As we look ahead into the next century, leaders will be those who empower others”

                                                              -- Bill Gates, the great Master of Silicon Valley

“Tyrants have used information as a weapon by withholding it or distorting it”

"The Net lin

   Copyright 2008 Prof. P. Kaushik 

 1. HISTORICAL DEVELOPMENT OR PAST OF MICROBIAL TECHNOLOGY OR INDUSTRIAL MICROBIOLOGY 

What comes under broad domain of industrial microbiology?

Mainly designing, sterilization and use of fermenters to make products of commercial importance come under it but we cannot overlook sterilization, deterioration and quality

Control concerning production and handling of food and beverages as well as the devices leading to protection against deterioration of fabrics, metals, concrete, wood, jet fuels, animal feeds and products of pharmaceutical value. Living and fossilized microorganisms are employed to discover new oil reserves and to improve and enhance oil recovery. It includes problems and prospects to disposal of municipal and industrial wastes. It includes production of biofertilizers to increase production of crops and get rid of toxic effect of chemical pesticides and other pollutants. The important products involving microorganisms are:

*Antibiotics: Ampicillin, bacitracin,carbenicillin,cephalosporins,chloramphenicol,ciprofloxacin, clindamycin, dapsone, erythromycin, gentamycin, isoniazid, methycillin, penicillin, polymyxin, rifampin, streptomycin, sulphonamides, tetracyclines, trimethoprim, and vancomycin.

*Organic solvents: Acetone, butanol, ethanol, amyl alcohol.

*Beverages:Wine,beer and other beverages.

*Foods:Bread, cheese fermented milk, pickles, sauerkraut,saue sauce,  yeast, vinegar, mushrooms and acidulants like citric acid.

*Flavouring agents: Monosodium glutamate and nucleotides.

*Organic acids: Acetic, butyric, gluconic, citric, lactic, fumaric,itaconic acids and koji.

*Glycerol

*Amino acids: L-glutamic acid and L-lysine.

*Steroids and biotransformation or microbial transformation of steroids:Hydroxilation of progesterone in 11  alpha  position by Rhizopus nigricans

*Baker’s yeast.

*Food and feed yeast and other single cell proteins.

*Mushroom cultivation.

*Biofertilizers.

*Biopesticides

*Vitamins and growth stimulants: B₁₂, riboflavin, vitamin A and gibberallins.

*Enzymes: amylase, protease, pectinases and invertase.

*Fats: The bacteria ferment carbohydrates to fatty acids, carbon dioxide and hydrogen.

*Insulin.

*Immunological diagnostic kits.

*vaccines, and recombinant DNA vaccines.

*Gases: carbon dioxide and hydrogen.

*Hybrid antibiotics.

*Biosurfactants and emulsifiers

*Biosensors with streptavidin-biotin-linked systems to detect important pathogens.

*Gene assay based on DNA recombinant technology to monitor gene expression.

*Bioprospecting.

*Forced evolution and adaptive mutations or “natural genetic engineering.

Purely theoretical research can well have applicability, e.g. a study regarding keto acid intermediates in the metabolism of a microorganism may provide information that addition of high amount of ammonium salt or urea to the growth medium may result accumulation of amino acids in the medium.

                                                                                                                                                                                                                                                                                                Who was the Pioneer in the field of Microbial Technology?                                                                                                                                                                           The people of almost all   civilizations might have used this or that product of fermentation process. But it is vedic and postvedic Sanskrit literature where we find descriptions about microorganisms of different categories and methodology of production of different types of ashavs, arishts and arks using different ingredients and raw materials and sources of inocula, using big earthen pots and wooden vats, which practice in production of ayurvedic preparations exist even today in most of pharmaceutical concerns. The potable alcohol and vinegar were the most common products before 1900.                                          Ancient Egyptians perhaps were the first to brew the beer, however, the really, large scale breweries, according H.S. Koran (1957) in “A History of Brewing” published by David and Charles, Newton Abbott, came into existence in 1700s with the introduction of wooden vats of capacity of 1500 barrels. The use of thermometers to control the process in early breweries was done in 1757, and that of primitive heat exchangers in1801.Though, Cagniard-Latour, Schwan and Kutzing     independently demonstrated role of yeasts in alcoholic fermentation by 1850 but it was Luis Pasteur who convinced the scientific community regarding obligatory role of yeasts in this process. Hansen in late 1800s            started his pioneer work at the Carlsberg brewery and developed process for isolation and propagation of single yeast cell and developed sophisticated techniques for production of starter cultures.. The use of pure cultures was not adopted by British ale breweries and it is interesting to note that many of the traditional ale making breweries even today use mixed yeast cultures and are maintaining good quality. In fact the vinegar was originally produced by leaving   wine in shallow bowls or partially filled barrels for slow oxidation of wine         to result vinegar as a result of development of natural flora. Importance of air in the process of oxidation   showed the way to development of the generator that comprised of a vessel packed with an inert material like coke, charcoal and various types of wood shavings on which beer or wine wad trickled. This type of vinegar generator may be regarded first aerobic fermenter developed.        Till the end of 19^th century and beginning of 20^th century the initial medium was pasteurized and was inoculated with one tenth of quality vinegar to make the medium acidic to prevent contamination as well as serving the purpose of a good inoculum. The baker’s yeast was produced on large scale in the early 1900s and in Germany yeast was produced            as a human food in First World War.                                                    The concept of control in the field of brewing and vinegar industries was thus well established by the beginning of 20^th century.

