Industrial Microbiology

Learning objectives
At the end of this topic you will be able to explain following:
• Definition
• Microorganisms used in Fermentation
• Industrial scale fermentation
• Fermentation techniques
• Types
• Biologicals obtained from fermentation
• Steps in preparation of antibiotics
• Production of penicillin, cephalosporin's, erythromycin, tetracycline, gentamycin, rifamycin
and Griseofulvin.
INTRODUCTION:
o Branch of Microbiology which deals with the study and use of various micro-
organisms that are responsible for the production of many products such as alcoholic
products, antibiotics, enzymes, vaccines.
o Use of Genetically Modified organisms – An advancement in the field of Industrial
Microbiology resulting in Increased productivity
Applications of Industrial Microbiology
o Bacteria, Fungi, Yeast etc. are used commonly in the production of various fermented
products like wine, yogurt etc.
o Strains of Corynebacterium glutamicum have been used in the production of the
amino acid, L-glutamate.
o Production of food and dairy products. Cheese, yogurt, alcoholic beverages, coffee,
tea, vitamins etc are some of the examples.
o Production of vaccines is another important application of industrial microbiology.
o Antibiotics are another important products produced by using micro-organisms.
Fermentation
o The word Fermentation is derived from Latin word fervere which means to boil.
o It is a process by which the living cell is able to obtain energy through the breakdown
of glucose without requiring oxygen
o Fermentation produce two ATP molecule per cycle
o Fermentation technology is the use of these microorganisms to create the products
on large scale for the mankind
• But the conventional definition of Fermentation is to break down of larger molecules
into smaller and simple molecules using microorganisms.
• In Biotechnology, Fermentation means any process by which microorganisms are
grown in large quantities to produce any type of useful materials.
• Fermentation produces 2ATP molecules per cycle
  It formation of alcohol (Ethanol) from glucose
in the absence of oxygen and presence of
microorganism
ATP
ATP

Alcoholic Glucose Glycolysis Pyruvic Acid

fermentati
NADH
on NAD+
NAD+ NADH

Ethyl Alcohol Acetaldehyde

Alcoholic fermentation of glucose

C6H12O6 → 2 C2H5OH + 2 CO2
glucose ethanol carbon dioxide
• Louis Pasteur in the 19th century used the term fermentation in a narrow
sense to describe the changes brought about by yeasts and other
microorganisms growing in the absence of air (anaerobically).

• He also recognized that ethyl alcohol and carbon dioxide are not the only
products of fermentation.
Fermentation products
(Some Examples)
 Microorganisms used in Fermentation
o Microorganisms used in Fermentation include bacteria, fungi, algae and
actinomycetes. The commonly used species are-
o Bacteria: Acetobacter lacti, Acetobacter woodi, Bacillus subtilis, Bacillus polymyxa,
Clostridium etc.
o Algae: Spirulina maxima, Chlorella sorokiniana etc.
o Fungi: Aspergillus oryzae, Aspergillus niger, Saccharomyces cervisae, Saccharomyces
lipolytica etc.
o Actinomycetes: Streptomyces griseus, Streptomyces noursei etc.
Industrial scale fermentation
• On industrial scale fermenter is used for fermentation
• It is a large tank used to create environment for fermentation
• It contains media for the process of fermentation
Requirements
1. Pure culture(bacteria, yeast etc) in appropriate quantity
2. Sterilized media for rapid and uniform growth of micro-organisms
3. Seed fermenter for growth of pure culture
4. Production fermenter for growth on industrial scale
5. Different machines and instruments for
a) Drawing culture media
b) Cell separation
c) Collection of cells
d) Product purification
Fermentation techniques
• Two type of techniques are used for fermentation
• 1. Surface technique
• 2. Submersion technique
1. Surface technique
o A complex process
o Micro-organisms are cultivated on the surface of liquid or solid media
o E.g., Mushrooms, bread are prepared by this method
2. Submersion technique

