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General Microbiology
BIO306

Assist. Prof. Betül AKCESME

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 Putting Microorganisms to Work

• Industrial Products and the Microorganisms

That Make Them
– Industrial microbiologu vs biotechnology ???
– Big amounts vs small amnounts!!!
– Natural enhancment vs genetic engineering!!

• Production and Scale

I. Industrial Products and the Microorganisms
That Make Them
• Industrial microbiology
– Uses microorganisms, typically grown on a large scale, to
produce products or carry out chemical transformation
• processes such as production of pharmaceuticals, food additives,
enzymes, and chemicals were developed

– Major organisms used are fungi and genus Streptomyces

• Classical genetic methods are used to select for high-yielding microbial variants

• goal is to increase the yield of the product to the point of being economically
profitable.
Industrial Products and the Microorganisms
That Make Them

• Properties of a useful industrial microbe include

– Produces spores or can be easily inoculated
– Grows rapidly on a large scale in inexpensive medium
– Produces desired product quickly
– Should not be pathogenic ( environment and human)
– Amenable to genetic manipulation
Industrial Products and the Microorganisms
That Make Them

• Microbial products of industrial interest include

– Microbial cells

– Enzymes

– Antibiotics, steroids, alkaloids

– Food additives

– Commodity chemicals
• Inexpensive chemicals produced in bulk

• Include ethanol, citric acid, and many others

Major Products of industrial microbiology
 Production and Scale
• Primary metabolite
– Produced during exponential growth
– Example: alcohol
– Ethanol is a product of the fermentative metabolism of
yeast or certain bacteria
– can grow only if they produce energy, ethanol grows in
parallel with growth
 Production and Scale
• Secondary metabolites
– Produced during end of the growth or stationary phase
– Not essential for growth
– Formation depends on growth conditions
– Produced as a group of related compounds
– Often significantly overproduced by spore-forming microbes during
sporulation, production is linked to the sporulation process itself.
– Antibiotics: Virtually all antibiotics, for example, are produced by either fungi
or spore-forming prokaryotes.
 Formation of alcohol by yeast—an example of Penicillin production by the mold
a primary metabolite. Penicillium chrysogenum— an
example of a secondary metabolite.
Note that penicillin is not made until
after the exponential phase.

Primary
metabolite Secondary

Penicillin, sugar, or cell number

Cells metabolite
Alcohol, sugar, or cell number

Alcohol
Sugar

Cells

Sugar

Penicillin

Time Time

Contrast between production of primary and secondary metabolites.

 Production and Scale

• Secondary metabolites are often large organic molecules that

require a large number of specific enzymatic steps for

production

– Synthesis of tetracycline requires at least 72 separate enzymatic steps

– Starting materials arise from major biosynthetic pathways

 Production and Scale
• Fermentor is where the microbiology process takes place.

• Any large-scale reaction is referred to as a fermentation!!

– whether or not it is, biochemically speaking, a fermentation.

– Most are aerobic processes

• Fermentors vary in size from 5 to 500,000 liters

– Aerobic and anaerobic fermentors

• Large-scale fermentors are almost always stainless steel

– Impellers and spargers supply oxygen
Fermentor sizes for various industrial
fermantations
Large-scale fermenters Motor

Steam pH pH controller
cylinder, closed at the top and bottom, Acid–base
Sterile
into which various pipes and valves have seal
reservoir and
pump
been fitted Viewing
port

Sterilization of the culture medium and Filter

removal of heat are vital for successful Exhaust

operation
Impeller
(mixing) External
A critical part of the fermentor is the cooling
water out
aeration system
Cooling
jacket
a high density of microbial cells, there is a External Culture
broth
tremendous oxygen demand by the culture cooling
water in
Sparger (high-
an aerator, called a sparger, and a stirring pressure air
for aeration)
device, called an impeller Steam in
Sterile air
Valve
monitored in real time for temperature,
oxygen, pH, and the levels of key
nutrients, such as ammonia and
Harvest
phosphate.
WHY necessary to alter the conditions
in the fermentor??
• It is often necessary to alter the conditions in the fermentor as the
fermentation progresses.

• Computers are used to process environmental data as the

fermentation proceeds and are programmed to respond by signaling
for nutrient additions, increases in the rate of cooling water,
impeller speed or sparger pressure, or changes in pH or other
parameters, at just the right time to maintain high product yield.