2.   CLASSIFICATION OF MICROORGANISMS

         What is a microorganism?

Taxonomy or Systematics consists of nomenclature, identification and classification.

Numerical taxonomy: Grouping by numerical methods.

Binomial system of nomenclature used by microbiologists was given by Swedish botanist

Carl von Linne or Carolus Linnaeus in which Latinized and Italicized names consist of two parts genus and species.

Informal names used by microbiologists instead of formal or hierarchical names are:

purple bacteria, spirochaetes, methane oxididizing bacteria, sulphate reducing bacteria and lactic acid bacteria.

The species is a basic taxonomic group or a collection of strains that have in common many stable properties and differ significantly from other groups of strains. Recently a species has also been called a genospecies, which is a collection of strains that have a similar G +C composition and 70% or greater similarity as judged by DNA hybridization.

A strain is a population of organisms which is distinguishable from other populations in a particular taxonomic category. A strain is considered to have descended from a single organism or pure culture isolate. A type strain  is generally  one of the first strains studied and is usually more fully described  and characterized than other strains. The strains within a species may differ from one another in many ways.

Biovars: Strains characterized by biochemical and physiological differences.

Morphovars: Which differ morphologically.

Serovars: With distinct antigenic properties

A genus is a group of one or more species with clear demarcation from other genera.

Phenetic classification: Based on overall similarity. The first edition of Bergey’s Manual of Systematic Bacteriology was largely phenotypic which divided prokaryotes into groups based on easily recognizable characteristics like shape, Gram staining characteristics, oxygen relationships and motility.

Phylogenetic or phyletic classification: Based on analysis of probable evolutionary relationships the results of that analysis are also summarized in the form of tree-like diagrams called dendrograms. The second edition of Bergey’s Manual of Sustematic Bacteriology was organised phylogenetically in which prokaryotes were divided into two domains and 35 phylla.

Classification of microorganisms is an arrangement of similar type of microorganisms in nonoverlaping hierarchical groups.

Our earth is 4.6 billions years old. There are reports of stromatolites, layered rocks formed as a result of incorporation of minerals into microbial mats. Thus the existence of fossilized stromatolites, believed to be 3.5 to 3.8 billion years old, is indicative of existence of microorganisms in history of the earth.

How did eukaryotic (also eucaryotic)   cells evolve from prokaryotes?

*Invaginaton of plasma membrane.

*Endosymbiotic hypothesis: probably fusion of bacteria and archaea.

Endosymbiosis has also been supported by discovery of biflgellate protist Cyanophora  paradoxa, inside which endosymbiotic cyanobacterium lives and works as a chloroplast. This endosymbiont has been called Cyanella that bears similarity to cyanobacteria for its photosynthetic system and is surrounded by a layer of peptidoglycan, however, it is devoid of the lippopolysaccharide, the  outer membrane  features of Gram negative bacteria.

E. H. Haeckel  in 1866 proposed a 3 Kingdom concept.

Robert H. Whittaker’s 5 kingdoms:

Carl Woese’s 3 Domains:On the basis of rRNA sequences divided organisms into  three primary groups-Archaea, Bacteria and Eucarya.These three primary groups are known as domains presumed above the level of Phyllum and Kingdom.

Eucaryotic organisms that primarily have glycerol fatty acyl diether membrane lipids and eucaryotic rRNA are placed under Eucaryota.

Six Kinngdom system: Archaeobacteria, Eubacteria, Protista, Plantae Fungi, and Animalia.

Eight Kingdom system: Eubacteria, Archaeobacteria, Archaezoa, Protozoa,Plantae, Chromista, Fungi, and Animalia.

