1. Assist. Prof. Betül AKCESME10
General Microbiology BIO306
Assist. Prof. Betül AKCESME

2. 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
These products include antibiotics, of course, but also a
wide variety of other products
The products typically originate from enhancements of metabolic reactions
that the microorganisms were already capable of carrying out, with
the main goal being the overproduction of the product of interest.

3. 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.

4. 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
the organism must be capable of growth and product formation in large-scale culture
it should produce spores or some other reproductive cell so that it can be easily inoculated into the large vessels used
to grow the producing organism on an industrial scale
especially
to humans or economically important animals or plants.
Because of the high cell densities in industrial microbial
processes and the virtual impossibility of avoiding contamination
of the environment outside the growth vessel, a pathogen would
present potentially disastrous problems.

5. 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

6. Major Products of industrial microbiology
Table 15.1 Major products of industrial microbiology

7. 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
A typical primary metabolite is alcohol. Ethanol is a product of the fermentative metabolism of yeast or certain bacteria. Because organism can grow only if they produce energy, ethanol grows in parallel with growth.
Secondary metabolite not coupled directly to growth are some of the most complex and important metabolites of industrial interest. Secondary metabolites typically share a number of characteristics.

8. 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.
Secondary metabolite not coupled directly to growth are some of the most complex and important metabolites of industrial interest. Secondary metabolites typically share a number of characteristics.

9. Contrast between production of primary and secondary metabolites.
Penicillin production by the mold Penicillium chrysogenum— an example of a secondary metabolite. Note that penicillin is not made until after the exponential phase.
Formation of alcohol by yeast—an example of a primary metabolite.
Primary metabolite
Secondary metabolite
Cells
Alcohol
Alcohol, sugar, or cell number
Penicillin, sugar, or cell number
Sugar
Cells
Formation of alcohol by yeast—an example of a primary
metabolite. (b) Penicillin production by the mold Penicillium chrysogenum—
an example of a secondary metabolite. Note that penicillin is not made
until after the exponential phase.
Sugar
Penicillin
Time
Time
Contrast between production of primary and secondary metabolites.

10. 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

11. 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
refers to any large-scale microbial process,
whether or not it is, biochemically speaking, a fermentation

12. Fermentor sizes for various industrial fermantations
Table 15.2 Fermentor sizes for various industrial fermentations

13. Figure 15.2 Fermentors.

14. Large-scale fermenters
cylinder, closed at the top and bottom, into which various pipes and valves have been fitted
Sterilization of the culture medium and removal of heat are vital for successful
operation
A critical part of the fermentor is the aeration system
a high density of microbial cells, there is a
tremendous oxygen demand by the culture
an aerator, called a sparger, and a stirring device, called an impeller
monitored in real time for temperature, oxygen, pH, and the levels of key nutrients, such as ammonia and phosphate.
Motor
Steam pH
pH controller
Acid–base reservoir and pump
Sterile seal
Viewing port
Filter
Exhaust
Impeller (mixing)
External cooling water out
Cooling jacket
External cooling water in
Culture broth
Figure 15.2 Fermentors.
Large-scale industrial fermentors are almost always constructed of stainless steel. Such a fermentor is essentially a large
cylinder, closed at the top and bottom, into which various pipes and valves have been fitted
A critical part of the fermentor is the aeration system. With large-scale equipment, transfer of oxygen throughout the growth medium is critical, and elaborate precautions must be taken to ensure proper aeration. Oxygen is poorly soluble in water, and in a fermentor with a high density of microbial cells, there is a tremendous oxygen demand by the culture. Because of this, two different devices are used to ensure adequate aeration: an aerator, called a sparger, and a stirring device, called an impeller
Sparger (high- pressure air for aeration)
Steam in
Sterile air
Valve
Harvest

15. 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.

16. The inside
of an industrial fermentor, showing the impeller and internal heating and cooling coils.
Figure 15.2 Fermentors.
Because sterilization
of the culture medium and removal of heat are vital for successful
operation, the fermentor is fitted with an external cooling jacket
through which steam (for sterilization) or water (for cooling) can
be run. For very large fermentors, sufficient heat cannot be transferred
through the jacket and so internal coils must be provided
through which either steam (for sterilization) or cooling water
(for growth) can be piped

17. 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

18. 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
If oxygen levels become limiting, even for a short period, the culture
may reduce—or even shut down—product formation.
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.

19. SCALE UP
Flask  laboratory fermentor(1-10 liter)  pilot plant( l)  commercial fermentor( 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.

20. A bank of small research fermentors used in process development
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.
Figure 15.3 Research and production fermentors.
(a) 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.
(b) A large bank of outdoor industrial-scale fermentors (each 240 m3) used in commercial production of alcohol in Japan.

21. Drugs, Other Chemicals, and Enzymes
Antibiotics: Isolation, Yield, and Purification
Industrial Production of Penicillins and Tetracyclines
Vitamins and Amino Acids
Enzymes as Industrial Products

22. Antibiotics: Isolation, Yield, and Purification
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

23. Some antibiotics produced commercially
Table 15.3 Some antibiotics produced commercially
b) EFB, endospore-forming bacterium; F, fungus; A, actinomycete.

24. 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).

25. 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

26. Method of testing an organism for its antibiotic spectrum of activity
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.

27. 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.

28. 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

29. 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
The parent structure of all penicillins is the compound 6-aminopenicillanic acid (6-APA), which consists of a thiazolidine ring with a
condensed -lactam ring (Figure 15.5).