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The inside
of an industrial
fermentor, showing the
impeller and internal
heating and cooling
coils.
 Production and Scale
• Industrial Fermentors
– Closely monitored during production run

– Growth and product formation must be measured

– Environmental factors must be controlled and altered as needed

• Including temperature, pH, cell mass, nutrients, and product
concentration

– Data on the process must be obtained in real time

 Production and Scale
• Scale-up from laboratory to commercial fermentor
– The transfer of a process from a small laboratory scale to large-scale
commercial equipment

– Major task of the biochemical engineer

• Requires knowledge of the biology of producing organism and the

physics of fermentor design and operation

– Many challenges in scale-up arise from aeration and mixing

• high cell densities, and this leads to high oxygen demand

 SCALE UP
• Flask  laboratory fermentor(1-10 liter)  pilot
plant(300-3000 l)  commercial fermentor(10000-
500000 l)

• In all stages of scale-up, aeration is the key variable that is closely

monitored;
• as scale-up proceeds, oxygen dynamics are carefully measured to
determine how increases in volume affect oxygen demand in the
fermentation.

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A bank of small research fermentors used in
process development. The fermentors are
the glass vessels with the stainless steel
tops. The small plastic bottles collect
overflow.

(b) A large bank of outdoor industrial-scale fermentors (each 240 m3) used in
commercial production of alcohol in Japan.
Drugs, Other Chemicals, and Enzymes

• Antibiotics: Isolation, Yield, and Purification

• Industrial Production of Penicillins and
Tetracyclines
• Vitamins and Amino Acids
• Enzymes as Industrial Products
 Antibiotics: Isolation, Yield, and
Purification
• Antibiotics

– Compounds that kill or inhibit the growth of other microbes

– Typically secondary metabolites

– Most antibiotics in clinical use are produced by filamentous fungi or

actinobacteria
– Modern drug discovery relies heavily on computer modeling of drug–
target interactions
- Before discovered by laboratory screening
• Microbes are obtained from nature in pure culture

• Assayed for products that inhibit growth of test bacteria

Some antibiotics produced commercially

b) EFB, endospore-forming bacterium; F, fungus; A, actinomycete.

• Isolation of antibiotic producers.
• (a) Isolation using media selective
for Streptomyces and identification
of antibiotic producers by
screening using an indicator
organism. Photo:
• Most of the colonies are
Streptomyces species, and some are
producing antibiotics as shown by
zones of growth inhibition of the
indicator organism (Staphylococcus
aureus).

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 Antibiotics: Isolation, Yield, and Purification

• Cross-streak method: Method of testing an organism for its antibiotic

spectrum of activity.
– Used to test new microbial isolates for antibiotic production

– Most isolates produce known antibiotics

– Most antibiotics fail toxicity and therapeutic tests in animals

– Time and cost of developing a new antibiotic is approximately 15 years and

$1 billion
• Involves clinical trials and U.S. FDA approval

• Antibiotic purification and extraction often involves elaborate methods

Method of testing an organism for its antibiotic
spectrum of activity. (SECREENING)
• The producer was streaked across one-third of the plate and the
plate incubated.
• After good growth was obtained, the five species of test bacteria
were streaked perpendicular to the producing organism, and the
plate was further incubated.
• The failure of several species to grow near the producing
organism indicates that it produced an antibiotic active against
these bacteria.
• Photo: Test organisms streaked vertically (left to right) include
Escherichia coli, Bacillus subtilis, S. aureus, Klebsiella
pneumoniae, Mycobacterium smegmatis.

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 Once a new antibiotic has been characterized and proven medically effective
and nontoxic in tests on experimental animals, it is ready for clinical trials on
humans. If the new drug proves clinically effective and passes toxicity and
other tests, it is given FDA approval and is ready to be produced
commercially.

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 Yield and purification

• One of the major tasks of the industrial microbiologist is to

isolate high-yielding strains
– mutagenizing the wild-type organism to obtain mutant derivatives
that are so altered that they overproduce the antibiotic of interest

• The next challenge is to purify the antibiotic specifically

and efficiently, and elaborate methods for extraction and
purification of the antibiotic are often necessary

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Industrial Production of Penicillins and Tetracyclines

• Penicillins are -lactam antibiotics

• are produced by fungi of the genera Penicillium and Aspergillus and by

certain prokaryotes. Commercial penicillin is produced in the United States

using high-yielding strains of the mold Penicillium chrysogenum.

– Natural and biosynthetic penicillins

– Semisynthetic penicillins

• Broad spectrum of activity

 Industrial Production of Penicillins and
Tetracyclines
• Penicillin G is produced in fermantors of 40.000-200.000 liters.

• Highly aerobic process.