• Micro-organisms are grown in liquid media

• E.g., antibiotics and enzymes are prepared by this method and sewage treatment are
carried out by this way
•
Types of fermentation
1. Batch fermentation
2. Continuous fermentation
3. Fed batch fermentation
Batch fermentation
o Pure, clean and sterilized fermenter is set ready for the process
o Sterile nutrient substrate is prepared having all nutrients for specific organism
growth
o Substrate is inoculated with specific organism and is incubated
o Incubation time is specific for specific organism
o Organism is grown and produce product
o When substrate get exhausted of nutrient, cells go on dying and no growth is evident
now
o It is then cleaned and harvested
o Fermenter is again set for another batch
Continuous fermentation
o It is similar to batch fermenter
o Here nutrient substrate is continually added to fermenter
o The product is also removed continually from the system
o Due to continuously supply of nutrients the cell extends its stay in LOG phase of the
growth cycle
o Thus unlike batch fermentation, in continuous fermentation, the fermentation
process never stops in between and it continues to run for long period of time with
the addition of nutrients.
Fed batch fermentation
o Most advance and recent way of fermentation
o It is the blend of batch fermentation and continuous fermentation
o Initially the batch is run on nutrient substrate
o When cells are in stationary phase, more substrate is added with the removal of the
product at the same time
o Product is also collected from time by time
o Cell remains in the log phase of growth cycle
Schematic Representation
Common features of typical fermenter:
• 1. They should be strong enough to withstand the pressure exerted by large volume
of the medium.
• 2. The materials used for the construction of fermenter should not be corroded by
the fermentation product and it should not yield toxic ion to the medium.
• 3. The fermenter should have provision for the control and prevention of the growth
of contaminating microorganisms because industrial fermentation requires pure
culture.
• 4. If aerobic organisms are used in the process, there should be provision for rapid
incorporation of sterile air into the medium so that the oxygen is immediately
dissolved in the medium and available to the microorganisms.
• 5. The Carbon dioxide produced by the microorganisms should be removed from the
medium.
• 6. Stirring is necessary to mix the organisms with the medium and to make
nutrients and oxygen available to individual microbe.
• 7. The fermenter should provide provision for the addition of antifoaming agents
intermittently depending on the foaming status of the medium.
• 8. Thermostatic system should be available to maintain constant temperature in
the fermenter.
• 9. There should be provision for aseptic withdrawal of culture during
fermentation and also for the aseptic introduction of inoculum at the starting of
the fermentation process.
• 10. A system should be available for detection of pH of the culture medium and
also for its adjustment.
Biologicals obtained from fermentation:

• 1. Antibiotics • 3. Vitamins
• Penicillin • Riboflavin
• Streptomycin • Cyanocobalamine
• Tetracycline • 4. Organic acids
• 2. Solvents • Lactic acid
• Ethanol • Citric acid
• Glycerol
Advantages
o Important contributor to human nutrition
o Used to improve digestibility, taste and flavor of the food
o Low operating cost
o Low production energy use
Disadvantages
o Microbial contamination may exist in the food all the time
o Rarely causes death
ANTIBIOTICS
o Substances which kill or inhibit the growth of micro-organisms.
o Penicillin is an example discovered by Alexander Fleming in 1929.
o Action of the antibiotics:
They can either destroy the bacterial cells completely or can affect their metabolism.
They can be used either externally as an ointment or can also be given for internal use
either orally or through injections.
Types of antibiotics:
o Penicillin(penicillin, amoxycillin)
o Cephalosporin (cephalexin)
o Macrolides(Erythromycin)
o Tetracycline(tetracycline, doxycycline)
o Broad range antibiotics(can kill both gram-positive and gram-negative bacteria eg.
Ampicillin)
o Narrow range antibiotics(can kill only a particular type of bacteria(eg. Azithromycin).
Raw materials for the production of antibiotics:
o Nutrient media like a nutrient broth(liquid media)
o Carbon source(such as molasses)
o Nitrogen sources(such as ammonia)
o Some of the trace elements like phosphorous , zinc etc. which help in the growth of
these microbes.
STEPS IN THE PREPARATION OF
ANTIBIOTICS
• Following steps are involved in the production of antibiotics