CYTOPLASMIC INCLUSIONS AND RESERVED FOOD MATERIAL IN PROCARYOTES

Poly-beta-hydroxy butyrate (PHB): It accumulates in aerobic and facultative bacteria deprived of oxygen but carry out fermentative metabolism and therefore, it has also been called polymeric, intracellular fermentation product, e.g. in Chromatium okenii and Bacillus megaterium. It serves as a source of carbon and energy on arrival of aerobic conditions and it takes part in oxidative metabolism. Some species of bacteria, however, bear capability to produce copolymers besides PHB. If propionic acid or b-hydroxyvaleric acid are given as substrate the polymers formed are b-hydroxybutyric acid and b-hydroxyvaleric acid.

 Sometimes g-hydroxy fatty acids and long chain (C 8, C10, C 12) are also formed ,and these reserved materials are called poly (hydroxy) fatty acids, or poly-hydroxy-alconates.

The applied aspect of PHAs lies in the fact that these can be thermoplastically moulded and can be used as new plastics.These being biodegradable  are preferred to be better in comparison to polypropylene or polyethylene and thus are environment friendly.

Polyphosphates:    The polyphosphates or polyphosphate granules have also been called volutin or metchromatic granules because of their  staining characteristics.These are the reserves of inorganic phosphates which can be used in synthesis of ATP.and formation of condtituents of cell. The polyphosphate accumulates in bacterial cells of Pseudomanas aeruginosa 

Cyanophycin granules: The cyanophycin granules, comprised of specific polypeptide s which are stores of bound oxygen, among prokaryotes are met in cyanobacteria alone.

Sulphur storage: Among a number of sulphide oxidizing bacteria, transient accumulation of sulphur in the shape of highly refractive spheres take place.Intracelluraly stored as well as extracellular excretory sulphur is initially in liquid form which gradually changes to orthorhombic form. The amount of sulphur stored depends on hydrogen sulphide content present in the medium. However, in the absence of hydrogen sulphide elementary sulphur is oxidized to sulphate.

Thus the sulphur stored is the source of energy for hydrogen sulphide oxidizing bacteria like Acromatium, Beggiatoa, Thiothrix and Thiovulum. In anaerobic  phototrophic purple sulphur bacteria as Chromatium the sulphur so stored works as electron donor.The sulphur inclusions occasionally found in cyanobacteria and Sphaerotilus natans may be called as detoxification products of  hydrogen sulphide usually found in their ecological niche or natural environment.

Gas vacuoles: The gas vacuoles are found in many aquatic bacteria particularly in phototrophic ones with an exception to non pigmented species like Pelonema and Peloploca, and halobacteria like Halobacterium halobium and a few clostridia. The gas vacuoles enable bacterial cells to change their specific mass to float in water. These vacuoles help bacteria to remain in fixed stratum with optimal growth conditions in absence of  motility providing organelles as flagella.

It is interesting to note  that anoxogenic phototrophs including red bacteria as, Amoebobacter, Lamprocystis and Thiodictyon; and green bacteria such as Pelodictyon

grow in anaerobic zone called hypolimnion just below the thermocline. This buoyancy is sufficient to keep the organisms in the cold heavy water layer of hypolimnion and does not permit them to rise into warmer lighter layer above. The oxygenic phototrophs ,cyanobacteria like, Aphanizomenon flos-aquae, Microcystis aeruginosa and Oscillatoria agardhi flourish above the thermocline.Thus the photosynthetic behaviour of these organisms is controlled  by the combined effect of photosynthesis, cellular turgor, and number and size of gas vesicles.Each gas vacuole comprise of few to  many spindle shaped vesicles. The walls of gas vesicles do not have the usual membrane structure rather comprise of pure protein in leaflet arrangement. Their walls are only 2 nm thick. Arrangement of rib like hoops around the barrel can be seen clearly in the electron micrographs. The protein of the wall is made of subunits with a relative molecular mass of 14x10 raised to power 3.The proteins are arranged in such a way that the hydrophobic surface is on the inside of the vesicle and the hydrophilic surface on the outside.

Lipid-like substances: Intracellular fat granules and oil droplets are found in many microorganisms including bacteria which can be stained with lipophylic dyes as Sudan III or Sudan black B. These can also be seen in unstained preparations under light microscope because of their high refractivity.

Magnetosomes: Some bacteria in the form of rods, spirilla and cocci isolated from surface layers of sediments in fresh water ponds and the sea have been observed to orient themselves in a magnetic field and swim in direction of field lines. They contain unusual amounts of iron, approximately 0.4 % of their dry weight. The iron may be in the form of ferromagnetic iron oxide (magnetite ) in the form of grana called magnetosomes, localized close to the area of insertion of flagella. The bacteria isolated in the northern hemisphere seek the north and the field linesare directed downwards with a gradient of about 70 º.It is apparent that their magnetostatic nature enables them to migrate downwards into the oxygen poor or oxygen free sediments . It is because that magnetotactic bacteria are anaerobic or microaerophilic.If such organisms are transferred to  the southern hemisphere they will not survive.