• Typical secondary metabolite, very little is produced during the growth phase.

• Can be extended several days by additions (carbon, nitrogen)

• High levels of glucose repress penicillin production but high levels of lactose do not, so lactose

is added

• At the end of the production phase, the cells are removed by filtration and the pH is made acidic.

• The penicillin can then be extracted and concentrated into an organic solvent and, finally,
crystallized.

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 Penicillin
Add
precursor I fermentation
Biosynthetic
penicillin I
Chemical or
Add enzymatic
precursor II Add
precursor treatment
Biosynthetic III of penicillin G
penicillin II 6-Aminopenicillanic acid
Biosynthetic
penicillin III
Add side
chains
Natural penicillins chemically
(for example,
penicillin G)
Semisynthetic penicillins
(for example, ampicillin,
amoxycillin, methicillin)
Industrial production of penicillins. The β-lactam ring is circled in red. The normal
fermentation leads to the natural penicillins. If specific precursors are added during the
fermentation, various biosynthetic penicillins are formed. Semisynthetic penicillins are
produced by chemically adding a specific side chain to the 6-aminopenicillanic acid
nucleus on the “R” group shown in purple. Semisynthetic penicillins are the most widely
prescribed of all the penicillins today, primarily because of their broad spectrum of activity
and ability to be taken orally.
 Glucose
feeding
Figure :Kinetics of the penicillin Nitrogen
feeding
fermentation with Penicillium 100

ammonia, penicillin (g/liter  10)

chrysogenum. 90

Biomass (g/liter), carbohydrate,

Kinetics of the penicillin 80 Penicillin
fermentation with Penicillium 70
chrysogenum. Note that penicillin is 60
produced as cells are entering the 50
stationary phase, when most of the
40
carbon and nitrogen has been Cells
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exhausted. Nutrient “feedings” keep
20 Lactose
penicillin production high over
10 Ammonia
several days.
0
20 40 60 80 100 120 140
Fermentation time (h)
Industrial Production of Penicillins and
Tetracyclines

• Biosynthesis of tetracycline has a large number of

enzymatic steps
– More than 72 intermediates
– More than 300 genes involved! (studies of Streptomyces
aureofaciens)
– Complex biosynthetic regulation
– Glucose and phosphate repress the synthesis, need for low phosphate
concentration.
 Inoculum
(spores on Production
agar slant or in
sterile soil) scheme for
Medium Growth in
chlortetracycline
optimal medium
2% Meat extract; 0.05%
Agar plates asparagine; 1% glucose; using
Spores as
0.5% K2HPO4; 1.3% agar Streptomyces
inoculum aureofaciens.
2% Corn steep liquor;
Shake flask 3% sucrose; 0.5% CaCO3
some key Medium mimics
24 h production
regulatory signals medium
Prefermentor Same as for shake culture
are known
and are accounted 19–24 h
pH 5.2–6.2
for in the
production Fermentor 1% Sucrose; 1% corn steep
scheme liquor; 0.2% (NH4)2HPO4; Production
0.1% CaCO3; medium, no
60–65 h
0.025% MgSO4 glucose, low
pH 5.8–6.0 phosphate
0.005% ZnSO4
0.00033% and each of
CuSO4, MnCl2 Glucose is used to
Antibiotic
purification from grow the
broth after cell
removal inoculum, but not
for commercial
production.
Chlortetracycline
 Vitamins and Amino Acids
• Production of vitamins is second only to antibiotics in terms of total
pharmaceutical sales

• Most of them are made commercially by chemical synthesis.

• Vitamin B12 produced exclusively by microorganisms 10,000 tons per

year.
• Deficiency results in pernicious anemia
– low production of red blood cells and nervous system disorders

• Cobalt is present in B12

– vitamin are greatly increased by addition of small amounts of cobalt to the
culture medium

– Riboflavin can also be produced by microbes, 1.000 tons per year.