• 1. Starting the culture

• 2. Fermentation
• 3. Isolation and purification
• 4. Refining
• 5. Quality control
1. Starting the culture:
• To initiate the growth of the desired micro-organism, A starter culture is taken from
the previous cold- stored organism
• and is grown on the agar plate.
• A pure culture is isolated and is transferred along with the required nutrients and
food into a shake flask to obtain the desired suspension
• which is then transferred into the seed tank.
• The seed tanks contain all the necessary vitamins, nutrients, carbon, nitrogen
sources for the growth of the cultures.
• A proper supply of air is also done to these tanks along with constant mixing of the
contents and after 24-48 hours,
• these contents are transferred to the primary fermentation tank.
2. Fermentation:
• The fermentation tank has an environment where the micro-organisms can excrete
the desired antibiotic. These tanks have a capacity of about 30,000 gallons and to
maintain proper growth, and pH, the required nutrients and either acids or bases are
added to it. The temperature of these tanks is maintained around 23-27.2 ° C.
3. Isolation and purification:
• The desired antibiotic will be produced in maximum amounts after 3-5 days. Water
soluble antibiotics are isolated by using Ion-exchange method and the Oil-soluble
ones are extracted by using solvent- exchange methods.
4. Refining:
• The antibiotics can be used in various forms such as the Intravenous bags or syringes,
ointments, gels, capsules etc. Based on the usage of the antibiotic, the powdered
antibiotic is converted to any of the above said forms and finally sent for packing and
delivery to the pharmacy, hospitals etc.
5. Quality control
• The equipment used in the entire process are steam sterilized and also frequent
checks are made about the purity of the microbial culture. pH, melting point and
moisture content of the finished product are checked before dispersing the final
product to ensure purity and safety.
Production of penicillin
o In 1928, Alexander Fleming was studying the growth and characteristics of
staphylococci at Saint Marry’s Hospital, London
o He accidently found that some molds coming from the contaminated material inhibit
the growth of the staphylococci
o He gave interpretation that, this fungus might have produced some bacteriolytic
agents that cause inhibition of the growth of the microorganism
o Later on in 1949, he called this fungus Penicillium rubrum, so it become the reason
of the naming penicillin
o Penicillin is produced by the fungus Penicillium chrysogenum
o Growth medium required is lactose, sugars and source of nitrogen
o It is secondary metabolite and produced only in the stationary phase
o It require batch fermenter or fed batch fermenter
Growth curve of Fungus used for Penicillin production
Fermenter for penicillin production
Production of penicillin procedure

o After everyday about 30% culture is removed and replaced with the fresh medium
o Fungal cells are separated from the culture through rotating filters
o Filtrate is then dissolve in butyl acetate
o Potassium ions are added to precipitate the salt of the penicillin
o Precipitate are wash, filter and dried
o Penicillin obtain is called as Penicillin G
o It is further treated enzymatically and chemically to produce new antibiotics i.e.
Penicillin V, ampicillin and amoxicillin etc.
Production of Cephalosporin
o In 1945, Prof. Brotzuin Italy questioned that typhoid is less prevalent in his city then
that of surrounding cities
o He investigate it and found that many of the young people take regular bath in the
sea exactly at the same point where city sewer system drained into the sea
o Still they are free from disease??
o He took water sample from that place and tests its effect on Salmonella typhi
o As a result he isolate a fungus that produced an effective substance against gram-
negative Salmonella
o Cephalosporin is produced by the fungus Acremonium chrysogenum
o Major growth medium required is sucrose and methionine
o Like penicillin, it is also secondary metabolite and produced only in the stationary
phase
o It require batch fermenter or fed batch fermenter
Production of cephalosporin procedure