30. Industrial Production of Penicillins and Tetracyclines
Penicillin G is produced in fermantors of 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.

31. Penicillin fermentation
Add precursor I
Biosynthetic penicillin I
Chemical or enzymatic
Add precursor II
Add precursor III
treatment of penicillin G
Biosynthetic penicillin II
6-Aminopenicillanic acid
Biosynthetic penicillin III
Add side chains chemically
Natural penicillins
(for example, penicillin G)
Semisynthetic penicillins
(for example, ampicillin, amoxycillin, methicillin)
Figure 15.5 Industrial production of penicillins.
Industrial production of penicillins. The-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.
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.

32. Glucose feeding
Figure :Kinetics of the penicillin fermentation with Penicillium chrysogenum.
Kinetics of the penicillin fermentation with Penicillium chrysogenum. Note that penicillin is produced as cells are entering the stationary phase, when most of the carbon and nitrogen has been exhausted. Nutrient “feedings” keep penicillin production high over
several days.
Nitrogen feeding
100 90 80
Penicillin 70
Biomass (g/liter), carbohydrate, ammonia, penicillin (g/liter  10) 60 50 40
Cells
Figure 15.6 Kinetics of the penicillin fermentation with Penicillium chrysogenum.
Kinetics of the penicillin fermentation with Penicillium chrysogenum. Note that penicillin is produced as cells are entering the stationary phase, when most of the carbon and nitrogen has been exhausted. Nutrient “feedings” keep penicillin production high over
several days. 30 20
Lactose 10
Ammonia 20 40 60 80
100
120
140
Fermentation time (h)

33. 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.

34. some key regulatory signals are known
Inoculum (spores on agar slant or in sterile soil)
Production scheme for chlortetracycline using Streptomyces aureofaciens.
Medium
Growth in optimal medium
2% Meat extract; 0.05% asparagine; 1% glucose;
0.5% K2HPO4; 1.3% agar
Agar plates
Spores as inoculum
2% Corn steep liquor; 3% sucrose; 0.5% CaCO3
some key regulatory signals are known
and are accounted for in the production scheme
Shake flask
Medium mimics production medium
24 h
Prefermentor
Same as for shake culture
19–24 h pH 5.2–6.2
1% Sucrose; 1% corn steep liquor; 0.2% (NH4)2HPO4; 0.1% CaCO3; 0.025% MgSO % ZnSO % and each of CuSO4, MnCl2
Fermentor
Production medium, no glucose, low phosphate
60–65 h pH 5.8–6.0
Figure 15.7 Production scheme for chlortetracycline using Streptomyces aureofaciens.
Production scheme for chlortetracycline using Streptomyces aureofaciens. The structure of chlortetracycline is shown on the bottom right. Glucose is used to grow the inoculum, but not for commercial production.
Glucose is used to grow the inoculum, but not for commercial production.
Antibiotic purification from broth after cell removal
Chlortetracycline

35. 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, tons per year.
As a coenzyme, vitamin B12 plays an important role in
microorganisms and animals in certain methyl transfers and
related processes.
Riboflavin (Figure 15.8b) is the parent compound of the flavins
FAD and FMN, coenzymes that play important roles in enzymes
for oxidation–reduction reactions

36. Vitamins produced by microorganisms on an industrial scale.
Figure 15.8
B12
Vitamins produced by microorganisms on an industrial scale.

37. Vitamins and 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.

38. Amino acids used in the food industry
Table 15.4 Amino acids used in the food industry

39. 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
digest insoluble polymers such as cellulose, protein, lipids, and starch, and because of this, have commercial applications in the food and health industries and in the laundry and textile industries

40. Microbial enzymes and their applications
Table 15.5 Microbial enzymes and their applications

41. 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.
These enzymes help remove stains from food, blood, and other organic rich substances by degrading the polymers into water-soluble components that wash away in the laundry cycle.
Other important enzymes manufactured commercially are amylases and glucoamylases, which are used in the production of glucose from starch. The glucose is then converted by a second enzyme, glucose isomerase, to fructose, which is a much sweeter sugar than glucose.

42. 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
Hypertermophiles

43. 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

44. Percent enzyme activity remaining
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
Percent enzyme activity remaining 10
Pullulanase
Starch
oligosaccharides
90°C 100°C 110°C 110°C plus Ca2
Figure Examples of extremozymes, enzymes which function under environmentally extreme conditions. 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.

45. 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

46. Enzyme inclusion in fibrous polymers Enzyme inclusion in microcapsules
Carrier-bound enzyme
Cross-linked enzyme
Procedures for the immobilization of enzymes.
Figure Procedures for the immobilization of enzymes.
Enzyme inclusion in fibrous polymers
Enzyme inclusion in microcapsules

47. 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.

48. 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

49. 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.

50. 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.

51. http://science. howstuffworks

52. A bioethanol production plant in Nebraska (USA)
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.
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

53. (c) The petroleum-producing colonial green alga, Botryococcus braunii
(c) The petroleum-producing colonial green alga, Botryococcus braunii. Note the excreted oil droplets that appear as bubbles along the margin of the cells.
Figure Biofuels.
Biofuels. (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 butanol. (c) The petroleum-producing colonial green alga, Botryococcus braunii. Note the excreted oil droplets that appear as bubbles along the margin of the cells.