PPT on Antibiotics Production i.e. Penicillin

PPT on Antibiotics Production

1. •Antibiotics are antimicrobial agents
produced naturally by other microbes
(usually fungi or bacteria).
•The first antibiotic was discovered in 1896 by
Ernest Duchesne and "rediscovered" by
Alexander Flemming in 1928 from the
filamentous fungus Penicillium notatum.
Production of penicillinProduction of penicillin

2. •The antibiotic substance, named The antibiotic substance, named
penicillin, was not purified until the penicillin, was not purified until the
1940s (by Florey and Chain), just in 1940s (by Florey and Chain), just in
time to be used at the end of the time to be used at the end of the
second world war.second world war.
•Penicillin was the first important Penicillin was the first important
commercial product produced by an commercial product produced by an
aerobic, aerobic, submerged submerged fermentationfermentation

3. •When penicillin was first made at the When penicillin was first made at the
end of the second world war using the end of the second world war using the
fungus fungus P. notatumP. notatum, the process made 1 , the process made 1
mg dmmg dm
-3-3
. .
•Today, using a different species (Today, using a different species (P. P.
chrysogenumchrysogenum) and a better extraction ) and a better extraction
procedures the yield is 50 g dmprocedures the yield is 50 g dm
-3-3
. .
•There is a constant search to improve There is a constant search to improve
the yield. the yield.

4. •Antibiotics can be selectively toxic by Antibiotics can be selectively toxic by
targeting such features as the bacterial targeting such features as the bacterial
cell wall, 70S ribosomes, and enzymes cell wall, 70S ribosomes, and enzymes
that are specific to bacteria. that are specific to bacteria.
•In this way the human eukaryotic cells In this way the human eukaryotic cells
are unaffected. are unaffected.

5. For example:For example:
•penicillin, ampicillin, amoxycillin, penicillin, ampicillin, amoxycillin,
methicillinmethicillin
•Inhibits enzymes involved in synthesis Inhibits enzymes involved in synthesis
of peptidoglycan for bacterial cell wall, of peptidoglycan for bacterial cell wall,
causing cell lysis.causing cell lysis.
•BacteriocidalBacteriocidal
•Narrow spectrum- little effect on Gram Narrow spectrum- little effect on Gram
negative cells.negative cells.

6. Antibiotic Production MethodsAntibiotic Production Methods
•Antibiotics are produced on an industrial scale
using a variety of fungi and bacteria.
•Penicillin is produced by the fungus Penicillium
chrysogenum which requires lactose, other
sugars, and a source of nitrogen (in this case a
yeast extract) in the medium to grow well.
•Like all antibiotics, penicillin is a secondary
metabolite, so is only produced in the
stationary phase.
•It requires a batch fermenter, and a fed batch
process is normally used to prolong the
stationary period and so increase production.

8. •Downstream processing is relatively Downstream processing is relatively
easy since penicillin is secreted into easy since penicillin is secreted into
the medium i.e. extracellular productthe medium i.e. extracellular product
•However, the product needs to be very However, the product needs to be very
pure, since it being used as a pure, since it being used as a
therapeutic medical drug, so it is therapeutic medical drug, so it is
dissolved and then precipitated as a dissolved and then precipitated as a
potassium salt to separate it from other potassium salt to separate it from other
substances in the medium. substances in the medium.

9. The typical fermentation conditions for the
 
Penicllium 
mold,
usually requires:-
• temperatures at 20-24
o
C
•pH conditions - 6.0 to 6.5.
•The pressure in the bioreactor is usually much higher than the
atmospheric pressure(1.02atm) this is to prevent contamination
from occurring as it prevents external contaminants from
entering.
•Production fermenter- 200-500 cubic metres.
•Sparging of air bubbles is necessary to provide sufficient
oxygen the viability of the fungus. Depending on the volume of
medium, for 2 cubic metres of culture, the sparging rate should
be about 2.5 cubic metres per minute.
•The impeller is necessary to mix the culture evenly throughout
the culture medium, fungal cells are much hardy and they are
able to handle rotation speed of around 200rpm.

