The presentation enlists industrial microbiology description and industrial production of penicillin, citric acids and enzymes

1. INDUSTRIAL MICROBIOLOGY

2. • Industrial microbiology uses microorganisms, typically
grown on a large scale, to produce valuable commercial
products or to carry out important chemical
transformations.
• The actual reactions carried out by microorganisms in
industrial microbiology are called biocatalysis.
• Originated with alcoholic fermentation processes.
– Later on, processes such as production of
pharmaceuticals, food additives, enzymes, and
chemicals were developed

4. • An industrial microorganism must:
– produce the product of interest in high yield;
– grow rapidly on inexpensive culture media available in bulk
quantities;
– be amenable to genetic manipulation; and,
– if possible, be nonpathogenic.
• Microbial products of industrial interest include
– Enzymes
– Antibiotics, steroids, alkaloids
– Food additives
– Commodity chemicals
• Inexpensive chemicals produced in bulk
• Include ethanol, citric acid, and many others

5. • A primary metabolite is a kind of metabolite that is
directly involved in normal growth, development, and
reproduction. Eg: alcohol, lactic acid etc
• Secondary metabolites are organic compounds that
are not directly involved in the
normal growth, development, or reproduction of an
organism. Unlike primary metabolites, absence of
secondary metabolites does not result in immediate
death.
• Primary metabolites are produced during active cell
growth, and secondary metabolites are produced near
the onset of stationary phase

6. • Secondary metabolites
– Not essential for growth
– Formation depends on growth conditions
– Produced as a group of related compounds
– Often produced by spore-forming microbes during
sporulation
Primary
metabolite Secondary
metabolite
Alcohol
Penicillin
Cells
Sugar
Cells
Sugar
Time Time
Alcohol,sugar,orcellnumber
Penicillin,sugar,orcellnumber

7. • Many economically valuable microbial products are
secondary metabolites.
• Humans use secondary metabolites as food flavours,
medicines etc.
Antibiotic Producing microorganism
Cephalosporin Cephalosporium acrimonium
Chloramphenicol Streptomyces venezuelae
Erythromycin Streptomyces erythreus
Griseofulvin Penicillium griseofulvin
Penicillin Penicillium chrysogenum
Streptomycin Streptomyces griseus
Tetracycline Streptomyces aureofaciens
Gentamicin Micromonospora purpurea

8. INDUSTRIAL USES OF MOLDS

9. PENICILLIN
• A class of antibiotics that comes from mold.
• All penicillin like antibiotics inhibit synthesis of peptidoglycan,
an essential part of the cell wall and lyses it.
• They do not interfere with the synthesis of other intracellular
components.
• These antibiotics do not affect human cells because human
cells do not have cell walls.
• Penicillin include ampicillin, phenoxymethylpenicillin,
amoxicillin etc.
• Penicillin is active against Gram positive bacteria

10. • Some members (e.g. amoxicillin) are also effective
against Gram negative bacteria
• Penicillin was the first important commercial product
produced by an aerobic, submerged fermentation
• Used as input material for some semi synthetic
antibiotics.
• Aerobic processes require mechanisms for stirring and
aeration.
• Industrial fermentors can be divided into two major
classes, those for anaerobic processes and those for
aerobic processes

12. • Table 30.1 shows fermentor sizes for various industrial processes.

13. • The major steps in the commercial
production if pencillin are:
– Preparation of inoculum
– Preparation of sterilization of medium
– Inoculation of medium of the fermenter
– Forced aeration with sterile air during
incubation
– Removal of the mold mycelium after
fermentation
– Extraction and purification of pencillin
PRODUCTION OF PENICILLIN

14. • Raw Materials
– Carbon Sources: lactose acts as a very satisfactory compound.
6% is used. Glucose and sucrose can be used.
– Nitrogen Sources: Corn steep liquor can be used. Ammonium
acetate and ammonium sulphate can be used.
– Mineral Sources: minerals such as magnesium, phosphorous,
sulphur, pottasium, zinc and copper are essential for penicillin
production. Some of these are supplied by corn steep liquid.