o Culture is taken from the fermenter and filtered with help of filter aid
o Its is then passed through the resin column containing amberlite400 to separate all
materials other then cephalosporin C
o The resultant products are then freeze dried to get powdered cephalosporin
Production of Erythromycin

o In 1949, AbelardoB. Aguilar, a Filipino scientist, sent some soil samples to his
employer Eli Lilly
o Eli Lilly’s research team, led by J. M. McGuire, managed to isolate erythromycin from
the metabolic products of a strain of Streptomyces erythreus found in the samples
o Lilly filed its patent in 1952 and produced brand name Ilosone
• Erythromycin is produced by the bacterium Streptomyces erythraeus
• Growth medium required for its growth must contain sucrose, Corn steep liquor,
Soyabean oil, Yeast, NaCl and CaCO3
• It is secondary metabolite and produced only in the stationary phase
• It require stirred tank fermenter or continuous fermenter
Production of erythromycin procedure

o The sporulating culture of Streptomyces erythraeusis done on Tryptone agar slant

o Cells are harvested from agar plates and the suspension is taken in sterile water
o The suspension is then store at 4°C
o Fermentation is carried out in stirred tank fermenter
o Mycelium is separated from the broth by centrifugation or drum filtration
o Acidic condition is maintained by addition of butyl acetate which favors the
separation of the mycelium
o Erythromycin is water soluble so it will go into the acidic media
Production of Tetracycline

o It is a large family of antibiotics

o Discovered as natural products by Benjamin Minge Duggarin 1945
o First prescribed in for the treatment in 1948
Production of Tetracycline Procedure

