Microbial Microbial Growth Growth KineticsKinetics

Chapter 2Chapter 2

IntroductionIntroduction• Growth of microbes results of:Growth of microbes results of:

- replication- replication- change in cell size- change in cell size

• Can grow under variety physical, chemical and nutritional Can grow under variety physical, chemical and nutritional conditions.conditions.

• Convert nutrient from medium into biological compoundsConvert nutrient from medium into biological compoundssubstrates + cells product + more cellssubstrates + cells product + more cells

• Rate of growth Rate of growth ∞ cell concentration∞ cell concentration

FermentationFermentation Traditionally-the process for the production Traditionally-the process for the production

of alcohol or lactic acid from glucoseof alcohol or lactic acid from glucose Broadly-an enzymatically controlled Broadly-an enzymatically controlled

transformation of organic compound.transformation of organic compound. Fermentation may be carried out as:Fermentation may be carried out as: Batch processBatch process Continuous processContinuous process Fed-batch processFed-batch process

Types of ProcessesTypes of Processes

Batch: (A)Batch: (A) Continuous: (B)Continuous: (B) Fed-batch: (C)Fed-batch: (C)

Batch GrowthBatch Growth

Culturing a cell without further addition or Culturing a cell without further addition or removal of nutrientremoval of nutrient

Cell must be quantified either:Cell must be quantified either: directly- due to presence of presence of directly- due to presence of presence of

suspended solidssuspended solids Indirectly (cell number, cell mass)Indirectly (cell number, cell mass)

Determining Cell Number DensityDetermining Cell Number Density

Petroff-Hausser slide/hemocytometerPetroff-Hausser slide/hemocytometer

- 20 grid squares in counted using - 20 grid squares in counted using microscope-averagemicroscope-average

- Disadvantages:- Disadvantages: medium must be free from particlesmedium must be free from particles stain is used to differentiate between stain is used to differentiate between

dead/live cellsdead/live cells not suitable for aggregated culturesnot suitable for aggregated cultures

Cont..Cont.. Plate count:Plate count:

- used for counting viable cell- used for counting viable cell- unit: colony forming unit (CFU)- unit: colony forming unit (CFU)- cultures are diluted and pipette or spread on - cultures are diluted and pipette or spread on

agar surfacesagar surfaces- plates are incubated and viable colonies are - plates are incubated and viable colonies are

countedcounted- a good plate count must consist between 30-- a good plate count must consist between 30-

200 colonies200 colonies- suitable for yeast and bacteria- suitable for yeast and bacteria- selection for best medium growth is crucial- selection for best medium growth is crucial

Cont..Cont.. Electrical resistance of cellsElectrical resistance of cells

- cells pass the orifice causes resistance - cells pass the orifice causes resistance

and provide pulsesand provide pulses

- number pulses is a measure - number pulses is a measure ∞ number of ∞ number of

cellscells

- height of pulses- measure cell size- height of pulses- measure cell size

Cont..Cont..

Light intensity measurementLight intensity measurement

- Intensity of light - Intensity of light ∞ cell concentration∞ cell concentration

- only for dilute suspension- only for dilute suspension

Determining Cell Mass Determining Cell Mass ConcentrationConcentration

Direct method:Direct method:

- Dry weight - biomass determination- Dry weight - biomass determination

- OD - spectrophotometer- OD - spectrophotometer Indirect method:Indirect method:

- measurement of cellular component- measurement of cellular component

- e.g: enzyme, chlorophyl- e.g: enzyme, chlorophyl

Growth Patterns & Kinetics in Growth Patterns & Kinetics in Batch*Batch*

Lag PhaseLag Phase

Delay before rapid growthDelay before rapid growth Occur immediately after inoculationOccur immediately after inoculation Cell mass increase, number of cell remained Cell mass increase, number of cell remained

constantconstant Cells may be damagedCells may be damaged Cells may be adapting to mediaCells may be adapting to media Cells may be old/coldCells may be old/cold Cells make new ribosomesCells make new ribosomes Cell make new proteinsCell make new proteins Cell begin to make cellsCell begin to make cells

Log/Exponential PhaseLog/Exponential Phase Cells divide at or close to maximumCells divide at or close to maximum Cell adjusted to new environmentCell adjusted to new environment Biomass increases quicklyBiomass increases quickly Nutrients consumed rapidlyNutrients consumed rapidly Oxygen (if needed) consumed rapidlyOxygen (if needed) consumed rapidly Heat produced in some culturesHeat produced in some cultures Changes in pH due to organismChanges in pH due to organism Protein in media may produce foamProtein in media may produce foam Cell mass & numbers multiply rapidlyCell mass & numbers multiply rapidly Balance growth-all component growth at same rateBalance growth-all component growth at same rate Growth rate independent of nutrient concentrationGrowth rate independent of nutrient concentration

Deceleration PhaseDeceleration Phase

Also known as “late log phase”Also known as “late log phase” Growth decelerates due to:Growth decelerates due to:

- depletion of essential nutrient- depletion of essential nutrient

- accumulation of toxic by product- accumulation of toxic by product

- unbalanced growth-restructuring of cell- unbalanced growth-restructuring of cell

Stationary PhaseStationary Phase

Resting phaseResting phase Zero growth rate (no cell division) or growth rate equal to Zero growth rate (no cell division) or growth rate equal to

the death ratethe death rate Nutrients depletedNutrients depleted Oxygen may be limitedOxygen may be limited Release of cellular chemicals e.g. toxinsRelease of cellular chemicals e.g. toxins Cell growth~= cell deathCell growth~= cell death Production of secondary metabolites (non growth related). Production of secondary metabolites (non growth related).