15. • Fermentation Process
– The medium is inoculated with a suspension of
penicillium chrysogenum spores.
– The medium is constantly agitated, aerated and the
molds grows throughout.
– After above 8 days, pH rises above 8.0 and the
growth is complete and the penicillin production
ceases.

17. • Downstreaming process
– The product should be so pure so that it is dissolved
and separated as potassium salts to separate it from
other substances in the medium.
– The first step in product recovery is the separation of
whole cells and other insoluble ingredients from the
culture broth by centrifugation and filtration.
– pH is adjusted to 2-2.5 with the help of phosphoric
acids or sulphuric acids.
– This pH will help certain organic solvents to separate
from aqueous solutions.

18. • This step has to be carried out quickly as penicillin is
very unstable at low pH
• Antibiotic is then extracted back to a buffer solution with
pH 7.5. These shifts between the water and the solvent
purifies penicillin.
• The crude penicillin from the solvent is then treated with
sodium hydroxide and charcoal and then further
sterilized.
• Pure metal salts of penicillin can be sterilized by dry heat
method.

19. • Further Processing
– For use the antibiotic is packed in sterile vials as
powder or suspension.
– For oral use it is now tabletted now with a film coating

20. Main Stages of Penicillin Production
• A medium of corn steep liquor, lactose, salts and other
ingredients is mixed, sterilised, cooled and pumped into
the fermenter
• After 40 hrs, penicillin starts to secreted by the fungus.
• The mould mycelium is filtered from the harvested
product.
• Penicillin is extracted in the organic solvent, butyl
acetate, in which it is dissolved.

21. • Potassium
salts are
added and a
penicillin
precipitate is
formed, this is
washed and
dried.

22. PRODUCTS
• The resulting penicillin can be chemically and
enzymatically modified to make a variety of penicillins
with slightly different property.
• These semi synthetic penicillins include penicillin V,
penicillin O, ampicillin and amoxycillin.

24. CITRIC ACID
• Weak organic acid found in fruits.
• Produced by fermentation and suitable pH is around 3-6.
• Application In Industry
– Beverages
– Food
– Pharmaceutical
– Agriculture
– Metal Industry Structural Formula of Citric Acid

25. SOURCE OF RAW MATERIALS
Beet Molasses
• the source of sugar for
microbial production of citric
acid
• low cost and high sugar
content
• low content of trace metals
• acts as carbon source of the
fermentation
Microorganism
• mycomycetes of A.niger
species can produce high yield
• consequence of incomplete
respiration
Aspergillus Niger
• filamentous ascomycete fungus
• maintained at pH 4.5 and
temperature at 5 °C
• The best strain for citric acid
production

26. INDUSTRIAL PRODUCTION OF CITRIC
ACID
• Citric acid production is mixed growth associated mainly
takes place under nitrogen and phosphate limitations
after growth has ceased.
• Medium requirements for high production:
– Carbon Source: molasses or sugar solution
– Na-ferrocyanide is added to reduce iron and manganese
– High DO concentration
– High sugar concentration
– pH < 2
– Temperature : 30 degree celsius

27. • 5.25X106 A. niger spores/L may be introduced to the
fermentor.
• Aeration is provided to the fermentator. Temperature is
controlled by cooilng coils.
• Agitated at a speed of 50-100 rpm to avoid damage to
the spores.

28. • Downstreaming Process
– Two major purification processes involved:
– Filtration and precipitation
React citric acid with
calcium carbonate
Filter precipitate
calcium citrate
React precipitate
with sulphuric acid
Filter precipitate
calcium sulphate
Purified citric acid

29. • Citric acid broth from the fermentator is highly
contaminated with left over biomass, sugars, salts and
water.
• First the citric acid must be reacted with calcium
carbonate to neutralize the broth and form the insoluble
precipitate calcium citrate.
• Calcium citrate contains 74% of citric acid.
• It is then washed, heated and filtered to remove any no:
of contaminats.
• To crack calcium citrate, sulphuric acid is used.