o Tetracycline is produced by the bacterium Streptomyces aurofaciens

o Growth medium required for its growth must contain sucrose, ammonium sulphate,
acetic acid and other trace elements like Mn(manganese), Zn, Ca and Cr(chromium).
o Growth medium must contain low chloride contents otherwise it will produce
chlortetracycline
o It is secondary metabolite and produced only in the stationary phase
o It require Shaker flask fermenter
o For fermentation medium is set at 30°C
o Rotation is set at 200 rpm
o Incubation is carried out for 4-6 days
o For isolation whole broth is acidified to pH 2.0 to 2.5 with 5N H2SO4
o It is then filtered with Whatman filter paper
o Tetracycline is separated and purified
Production of gentamicin
o Fermentation by micromonospora species echinospora and purpurea produces a
family of aminocyclitol antibiotic called gentamicin.
o Gentamicin is board spectrum basic and water soluble antibiotic first reported by
weinstein.
o Gentamicin is highly effective against gram negative bacteria and mycobacterium
tuberculosis.
o Gentamicin is aminocyclitol containing aminoglycoside antibiotic complex consisting
predominantly three major components gentamicin C1, C2 and C1a.
o Process is batch fermentation.
o Echinospora strain is used as production strain. Culture is maintained in agar slant
containing: Yeast extract, Glucose, CaCO3, Agar
o Inoculation Medium contain beef extract, Glucose, Soluble starch, Yeast extract,
CaCO3 at pH 7.6
o Culture is inoculated with M. echinospora under specific conditions.
o The inoculated flask is kept on rotary shaker at 200 rpm at 27°C for 72 hours to
obtain growth.
Procedure
o These are inoculated with 5% (v/v) of 48 hours inoculum.
o Inoculated flasks is incubated for 10 days and observe pH concentration.
o Production of antibiotic determined by disc diffusion using staphylococcus aureus
MTCC as assay organisms.
o pH value of culture medium is increased from 7.3 - 8.8.
o Biosynthesis of gentamicin starts during 3 days and reached maximum at stationary
phase at day 6 of fermentation period.
o It is filtered by vaccum filteration and drying at 100°C for 4 hours.
Production of Rifamycin
o This group of antibiotics synthesized either naturally by bacterium Amycolatopsis
rifamycin or artificially.
o It is used to treat mycobacterium disease such as T.B
o Gram positive bacteria in Amycolatopsis genus produces rifamycin
o Rifamycin SV show good activity, tolerability and solubility properties and used to
treat infections from gram positive bacteria
o Stock cultures of Nocardia mediterranei is maintained in sealed ampoules in
lyophilized state.
o Vegetative medium includes Peanut meal, Soya bean meal, Ammonium sulphate,
Propylene glycol and Glucose
o Yield can be obtain when 5 % inoculum of pre-culture grown for 24 hours is used in
aerated fermenters at 28°C
o It gives a yield of 6-7 g/l
o An air flow ranging from 0. 8-1.5 l/min is essential
o Fermentation is carried out in conventional bioreactor of 20-100 m3 volume
o In fermenter consumption of glucose, growth of mycelium and production of
antibiotic occur at the same time
o An initial stage consumption of glucose and nitrogen is slow and fast when mycelial
growth starts
o pH changes to 6.2-6.4 which is optimal pH
o Fermentation last for 300 hours
o Purification is done at pH 7.5-8.0
o Filterate is acidifies having 2.0 pH
o Extraction of product is carried out by chloroform
Production of Griseofulvin
o Griseofulvin is antifungal antibiotic first isolated from a penicillium species in 1939
o Secondary metabolite produced by fungus penicillium griseofulvin
o Compound is insoluble in water and slightly soluble in ethanol, methanol, acetone,
benzene, CHCl3 and acetic acid..
o The pH of Czapek Dox medium was adjusted between 6.0-7.2. The medium was
dispensed in fermenter.
o Fresh sample of mycelial suspension of fungus penicillium griseofulvin from fresh
slope on raper steep agar(Czapek Dox medium + cornsteep + agar) was obtained.
o Solution was autoclaved for 200 minutes at 120°C at 15 lbs pressure and fermented
for 14 days at 24°C
 Pretreatment of fermentation broth
o Broth is heated above 60°C for 20-30 minutes.
o After heating sufficient coagulation of material occur to produce valuable improvement in
separation characteristics of broth.
o Period of heating is short 5-10 minutes at 80°C having been found to provide satisfactory
increase in filtration rate.
Filtration
o Drum covered with diatomaceous earth matter and allowed to rotate under vacuum with half
immersed in slurry tank.
o Small amount of coagulation agent added to broth and pumped into slurry tank.
o As drum rotates in slurry tank under vacuum thin layer of coagulated particle adhere to drum.
o The layer thicken to form cake. As cake portion in drum come to upper region which is not
immersed in liquid it is washed with water and dewatered immediately by blowing air over it.
o Then before dried portion is again immersed into liquid it is cut off from drum by knife.
o Griseofulvin is extracted by cold acetone when it is used as an extracting agent.
o The extraction with cold acetone may be carried out with efficiencies between 75-96% or even
up to 99.5% the quantity of solvent used in extraction at large scale production should be kept
minimum.
o The volume of acetone should be 3-5 time of mycelial felt.
o The color of extract can be improved by addition of calcium hydroxide usually
2.5-50g/liter preferably 5-30g/liter.
o The pH of extract should be above 10.
o It can be neutralize by removal of lime or by using mineral acid.
o The impurities or waxy substance are removed by washing extract with solvent in
which extract is immiscible and also griseofulvin is insoluble.
o Hydrocarbon solvent such as hexane are in general suitable for that step.
o Griseofulvin are precipitated from solvent extract in various ways.
o One of the method is using the liquid solvent in which griseofulvin is insoluble.
o The alkaline water is more effective for removal of colored impurities present in
crystal of griseofulvin.
o Water is made alkaline with ammonia or an alkali metal carbohydrate or alkali
metal hydroxide.
o The suitable pH is about 8.5
o The purity of precipitate is generally improved by washing with a solvent for small
quantities of impurities remaining.
o Marked Purification is obtained with use of methanol for this step.
Reference:-
• Hugo and Russell’s Pharmaceutical Microbiology, EDITED BY Stephen P Denyer
(Chapter 22 Manufacture of antibiotics)
• Prescott’s microbiology