Microbial growth stages

Lag phase
Adaptation, preparation for division, increase in size and density.
Log phase (logarithmic or exponential).
Max. growth rate, increase linearly with time.
Growth yield and growth rate.
Stationary phase
Depletion of nutrient, accumulation of toxic. materials, cell crowding.
Decline phase

1. Pharmaceutical
Biotechnology
Lecture (4)
Dr. Ahmed Abdellatif
College of Pharmacy, Qassim University

2. Five major commercially important groups can
be obtained by fermentation:
•Biomass or microbial cells
•Microbial enzymes and hormones
•Microbial metabolites
•Recombinant products
•Transformation products
Major Biotechnological Products

3. 1. Trophophase (primary products)
2. Idiophase (secondary products)
Microbial
growth stages

4. Bacterial growth

5. Asexual reproduction or Binary fission (Two
identical daughter cells)
1-doubling of bacterial chromosome
2-attached to cell membrane (or mesosome)
3-segregation
4-detached or remain
Bacterial growth

6. qPopulation growth
•Increase number or cell mass per unit time
qGeneration time, G, (doubling time)
•Minute range (rapid growers)
•Hours range (24hr) (slow growers)
ØM. tuberculosis
Bacterial growth

7. qLag phase
ØAdaptation, preparation for division, increase in size
and density.
qLog phase (logarithmic or exponential).
ØMax. growth rate, increase linearly with time.
ØGrowth yield and growth rate.
qStationary phase
ØDepletion of nutrient, accumulation of toxic.
materials, cell crowding.
qDecline phase
Bacterial growth

9. •Continuous growth
§Constant volume flow
system, fresh media,
excess cells are
removed.
•Synchronous growth
§Filtration, nutrient
removal.
§Heating spores.
Bacterial growth

10. qMeasurement of bacterial growth
§Total count.
üCounting chambers (Petroff-Hausen counter or
hemocytometer).
üElectrical counters (Coulter counter).
§Viable count
√Pour plate method.
qMeasurement of bacterial mass
üDry weight, turbidimetic method
Bacterial growth

11. 1. Trophophase (primary products)
2. Idiophase (secondary products)
Microbial
growth stages

12. The growth of a microbial culture can
be divided into a number of stages.
A.During the log (or exponential) phase:
ØThe products produced are essential for
growth of the cells and include:
§amino acids,
§lipids,
§nucleotides,
§proteins,
§carbohydrates and
§nucleic acids etc.

13. ØThese products are called primary productsof
metabolism and the phase is referred to as
trophophase.
ØMany products of primary metabolism are of
substantial economic valueand are being
produced by fermentation.
ØPrimary metabolites formed by wild strain of a
microorganism are satisfactory to meet the
necessities of the organism.
ØThus, it is the role of biotechnologists to increase
the yield to industrial level.

14. B.Duringstationaryphase:
Øsomemicroorganismssynthesize
compoundswhicharenotproducedduring
thetrophophaseandwhichdonotappear
tohaveanyclearfunctionincell
metabolism.
ØTheseproductsarereferredtoasthe
secondarycompoundsofmetabolism
andthisphaseiscalledidiophase.

15. ØCompounds yielded in the idiophase have
no direct relationship to the synthesis of
cell materials and normal growth.
ØMostantibioticsandthemycotoxinsfall
intothiscategory.
ØIt is important to notes that secondary
metabolism may occur in continuous
cultures at low growth rates and is a
property of slow-growing as well as non-
growing cells.

16. ØGenerally,notallmicroorganisms
undergosecondarymetabolism.
ØItiscommonamongstthefilamentous
bacteriaandfungiandthespore
formingbacteriabutitisnotfoundin
Enterobacteriaceae.

17. ØMany of secondary metabolisms have ;
üantimicrobial action,
üothers are specific enzyme inhibitors,
üand some are growth promoters,
üand many have pharmacological
activities.
ØAs in case of primary metabolites, wild
organism tends to produce only low
concentration of secondary
metabolites, thus it is the role of
industry to increase the production.

18. Antibiotics

19. 1. Antibiotics: Isolation, Yield, and Purification
•Antibiotics
–Compounds that kill or inhibit the growth of other
microbes.
–Typically secondary metabolites.
–Most antibiotics in clinical use are produced by
filamentous fungi or actinomycetes.
–Still discovered by laboratory screening.
•Microbes are obtained from nature in pure culture.
•Assayed for products that inhibit growth of test
bacteria.
© 2012 Pearson Education, Inc.
Animation: Isolation and Screening
of Antibiotic Producers

20. I. Isolation
Sterile glass spreader
Colonies of
Streptomyces
species
Nonproducing
organisms
Zones of
growth inhibition
Producing
organisms
Spread a soildilution on a plate
of selective medium
Incubation
Overlay with anindicator organism
Incubate

21. 1. Antibiotics: Isolation, Yield, and Purification
•Cross-streak method
–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

22. Figure 15.4bII. Testing Activity Spectrum
Streak antibiotic produceracross one side of plate
Incubate to permit growthand antibiotic production
Cross-streak with test organisms
Incubate to permit test organisms to grow
Antibiotic diffuses
into agar
Streptomycescell mass
Growth of test organism
Inhibition zones where
sensitive test organisms
did not grow
© 2012 Pearson Education, Inc.

