemical and physical characteristics of different compounds. These cyclic turnover of elements are brought about by different types of microorganisms resulting into continuous change in chemical states of matter.

The life of earth depends on cyclic conversion of chemicals from inorganic state to organic (complex state) to the elemental state. The break in the cycle at any point would dramatically affect all life forms. Various processes carrying out these transformation include :

Mineralization : 

It is a process of conversion of complex organic compounds to simple inorganic forms. Many heteroprophic microbes play role in mineralization.
The resultant simple compounds are made available to plants and microbes. Energy is released in the process. The process of mineralization is very important as it increases soil fertility.

Carbon mineralization :

• Organic carbon is mineralized to inorganic state.
• Under aerobic condition the main products of carbon mineralization are CO₂ and water.
• In absence of O₂ organic carbon is incompletey metabolized to produce organic acids, alcohols and gases.

Assimilation :

• It is the process of conversion of substrate elements to protoplasmic elements. 
• Microbes take up the simple materials from the environment (soil) and convert them into cellular materials. It is known as assimiliation or biosynthesis.
• In this process synthesis of energy and cellular material takes place. Through assimilation microbes store the excess of simple inorganic chemicals, and prevent their loss due to erosion.

Immobilization :

It is a process in which the quantity of plant available nutrients are reduced in soil by microorganisms. The nutrient assimilation is an important method of immobilization.
The uptake of various elements like carbon, nitrogen, phosphorus, sulphur, etc. cause immobilization.

  Intermediary substances accumulate abundant quantities of CH4 and smaller amount of H₂ is evolved. The factors affecting mineralization are level of organic matter, temperature, moisture, pH, depth and aeration.
  All these factors affect the growth and metabolism of microbes and the process of mineralization.

• In nitrogen mineralization ammonium, nitrite and nitrates are accumulated from organic nitrogenous compounds like proteins, nucleic acids, etc.
• In phosphorus mineralization organic phosphorus present in nucleic acid, phytin, lecithin, etc. are converted to inorganic phosphorus.
• Sulfur mineralization involves aerobic breakdown of sulfur containing amino acids : cystine, cysteine, methionine, and vitamins, thiamine, biotin, thioctic acid releasing sulfates. Whereas in absence of oxygen , H₂S and odoriferous mercaptans accumulate in soil.

Read More

Lytic cycle: Multiplication of T4 Bacteriophage

T4 coliphage is a phage that infect coliform bacteria especially E.coli. T4 is a double stranded DNA phage, it has a contractile sheath and an unique base 5-hydroxyl methyl cytosine (5-HMC). It is the best known member of large virulent phages.

The lytic cycle of T4 phage involve following steps: ADSORPTION
PENETRATION
BIOSYNTHESIS
ASSEMBLY
RELEASE

1]. Adsorption :

• The lytic cycle begins when a bacteriophage comes in contact with a susceptible host cell by random collision.
• Phage possess an adsorption organs or anti-receptors and host cells possess receptors.
• Host cell surface components -Flagella, Pilli , Teichoic acids, Proteins, Carbohydrates , LPs and Lipopolysaccharides serves as receptors.
• Phage components such as tail fibers, tail proteins and spikes serves as adsorption organs or anti-receptors.
• Each phage has its specific receptor to which it adsorbs.
• Adsorption takes place only when the anti-receptor is chemically complementary to the receptor.
• T4 phage possess tail fibers that serves as an adsorption organ or anti-receptor.
• Normally, tail fibers are present in folded form around the tail.
• Whiskers hold the tail fibers in folded form.
• When phage come in contact with host, tail fibers unfold.
• Unfolding of fibers requires tryptophan & co-factors - Mg++ & Ca++.
• Thus, the phage and host binding is favoured by ionic environment.
• T4 host E.coli possess outer membrane protein C (OmpC) – lipopolysaccharide complex as receptor.
• Initial attachment occurs when tail fibers attach to the OmpC- lipopolysaccharide complex.
• Initial adsorption is weak and reversible.
• It becomes irreversible when tail pins attach to lipopolysaccharide.

