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APPLICATION OF
MICROBES IN INDUSTRIAL
BIOTECHNOLOGY
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Small molecules of living cells
Intermediates or end products of the
pathway
Related to synthesis of microbial cells in
the growth phase, essential for G, D and R
Include alcohols, amino acids, nucleotides, organic acids,
polyols, vitamins, and enzymes
Involved in maintaining normal physiological processes, thus
often referred to as central metabolites 3
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Accumulate following active growth
Have no direct relationship to
synthesis of cell material and
natural growth
Not absolutely required for cell
survival
But, has ecological function
Include antibiotics and toxins 5
Primary metabolic pathway
• for the synthesis of aromatic
amino acids
The secondary metabolite
• antibiotics containing aromatic
rings
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▪ A large number of organic acids with actual or potential uses are
produced by microorganisms
▪ Such as, citric, itaconic, lactic, malic, tartaric, gluconic, mevalonic,
salicyclic, gibberelic, diamino-pimelic, and propionic acids
▪ Citric acid is a tribasic acid used in the food industry, in
medicine, pharmacy and in various other industries
▪ Citric acid is an intermediate in the citric acid cycle (TCA) or
Kreb cycle of glycolysis
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a) By mutation:- giving rise to mutant organisms which may only
use part of a metabolic pathway, or
▪ regulatory mutants; that is using a mutant lacking an enzyme
of the cycle
b) By inhibiting the free-flow of the cycle through altering the
environmental conditions
▪ Many of the enzymes of the TCA can be directly inhibited
by various compounds and this phenomenon is exploited to
increase citric acid production
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▪ For a long time the production of citric acid has been based on the
use of molasses and various strains of Aspergillus niger and
occasionally Asp. wenti
▪ The industrial production is performed using carbohydrates or
agro-industrial residues as substrates
▪ Submerged, surface and solid fermentation process can be used
▪ Several reports of citric acid production by Penicilliumare spp.
available, but they have low productivity
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▪ Lactic acid is produced by many organisms
▪ The organisms which produce adequate amounts at industrial
level are:
▪ Homofermentative lactic acid bacteria, Lactobacillus spp.,
especially L. delbrueckii
▪ Rhizopus oryzae
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▪ Amino acids can be produced by microbial organisms by utilizing
several carbon sources
▪ Can be produced by different processes such as extraction from
protein hydrolysates, chemical synthesis or enzymatic and
fermentation pathways
▪ The most important method for producing amino acids
microbiologically is by direct fermentation
▪ The production via by fermentation was stimulated by the discovery
of an efficient L-glutamic acid producer C. glutamicum
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▪ Many microorganisms have been reported to produce amino acids
▪ They are mainly bacteria, but they also include some molds and
yeasts.
▪ The four most widely reported bacteria belong to the following
four genera
▪ Corynebacteriumspp. (C. glutamicum; C. lilum)
▪ Brevibacterium spp. (B. divericartum: B. alanicum)
▪ Microbacterium spp. (M. flavumvar. glutamicum)
▪ Arthrobacter spp. (A. globiformis; A. aminofaciens)
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▪ Glutamic acid production is greatest when biotin is limiting; that
is, when it is suboptimal
▪ When biotin is optimal, growth is luxuriant and lactic acid, not
glutamic acid, is excreted
▪ Wild type strains of the organisms of the four genera mentioned
above are now used for the production of glutamic acid
▪ This increased permeability to the acid can
be achieved in the following ways:
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1. Ensuring biotin deficiency in the medium.
2. Treatment with fatty acid derivatives
3. Addition of penicillin during growth of glutamic acid
bacteria
▪ Cells treated in one of above method have cell membranes in
which the saturated to unsaturated fatty acid ratio is abnormal,
therefore the permeability barrier is destroyed and glutamic acid
accumulates in the medium
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▪ Most industrial enzymes are obtainable from microorganisms
▪ Due to rapid growth of microbes in short periods of time, used to
obtain desired amount of enzymes for industrial use
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▪ Antibiotics are chemicals produced by microorganisms and which
in low concentrations are capable of inhibiting the growth of, or
killing, other microorganisms
▪ Antibiotics may be wholly produced by fermentation
▪ Action in different ways
disrupting the plasma membrane of microbial cells
inhibiting cell wall synthesis
inhibiting synthesis of important metabolites
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▪ Several methods of antibiotic classification have been adopted
by various authors based on:
▪ mode of action
▪ based on the producing organisms
▪ routes of biosynthesis
▪ spectra of organisms
▪ chemical structure of the antibiotics attacked
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▪ Chemical groups ▪ Examples of antibiotics
• Aminoglycosides • Streptomycin
• Ansamacrolides • Rifamycin
• Beta-lactams • Penicillin
• Chloramphenicol and
• Chloramphenicol
analogues
• Linocomycin
• Linocosaminides
• Macrolides • Erythromycin
• Nucleosides • Puromycin
• Puromycin • Curamycin
• Peptides • Neomycin
• Phenazines • Myxin
• Polyenes
• Amphothericin B
• Polyethers
• Nigericin
• Tetracyclines
• Tetracycline
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▪ The Beta-lactam antibiotics named from their structure the four
membered lactam ring
▪ Penicillins and Cephalosporins are common groups
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▪ Inhibit cell wall synthesis
▪ Bactericidal (except against Enterococcussp.); time
dependent killers
▪ Short elimination half-life
▪ Primarily renally eliminated
▪ Cross-allergenicity - except aztreonam.