30. • The reaction will produce free citric acid and a new
product, calcium sulphate, which will be removed later.
• In the filter calcium sulfate is washed away and left over
biomass is removed.
• Again the contamination that were present in the
fermentation broth can be removed by ultrafiltration or
nanofiltration.
• Citric acid can be produced in two forms: anhydrous and
monohydrate.
• These forms may require additional purification steps to
reach the desired purity.

31. • Monohydrate:
– Contains one water molecule for every citric acid
molecule
– Requires repeated crystallisation until the water
content reaches 7.5-8.8%.
• Anhydrous:
– Processed to remove all water from final product.
– Prepared by dehydrating monohydrate citric acid at a
temperature above 36.6 degree celsius

32. • Once the product has achieved desired impurity, it can
be sent for packaginf and distribution
• APPLICATIONS
– Flavour and preservative in food items
– Used in cleaning product and sodas
– Used to remove scale from boilers
– Used to soften water
– Emulsifying agent

33. Main stages in the process
1. Inoculation of Aspergillus Niger
2. Fermentation of Citric Acid
3. Biomass Removal
4. Liquid-liquid extraction
5. Crystallization
6. Drying

34. ENZYMES
• It is industrially feasible to concentrate and purify
enzymes from cultures of molds of Aspergillus,
Pencillium and Rhizopus.
• Mold enzymes; amylases, invertase, proteases and
pectinases are useful in processing wide variety of
materials

35. PRODUCTION OF ENZYMES
• The large scale production of enzymes involves culturing
micro-organisms in chambers called FERMENTERS or
BIOREACTORS
• COMMERCIAL ENZYME PRODUCTION - AN EXAMPLE
• Pectin is an insoluble substance found in the cell walls of
plants
• Pectinase is obtained from the fungus Aspergillus niger
PRODUCTION OF PECTINASE

36. PRODUCTION OF PECTINASE
Aspergillus niger is grown in
a fermenter with a source of
nitrogen, with sucrose as the
arbon source and the substrate
pectin to stimulate pectinase
production by the fungus
Filtration or centrifugation to obtain
a cell-free system containing
pectinase in solution
Evaporate to concentrate
the enzyme
Precipitate the pectinase
out of the solution and
filter the solid
Dry and purify the crude
pectinase
Pure, powdered
pectinase

37. IMMOBILIZATION OF ENZYMES
• Immobilisation of enzymes is an important technique
used in industry as it enables economical operation of a
process and protection of enzymes during their use
• The costs associated with the use of enzymes for
industrial purposes can also be reduced by immobilising
the enzymes
• Enzymes are immobilised by binding them to, or trapping
them in a solid support
• Various methods for immobilising enzymes are available

38. Enzymes are held on to a solid
support (matrix) by weak forces
such as hydrogen bonding
Enzymes are trapped within
the structure of a solid polymer
(usually in the form of beads)
– the enzyme is trapped rather
than bound
Methods for Immobilising Enzymes

39. Enzymes are covalently bonded
to a matrix such as cellulose
or collagen
Another more expensive method involves
enzymes which are both covalently bonded
to, and cross-linked within, a matrix
Cross-linking and covalent bonding may
cause some enzymes to lose their catalytic
activity especially if the active site is involve
in forming the linkages

40. ADVANTAGES OF IMMOBILIZED
ENZYMES
• Compared with free enzymes in solution, immobilised
enzymes have a number of advantages for use in industrial
processes
• The stability of many enzymes is increased when they are in
an immobilised state; they are less susceptible to changes in
environmental conditions such as temperature and pH
fluctuations
• Immobilised enzymes can be recovered and re-used,
reducing overall costs

41. • The products of the reaction are not contaminated with
enzyme eliminating the need to undertake costly
separation of the enzyme from the product
• Immobilising enzymes allows for continuous production
of a substance
• Biosensors are electronic monitoring devices that make
use of an enzyme’s specificity and the technique of
enzyme immobilisation