2]. Penetration :

• Once attached, the bacteriophage injects DNA into the bacterium.
• Bacteria possess rigid cell wall and therefore the phages directly cannot penetrate into the bacterial cells.
• They inject only their nucleic acids inside the host cell.
• In the T-even phage, irreversible binding of the phage to host results in the contraction of the sheath and the hollow tail tube is inserted through host cell wall.
• Some phages have enzymes like lysozyme that digest the cell wall components of the bacterial cell.
• The penetration of T4 phage DNA occurs when -

1. There is irreversible attachment of phage to host cell,
2. Contraction of sheath, pushing tail tube through cell envelope
3. Injection of DNA into cell like injection of vaccine/drug by a syringe

3]. Biosynthesis :

Biosynthesis divided into three steps:

1. Formation of immediate early and delayed early protein
2. Replication of phage genome
3. Formation of late proteins

i) Formation of immediate early and delayed early protein :

• Part of phage DNA is immediately transcribed by host RNA polymerase to form immediate early m-RNAS.
• These early m-RNA translate to following enzyme proteins -
• a) Nucleases - Breaks down host DNA & make nucleotides available for its own synthesis.
• b) α-subunit modifying enzyme - modifies α-subunit of host RNA polymerase.
• Modified host RNA polymerase transcribes part of viral genome to delayed early m-RNAS.

Delayed early mRNAs are translated to following enzymes-

•  a) Phage enzymes that produce 5-hydroxyl methyl cytosine (5-HMC), a unique base in phage DNA
• b) Polymerases and ligases - that play role in phage DNA replication and recombination.
• c) Glucosylation enzyme-adds glucose to HMC & protects phage DNA from host restriction endonuclease
• d) σ-subunit modifying enzyme - modifies σ-factor of RNA polymerase so that is transcribes late mRNAs.

ii) Replication of Phage Genome :

  Two modes have been proposed for the replication of T4 phage DNA.
By bi-directional mode - at early stage
By rolling circle mode - at later stage

• Initial replication is bi-directional and semi-discontinuous.
• Leading strand is synthesized continuously and lagging strand is synthesized discontinuously leading to the formation of eye structure.
• Bi-directional replication is initiated at several origins along the DNA and is catalyzed by phage coded enzymes.

• In the rolling circle mode of replication, a cut is made in one of the DNA strands by a specific endonuclease and 3'end is made free.
• DNA polymerase extends the free 3'OH end by adding complementary bases.
• Intact strand serves as template for addition of complementary bases.
• Due to extension of 3'OH end, the 5'end is displaced.
• Displaced strand is synthesized discontinuously by adding Okazaki fragments.
• This mechanism produces multi-genome length molecules.
• Such molecules are referred to as concatemers.
• The concatemers are later cleaved to head sized molecules by headful cutting mechanism.

III] Formation of late proteins 

• Soon after the replication of phage DNA, transcription of late m-RNAs occur.

• These late m-RNAs translate to different proteins.
• These proteins include the structural proteins.
• They are proteins involved in phage assembly and an enzyme lysozyme that degrades the peptidoglycan layer of bacterial cell wall.
• For example - head (capsid) proteins, tail tube protein, sheath proteins, collar, whiskers, base plate, tail fiber, tail pins, lysozyme etc.

4. Assembly of Phages :

• Assembly of new phage particles begins after accumulation of structural proteins and nucleic acid molecules in the cell.
• Process of assembling phage particles is known as known as maturation.
• There are four different pathways that lead to the formation of phage particles.
• These include base plate, tail tube & tail sheath, tail fibers and head.
• About 50 genes take part in the morphogenesis of T4 phage.
• Subunits of base plate assemble to form a base plate.
• Then tail tube and sheath subunits polymerize on base plate to form mature tail.
• The subunits of head assemble together to form prohead and then DNA is inserted in the prohead to form complete head.

5. Release :

• The release of newly synthesized phages occurs by sudden explosion or bursting (lysis) of bacterial cell.
• Lysis begins after about 22 minutes.
• One of the gene products involved in the process include lysozyme.
• It cleaves glycosidic bonds in the peptidoglycan making the cell wall susceptible to the rupture.
• There is another protein termed as holin that make holes in the cell membrane and makes the way for lysozyme action.

Read More