10. •Filtration is necessary at this point of the bioprocess flow, as bioseparation is
required to remove the biomass from the culture such as the fungus and other
impurities away from the medium which contains the penicillin product. There are
many types of filtration methods available today, however, the Rotary vacuum filter
is commonly employed as it able to run in continuous mode in any large scale
operations. Filtrate is cooled to 2-4
o
C to avoid enzymatic degradation.
•penicillin rich solution is then treated with 0.25-5% activated carbon to remove
pigments and impurities.
•In order to extract the penicillin present in the filtrate, organic solvents such as amyl
acetate or butyl acetate are used as they dissolve penicillin much better than water
at physiological pH. At this point, penicillin is present in the solution and any other
solids will be considered as waste. Solvent extraction is achieved at low pH 2.5-3.0.
Continuous, countercurrent, multistage centrifugal extractors are used.
•Crystallization- rotary vacuum filters are used to separate crystals.
•Drying is necessary to remove any remaining moisture present in the powdered
penicillin salt. In fluid bed drying, hot gas is pump in from the base of the chamber
containing the powdered salt inside a vacuum chamber.
Recovery:-

14. 1.1.What is the Carbon source?What is the Carbon source?
2.2.What is the nitrogen source?What is the nitrogen source?
3.3.What is the energy source?What is the energy source?
4.4.Is the fermentation aerobic or anaerobic?Is the fermentation aerobic or anaerobic?
5.5.What is the optimum temperature?What is the optimum temperature?
6.6.Is penicillin a primary or secondary metabolite?Is penicillin a primary or secondary metabolite?
7.7.What volume fermenter is used?What volume fermenter is used?
8.8.Why isn't a larger fermenter used?Why isn't a larger fermenter used?
9.9.When is penicillin produced?When is penicillin produced?
10.10.How long can it be produced for?How long can it be produced for?
11.11.What was the first fungus known to produce penicillin?What was the first fungus known to produce penicillin?
12.12.What species produces about 60mg/dm3 of penicillin?What species produces about 60mg/dm3 of penicillin?
13.13.How did scientists improve the yield still further?How did scientists improve the yield still further?
14.14.What is the substrate?What is the substrate?
15.15.Why is batch culture used?Why is batch culture used?
16.16.What are the processes involved in down-stream processing?What are the processes involved in down-stream processing?
a)a)
b)b)
lactoselactose
yeastyeast
glucoseglucose
aerobicaerobic
25 - 2725 - 27ºCºC
secondarysecondary
40 – 400 dm40 – 400 dm
33
To difficult to aerateTo difficult to aerate
40 hours – after main increase in fungal mass40 hours – after main increase in fungal mass
140 hours140 hours
Penicillin notatumPenicillin notatum
Penicillin chrysogenumPenicillin chrysogenum
Genetic modificationGenetic modification
Corn steep liquorCorn steep liquor
Secondary metaboliteSecondary metabolite
Filtration of liquidFiltration of liquid
Extraction from filtrate by counter current of butylacetateExtraction from filtrate by counter current of butylacetate

15. -Microorganism: Aspergillus niger (mainly), Candida yeast (from
carbohydrates or n-alkanes)
-Citric acid production is mixed growth associated, mainly take place
under nitrogen and phosphate limitation after growth has ceased.
-Medium requirements for high production
- Carbon source: molasses or sugar solution.
- Na-ferrocyanide is added to reduce Iron (1.3 ppm) and manganese
(<0.1ppm).
- High dissolved oxygen concentration
- High sugar concentration
- pH<2
- 30
o
C.
Industrial Production of Citric Acid

16. Bioreactor: batch or fed-batch (100m
3
)
-5-25X10
6
A. niger spores/L may be introduced to the fermentor.
-Aeration is provided to the fermenter by air sparging (0.1-0.4 vvm)
-Temperature is controlled by cooling coil.
-Agitation: 50-100rpm to avoid shear damage on molds.
-Fed-batch is used to reduce substrate inhibition and prolong the
production phase one or two days after growth cessation.
-Volumetric yield: 130 kg/m
3
Industrial Production of Citric Acid

17. Separation:
- The biomass is separated by filtration
- The liquid is transferred to recovery process:
- Separation of citric acid from the liquid:
precipitation
calcium hydroxide is added to obtain calcium
citrate tetrahydrate → wash the precipitate→
dissolve it with dilute sulfuric acid, yield citric
acid and calcium sulfate precipitate → bleach
and crystallization → anhydrous or monohydrate
citric acid.
Industrial Production of Citric Acid

18. The citric acid broth from the production fermenter is highly contaminated by
leftover biomass, salts, sucrose, and water.
First, the citric acid must be reacted with calcium carbonate to neutralize the
broth and form the insoluble precipitate calcium citrate. (pH 7.2 , 90
o
C) Calcium
citrate contains about 74% citric acid. The
stoichiometric equation is as follows:
CaCO
3 + Citric Acid → CO
2+ Calcium Citrate
The calcium citrate is then washed, heated, and filtered to remove any number of
the contaminants.
To crack the calcium citrate precipitate, sulfuric acid is needed. The temperature
of this reaction should stay below 60ºC. The reaction will produce free citric acid
and a new precipitate, calcium sulfate, which will need to be removed later.
The citric acid solution is concentrated by vacuum evaporation and crystallized
at low temperature.