▪ Interfere with cell wall synthesis by binding to penicillin-
binding proteins (PBPs) which are located in bacteria cell walls.
▪ Inhibition of PBPs leads to inhibition of peptidoglycan
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“EXTRACTION AND
PURIFICATION OF
PENICILLIN”
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f) Industrial Alcohol Production
▪ Ethanol may be produced by either synthetic chemical method
or by fermentation
▪ Due to the increase in price of crude petroleum, the source of
ethylene used for alcohol production, attention has turned
worldwide to the production of alcohol by fermentation
▪ Microbial production of ethanol from the organic feed
stocks and from plant substances such as molasses is
presently used for ethanol production
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▪ Biomining is the process of using microorganisms
(microbes) to extract metals of economic interest
from rock ores or mine waste
▪ Biomining techniques may also be used to clean up
sites that have been polluted with metals
▪ Bioleaching is the process by which metals are
dissolved from ore bearing rocks using microbes
▪ E.g. Metals from sulfide ores
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▪ Heap leaching: freshly mined material is moved
directly into heaps that are then bioleached.
▪ Dump leaching: low-value ore or waste rock is placed
in a sealed pit and then bioleached to remove more of
the valuable metals from the waste pile.
▪ Agitated leaching: crushed rocks are placed into a
large vat that is shaken to distribute the microbes and
material evenly and
speed up the bioleaching process.
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▪ Microbial bioremediation makes use of microorganisms
and/or their derivatives (enzymes or spent biomass) to
clean-up environmental contaminants
▪ Bioremediation using microbial bioreactors finds
application in soil, air and water environments including:
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▪ Waste water and industrial effluent treatment:
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▪ Microorganisms are the primary agents of any biological
wastewater treatment
▪ They feed on complex substances in the wastewater
converting them to simpler substances
▪ Soil and land treatment
▪ Contaminants successfully treated include diesel fuel, fuel
oils, oily sludge, wood-preserving wastes (PCP, PAHs, and
creosote), coke wastes, and certain pesticides
▪ Soil bioremediation has proven most successful in treating
petroleum hydrocarbons and other less volatile,
biodegradable contaminants.
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▪ Control of air pollution
▪ Microorganisms are used in the bioremediation of
organic and inorganic air pollutants
▪ Microorganisms oxidize pollutants such as H2S, SO2,
VOCs, and reduce pollutants such as NOx to nitrate
and this assist to prevent likely environmental,
health hazards and nuisances
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▪ Microorganisms can be isolated from almost any environmental
conditions
▪ Microbes will adapt and grow at subzero temperatures, as well as
extreme heat, desert conditions, in water, with an excess of
oxygen, and in anaerobic conditions, with the presence of
hazardous compounds or on any waste stream.
▪ The main requirements are an energy source and a carbon source
▪ Because of the adaptability of microbes and other biological
systems, these can be used to degrade or remediate
environmental hazards
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▪ Aerobic: degrade pesticides and hydrocarbons, both alkanes
and polyaromatic compounds
• Many of these bacteria use the contaminant as the sole source of
carbon and energy. E.g Pseudomonas, Alcaligenes,
Sphingomonas, Rhodococcus, and Mycobacterium
▪ Anaerobic: are not as frequently as aerobic, degrade
polychlorinated biphenyls (PCBs), dechlorination of the solvent
trichloroethylene (TCE), and chloroform
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▪ Methylotrophs: Aerobic bacteria that grow utilizing methane
for carbon and energy
• The initial enzyme in the pathway for aerobic degradation,
methane monooxygenase, has a broad substrate range and is
active against a wide range of compounds, including the
chlorinated aliphatics trichloroethylene and 1,2-dichloroethane
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Although the microorganisms are present in contaminated soil,
they cannot necessarily be there in the numbers required for
bioremediation of the site
Their growth and activity must be stimulated
▪ Bio-stimulation usually involves the addition of nutrients and
oxygen to help indigenous microorganisms
▪ These nutrients are the basic building blocks of life and allow
microbes to create the necessary enzymes to break down the
contaminants
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▪ All of them will need nitrogen, phosphorous, and carbon
▪ Carbon is the most basic element of living forms and is
needed in greater quantities than other elements. In
addition to hydrogen, oxygen, and nitrogen it constitutes
about 95% of the weight of cells
▪ Phosphorous and sulfur contribute with 70% of the
remainders. The nutritional requirement of carbon to
nitrogen ratio is 10:1, and carbon to phosphorous is 30:1
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1- Isolation of the microorganism
2- Purification of the obtained isolates
3- Identification of the microbial isolate
4- Optimization of the biodegradation
conditions
5- Determination of the biodegradation
efficiency
6- Identification of the biodegradation products.
7- Cell or enzyme immobilization.
8- Enzyme identification. 110
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