B12 Vitamins produced by microorganisms on an industrial scale.
 Vitamins and Amino Acids
• Amino acids

– Used as food additives in the food industry

– Used as nutritional supplements in nutraceutical industry

– Used as starting materials in the chemical industry

– Examples include

• Glutamic acid (monosodium glutamate, MSG)

– Over one million tons of this amino acid are produced annually by
the gram-positive bacterium Corynebacterium glutamicum.
Amino acids used in the food industry
Enzymes as Industrial Products

• Exoenzymes
– Enzymes that are excreted into the medium instead of being
held within the cell; they are extracellular
– Can digest insoluble polymers such as cellulose, protein, and
starch

• Enzymes are useful as industrial catalysts

– Produce only one stereoisomer
– High substrate specificity
Microbial enzymes and their
applications
 Enzymes as Industrial Products
• Enzymes are produced from fungi and bacteria
– Bacterial proteases are used in laundry detergents (can also contain amylases, lipases, and
reductases)
• Isolated from alkaliphilic bacteria

• Usually acttive pH between 9-10 (alkaline pH of laundary detergant)

• Amylases and glucoamylases are also commercially important

– Produce high-fructose syrup
– Production of glucose from starch
– then converted by a second enzyme, glucose isomerase, to fructose, which is a much
sweeter sugar than glucose.
– Widely used in the food industry to sweeten soft drinks, juices, and many other products.
– Worldwide production of high-fructose syrups is over 10 billion
kilograms per year.
 Enzymes as Industrial Products
• Extremozymes
– Enzymes that function at some environmental
extreme
– Produced by extremophiles
– This feature makes these enzymes of interest to a
variety of biotechnical applications
– Taq polymerase
• Cold-tolerant Extremozymes
– Food processor
– cold-wash detergent
• Acid-tolerant Extremozymes
– Catalyses for the synthesis of compounds in acidic solution
– additives for animal feed
• Alkali-tolerant Extremozymes
– Detergent (protease, lipase etc.)
– Dye
• Salt-tolerant Extremozymes
– Oil exploitation

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 Thermostability of the enzyme pullulanase from Pyrococcus woesei, a hyperthermophile
whose growth temperature optimum is 100°C. At 110°C the enzyme denatures, but calcium
improves the heat stability of this enzyme dramatically.

100

activity remaining
Percent enzyme
10 Pullulanase
Starch oligosaccharides

90°C
100°C
110°C
110°C plus Ca2

1
1 2 3 4
Time (h)

An acid-tolerant enzyme mixture used as a feed supplement for poultry.

The enzymes function in the bird’s stomach to digest fibrous materials in the feed, thereby
improving the nutritional value of the feed and promoting more rapid growth.
Enzymes as Industrial Products

• Immobilized enzymes are attached to a solid surface

– makes it easier to carry out the enzymatic reaction under large-
scale continuous flow conditions,

– also helps stabilize the enzyme to retard denaturation.

• Three ways to immobilize an enzyme

– Bonding of enzyme to a carrier

– Cross-linking of enzyme molecules

– Enzyme inclusion
 Carrier-bound Cross-linked
Procedures for the
enzyme enzyme
immobilization of enzymes.

Enzyme inclusion in Enzyme inclusion

fibrous polymers in microcapsules
 Biofuels
• A biofuel is a type of fuel whose energy is derived from
biological carbon fixation. 

• Fermentation of recently grown plant materials rather than

being of ancient origin (fossils).

– Biodisels –made from vegatable oils.

– Algal fuels- from green algea.
– Gasohol-produced by adding ethanol to gasoline.

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 Biofuels
• Ethanol Biofuels
– Ethanol is a major industrial commodity chemical

– In USA most ethanol is obtained from by yeast

fermentation of glucose obtained from cornstarch.

– Various yeast have been used but most ethanol is

produced by Saccharomyces.
– Over 60 billion liters of alcohol are produced yearly from

the fermentation of feed stocks

• The increased demand for corn as a biofuel feedstock
has driven up the price of human foods and livestock
feeds.
• Sugar cane, whey, sugar beets, and even wood chips
and waste paper are used as feedstocks for the
fermentation
• Cellulosic materials, the cellulose must first be treated
to release glucose, which is then fermented to alcohol.
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• Petroleum Biofuels
– Production of butanol

– Synthesis of petroleum from green algae

• during growth the colonial green alga Botryococcus

braunii excretes long-chain (C30–C36) hydrocarbons

that have the consistency of crude oil.

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http://science.howstuffworks.com/environmental/green-science/algae-
biodiesel3.htm
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(a)A bioethanol production plant in Nebraska (USA). In the plant,
glucose obtained from corn starch is fermented by Saccharomyces
cerevisiae to ethanol plus CO2. The large tank in the left foreground
is the ethanol storage tank, and the tanks and pipes in the
background are for distilling ethanol from the fermentation broth.
(b)Switchgrass, a promising feedstock for bioethanol production. The
cellulose from this rapidly growing plant can be treated to yield
glucose that can then be fermented to ethanol or butano
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(c) The petroleum-producing colonial green alga,
Botryococcus braunii. Note the excreted oil droplets that
appear as bubbles along the margin of the cells.
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