19. Yields 60-75%
Generic Platform Process for
Purification of MAbs
Upstream Downstream
Virus Removal
Nanofiltration
Formulation
UF
Aggregates
Degradatio
n products
Low pH virus
inactivation
Cells

20. Downstream processing in smallpox vaccine manufacturing

21. STREPTOMYCIN
•Streptomycin was the first aminoglycoside that was successfully used to treat tuberculosis.
It is isolated from the organism Streptomyces griseus. Usually aminoglycosides are
regarded as reverse antibiotics as resistance may develop easily. These are very potent
against Gram positive and negative bacteria besides mycobacteria. At the molecular level,
aminoglycosides bind to 30S ribosome and block protein synthesis. Prolonged use of
theseantibiotics causes damage to kidneys, and hearing impairment.
•PRODUCTION PROCESS OF STREPTOMYCIN
The medium used for streptomycin usually consists of soy meal or soy flour or corn
syrup that can supply glucose at a slow rate (amylase activity is poor in streptomyces
sp.). The initial supply of nitrogen and phosphate is also obtained from soy meal.
This is required since glucose, ammonia and phosphate in high quantities inhibit
streptomycin synthesis. The fermentation conditions are –temperature 27-30 C,

pH 6.5-7.5, and aeration rate 0.5-1vvm. The duration of fermentation process
depends on the strain used, and is between 6-8 days.

23. •The various salts of streptomycin are :(i)
Trihydrochloride(ii) Trihydrochloride-cadium(iii)
Pantothenate(iv) Sesquisulfate
•Medium for Growth: Medium for the fermentative process
of streptomycin production essentially comprises of :(a)
Carbon Source : e.g., dextrin, glycerol, glucose, starch,(b)
Nitrogen Source : cotton seed meal, soyabean meal,
casein- hydrolysate, yeast and its extracts ; pure
inorganic salts : ammonium, ammonium nitrate.(c)
Vegetable/Animal Fat : e.g. soyabean oil, linseed oil.

30. •4.2.2.4. RECOVERY OF STREPTOMYCIN
•Broth, after harvest, is filtered. Streptomycin or other aminoglycosides are basic in
nature. They can be recovered by weak cationic exchange resins in an ion
exchange column. Streptomycin is eluted from resin column as streptomycin
sulphate. Treatment with activated carbon is often necessary to remove impurities.
Streptomycin can be precipitated in the form of sulphate salt.
•VITAMIN B12
Vitamin B12 is present in animal tissue at a very low concentration. It occurs
mostly in the coenzyme forms methylcobalamin and
deoxyadenosylcobalamin. Isolation of vitamin B12 from animal tissue is
highly expensive. Deficiency causes pernicious anemia. It is water soluble
vitamin with complex structure. It consists of a corrin ring with a central
cobalt atom. The corrin ring is almost similar to the tetrapyrrole ring
structure found in porphyrin compounds. The corrin ring has four pyrrole
units and bonded with four pyrrole nitrogens with cobalt. Microorganisms
used for commercial synthesis of vitamin are Propionibacterium
freudenreichii, Pseudomonas denitrificans, Bacillus megaterium,
Streptomyces olivaceus.

31. •It is commercially produced by fermentation. It was first obtained as a byproduct of
•Streptomyces fermentation in the production of certain antibiotics (streptomycin,
chloramphenicol or neomycin).
•Production using Propionibacterium sp: P.freudenreichii and P.shermanii and their mutant
strains are used for vitamin synthesis. Process is carried out by adding cobalt in two phases.
•Anaerobic phase: this is preliminary phase that may take 2-4days. In the anaerobic phase 5’-
deoxyadenosylcobinamide is predominantly produced.
•Aerobic phase: in this phase, 5,6-dimethyl benzimidazole is produced from riboflavin which
gets incorporated to finally form coenzyme of vitamin B12 namely
5’-deoxyadenosylcobalamin. In recent years, some fermentation technologists have successfully
clubbed both an anaerobic and aerobic phases to carry out the operation continuously in two
reaction tanks. The bulk production of vitamin is done mostly by submerged bacterial
fermentation with beet molasses medium supplemented with cobalt chloride.
•4.3.2.2. RECOVERY OF VITAMIN B12
The cobalamins produced by fermentation are mostly bound to the cells. They can be solubilized
by heat treatment at 80-120 C for about 30minutes at pH 6.5-8.5. The solids and mycelium
are filtered or centrifuged and the fermentation broth collected. The cobalamins can be
converted to more stable cyanocobalamins.