23. Finding Antibiotic
Producers

24. Many antibiotics are produced by
microorganisms, mostly by
actinomyces, predominately the
genus Streptomycesand filamentous
fungi.

25. Penicillin

26. ØPenicillinisproducedinstirredfermentersby
Penicilliumchrysogenumisamoldthatiswidely
distributedinnature,andisoftenfoundlivingonfoods
andinindoorenvironments.Itwaspreviouslyknownas
Penicilliumnotatum.
ØPenicillin (sometimes abbreviated PCN or pen)
antibiotics are historically significant because it is the first
drugs that was effective against many previously serious
diseases such as syphilisand Staphylococcusinfections.
ØPenicillins are still widely used today, through many
types of bacteriaare now resistant.
ØAll penicillins are Beta-lactam antibioticsand are used in the
treatment of bacterialinfectionscaused by susceptible,
usually Gram-positive, organisms.

28. •Rapidproductionofcellsoccurswhenhighlevelsof
glucoseareusedasacarbonsourceandthisdoesnot
leadtomaximumantibioticsyields.
•Provisionoftheslowlyhydrolyzeddisaccharidelactosein
combinationwithlimitednitrogenavailabilitystimulatesa
greateraccumulationofpenicillinaftergrowthhasstopped.
•Productioncanbestimulatedalsobyslowcontinuousfeed
ofglucose.
•Forproductionofaparticularpenicillin,aspecificprecursor
isaddedtothemediume.g.phenylaceticacidisaddedto
maximizeproductionofpenicillinG.
•ThefermentationpHismaintainedaroundneutralitybythe
additionofsterilealkaliwhichstabilizethenewlysynthesized
penicillin.Thefermentationiscompletedin6-7days.The
basicproductcanbemodifiedbychemicalproceduresto
yieldavarietyofsemisyntheticpenicillins.

29. Streptomycin

30. ØItisasecondarymetaboliteproducedbyStreptomycesgriseus.
ØThemediumcontainssoybeanmeal(N-source),glucose(C-
source)andNaCl.
ØProcessiscarriedoutat28°Candthemaximumproduction
achievedatpHrangeof7.6-8.0.
ØHighagitationandaerationareneeded.Processlastsforabout
10days.
ØTheclassicfermentationprocessinvolvesthreephases.During
thefirstphasethereisrapidgrowthofthemicrobewith
productionofmycelialbiomass.
ØProteolyticactivityofthemicrobereleasesNH3tothemedium
fromthesoybeanmeal,causingariseinpH.Duringthisinitial
fermentationphasethereislittleproductionofstreptomycin.
Duringsecondphasethereislittleadditionalproductionof
mycelium,butthesecondarymetabolite,streptomycin
accumulatesinthemedium.TheglucoseandNH3releasedare
consumedduringthisphase.

31. ØThepHremainsfairlyconstant-between7.6and8.0.In
thethirdandfinalphase,whencarbohydratesbecome
depleted,streptomycinproductionceasesandthe
microbialcellsbegintolysepHincreasesandprocess
normallyendsbythistime.
Aftertheprocessiscompleted,myceliumisseparated
fromthebrothbyfiltrationandtheantibioticrecovered.
Inonemethodofrecoveryandpurification,streptomycin
isadsorbedontoactivatedcharcolandelutedwithacid
alcohol.Itisthenprecipitatedwithacetoneandfurther
purifiedbyuseofcolumnchromatography.

32. DEXTRANS
They are glucans (polymers of glucose)
Production:
MicroorganismslikeLeuconostocmesentroides.Theyare
producedbyextracellularenzymeslikedextransucrase
whichactonsucroseandbringpolymerizationofglucose
residuesandliberatesfreefructoseinmedium
Applications:
Blood plasma expanders for prevention of thrombosis. Wound
healing and dressing purification of biomolecules

33. Amino Acids

34. üAminoacidssuchaslysineandglutamicacidareusedinfood
industryasnutritionalsupplementsinbreadproductionand
asflavor-enhancingcompounds.
üGlutamicacidisproducedbymutantsofCorynebacterium
glutamicum.
üAcontrolledlowbiotinlevelandtheadditionoffattyacid
derivativesresultsinincreasedmembranepermeabilityand
excretionofhighconcentrationofglutamicacid.
Glutamic Acid

35. üLysine is an essential amino acid used as supplement
cereals and bread.
üIt is produced by Corynebacterium glutamicum, which
blocked in the synthesis of homoserine, accumulates
lysine. Over 44 g/liter can be obtained in a 3 days
fermentation.
Lysine

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