The document discusses the production of the antibiotic penicillin. It describes how penicillin is produced through the fermentation of Penicillium chrysogenum fungus. Key steps include using lactose and yeast extract as a carbon and nitrogen source, respectively, in an aerobic fermentation process at 25-27C. Downstream processing after fermentation involves filtering cells from the liquid and extracting and precipitating the penicillin product. The yield has increased from 1 mg/L originally to over 50 g/L today through strain and process improvements.

1. Production of penicillinProduction of penicillin
Learning objective:Learning objective:
 To be able to identify useful productsTo be able to identify useful products
from microorganismsfrom microorganisms
 To be able to identify theTo be able to identify the
microorganisms used and the mainmicroorganisms used and the main
stages in the production of penicillin.stages in the production of penicillin.
 To be able to describe how DownstreamTo be able to describe how Downstream
processing is carried out to extract andprocessing is carried out to extract and
purify the end-product of fermentation.purify the end-product of fermentation.

2. Downstream Processing
 Products in a fermenter are impureProducts in a fermenter are impure
and dilute, so need to be purified byand dilute, so need to be purified by
downstream processing.downstream processing.
 This usually involves filtration toThis usually involves filtration to
separate the microbial cells from theseparate the microbial cells from the
liquid medium, followed by chemicalliquid medium, followed by chemical
purification and concentration ofpurification and concentration of
the productthe product
 Downstream processing can accountDownstream processing can account
for 50% of the cost of a process.for 50% of the cost of a process.

3.  Antibiotics are antimicrobial agents produced naturally by otherAntibiotics are antimicrobial agents produced naturally by other
microbes (usually fungi or bacteria).microbes (usually fungi or bacteria).
 The first antibiotic was discovered in 1896 by Ernest Duchesne andThe first antibiotic was discovered in 1896 by Ernest Duchesne and
"rediscovered" byAlexander Flemming in 1928 from the filamentous"rediscovered" byAlexander Flemming in 1928 from the filamentous
fungusfungus Penicilium notatuPenicilium notatum.m.

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

5.  When penicillin was first made at the end of the second world warWhen penicillin was first made at the end of the second world war
using the fungususing the fungus Penicilium notatumPenicilium notatum, the process made 1 mg dm, the process made 1 mg dm-3-3..
 Today, using a different species (Today, using a different species (P. chrysogenumP. chrysogenum) and a better) and a better
extraction procedures the yield is 50 g dmextraction procedures the yield is 50 g dm-3-3..
 There is a constant search to improve the yield.There is a constant search to improve the yield.

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

7. For example:(For example:( Not incNot inc))
 penicillin, ampicillin, amoxycillin, methicillinpenicillin, ampicillin, amoxycillin, methicillin
 Inhibits enzymes involved in synthesis ofInhibits enzymes involved in synthesis of
peptidoglycan for bacterial cell wall, causing cellpeptidoglycan for bacterial cell wall, causing cell
lysis.lysis.
 BacteriocidalBacteriocidal
 Narrow spectrum- little effect on Gram negativeNarrow spectrum- little effect on Gram negative
cells.cells.

8. (N.I)(N.I)
Other antibiotics MO may affect:Other antibiotics MO may affect:
 Cell membraneCell membrane
 DNA replicationDNA replication
 TranscriptionTranscription
 TranslationTranslation

9. Antibiotic productionAntibiotic production
 There are over 10 000 differentThere are over 10 000 different
antibiotics known, but only aboutantibiotics known, but only about
200 in commercial200 in commercial
 use, since most new antibiotics areuse, since most new antibiotics are
no better than existing ones.no better than existing ones.
 There is a constant search for newThere is a constant search for new
antibiotics. Antibiotics are the most-antibiotics. Antibiotics are the most-
prescribed drugs and are bigprescribed drugs and are big
business.business.
 Finding a new antibiotic and gettingFinding a new antibiotic and getting
it on to the market is a very longit on to the market is a very long
process and can take 15 years.process and can take 15 years.

10. Antibiotic ProductionAntibiotic Production
MethodsMethods
 Antibiotics are produced on an industrial scaleAntibiotics are produced on an industrial scale
using a variety of fungi and bacteria.using a variety of fungi and bacteria.
 Penicillin is produced by the fungusPenicillin is produced by the fungus PenicilliumPenicillium
chrysogenumchrysogenum which requires lactose, other sugars,which requires lactose, other sugars,
and a source of nitrogen (in this case a yeastand a source of nitrogen (in this case a yeast
extract) in the medium to grow well.extract) in the medium to grow well.
 Like all antibiotics, penicillin is a secondaryLike all antibiotics, penicillin is a secondary
metabolite, so is only produced in the stationarymetabolite, so is only produced in the stationary
phase.phase.
 What sort of fermenter does it require?What sort of fermenter does it require?
 It requires a batch fermenter, and a fed batchIt requires a batch fermenter, and a fed batch
process is normally used to prolong the stationaryprocess is normally used to prolong the stationary
period and so increase production.period and so increase production.

12.  (N.I)(N.I)
 Downstream processing is relatively easy since penicillin is secretedDownstream processing is relatively easy since penicillin is secreted
into the medium (to kill other cells), so there is no need to break openinto the medium (to kill other cells), so there is no need to break open
the fungal cells.the fungal cells.
 However, the product needs to be very pure, since it being used as aHowever, the product needs to be very pure, since it being used as a
therapeutic medical drug, so it is dissolved and then precipitated as atherapeutic medical drug, so it is dissolved and then precipitated as a
potassium salt to separate it from other substances in the medium.potassium salt to separate it from other substances in the medium.

15.  The resulting penicillin (called penicillin G) can be chemically andThe resulting penicillin (called penicillin G) can be chemically and
enzymatically modified to make a variety of penicillins with slightlyenzymatically modified to make a variety of penicillins with slightly
different properties.different properties.
 These semi-synthetic penicillins include penicillin V, penicillin O,These semi-synthetic penicillins include penicillin V, penicillin O,
ampicillin and amoxycillin.ampicillin and amoxycillin.

16. 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)
c)c)
17.17. Why can't penicillin be taken orally?Why can't penicillin be taken orally?
18.18. Name the form of penicillin which can be taken orally.Name the form of penicillin which can be taken orally.
19.19. How does Penicillin kill bacteria?How does Penicillin kill bacteria?
20.20. Why are Gram negative bacteria not killed by penicillin?Why are Gram negative bacteria not killed by penicillin?
lactoselactose
yeastyeast
glucoseglucose
aerobicaerobic
25 - 27ºC25 - 27ºC
secondarysecondary
40 – 200 dm40 – 200 dm33
To difficult to aerateTo difficult to aerate
40 hours – after main increase in fungal mass40 hours – after main increase in fungal mass
140 hours (180 – 40 hours)140 hours (180 – 40 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
Precipitation by potassium saltsPrecipitation by potassium salts
Destroyed by stomach acidDestroyed by stomach acid
Penicillin V, ampicillinPenicillin V, ampicillin
Stops production of cell wallStops production of cell wall
Different cell wallDifferent cell wall