E.g: antibiotics, hormonesE.g: antibiotics, hormones Mixed growth and non-growth associated production.Mixed growth and non-growth associated production. Cell lysis, cryptic growth occurCell lysis, cryptic growth occur

Death PhaseDeath Phase

Cells decline exponentiallyCells decline exponentially Some may start during stationary phaseSome may start during stationary phase Cell autolysis may occurCell autolysis may occur Some residual survivors last a timeSome residual survivors last a time At the end occur because of nutrient At the end occur because of nutrient

depletion or waste accumulationdepletion or waste accumulation Sometimes death rate change after hours Sometimes death rate change after hours

or days.or days.

Product FormationProduct Formation

Primary metabolitesPrimary metabolites

- Growth associated- Growth associated Secondary metabolitesSecondary metabolites

- Stationary growth associated- Stationary growth associated

Primary MetabolitesPrimary Metabolites

Formed during active cell growthFormed during active cell growth During primary growth phaseDuring primary growth phase Product essential for the metabolic activity Product essential for the metabolic activity

& growth& growth Produced from growth substrate by the Produced from growth substrate by the

cell activitycell activity Ex: alcohol, amino acidEx: alcohol, amino acid

Secondary Metabolites*Secondary Metabolites*

Formed at the end or during stationary phaseFormed at the end or during stationary phase Not essential for growthNot essential for growth Growth conditions crucial to determine the Growth conditions crucial to determine the

synthesis rate of secondary metabolitessynthesis rate of secondary metabolites Over-production often achievable (not growth Over-production often achievable (not growth

related)related) Unpredictable - formation is not consistent Unpredictable - formation is not consistent

among all members of a speciesamong all members of a species Ex: penicillin, antibiotics.Ex: penicillin, antibiotics.

Microbial ProductMicrobial Product

Microbial growth, product formation and Microbial growth, product formation and substrate utilization rates are usually substrate utilization rates are usually expressed in the form of specific ratesexpressed in the form of specific rates

Classified to 3 categories:Classified to 3 categories: growth associatedgrowth associated non growth associatednon growth associated Mixed growth associatedMixed growth associated

Growth Associated ProductGrowth Associated Product

Produced simultaneously with growthProduced simultaneously with growth Specific rate product formation Specific rate product formation ∞ specific ∞ specific

growth rategrowth rate Product: primary metabolitesProduct: primary metabolites E.g.: Enzyme protease (E.g.: Enzyme protease (Bacillus subtilisBacillus subtilis), ),

amino acidamino acid

Non Growth Associated ProductNon Growth Associated Product

Production occur during stationary phaseProduction occur during stationary phase The specific rate of product formation is The specific rate of product formation is

constantconstant Product: secondary metaboliteProduct: secondary metabolite E. g: hormones, antibiotics (penicillin)E. g: hormones, antibiotics (penicillin)

Mixed Growth AssociatedMixed Growth Associated

Production occur during slow growth and Production occur during slow growth and stationary phasestationary phase

E. g: lactic acid & certain secondary E. g: lactic acid & certain secondary metabolites metabolites

Effect of Environmental Factors Effect of Environmental Factors On GrowthOn Growth

TemperatureTemperature pHpH Oxygen availabilityOxygen availability Osmotic pressure/salt concentrationOsmotic pressure/salt concentration Nutrient availabilityNutrient availability

Continuous CultureContinuous Culture

The importance of continuous culture:The importance of continuous culture:- maintenance of a culture in constant - maintenance of a culture in constant environmental conditions through continuous environmental conditions through continuous supply of nutrientsupply of nutrient- provision of nutrients and removal of wastes. - provision of nutrients and removal of wastes. - useful for:- useful for:

study in a certain growth phasestudy in a certain growth phase evolution studiesevolution studies study the effect of changes in the environmental study the effect of changes in the environmental

on cell physiologyon cell physiology

How Cell Grow in Continuous How Cell Grow in Continuous CultureCulture

Fresh medium continually supplied to well-stirred Fresh medium continually supplied to well-stirred cultureculture

Product (cell/culture medium) continuously Product (cell/culture medium) continuously withdrawnwithdrawn

During cultivation, growth & product formation During cultivation, growth & product formation can be prolongedcan be prolonged

At steady state: cell, product and substrate At steady state: cell, product and substrate concentration remain constantconcentration remain constant

An essential nutrient is in limiting quantitiesAn essential nutrient is in limiting quantities

Batch vs. Continuous CultureBatch vs. Continuous Culture

Batch culture; the culture environment continuously Batch culture; the culture environment continuously changeschanges

Growth, product formation and substrate utilization Growth, product formation and substrate utilization all terminate after a certain time intervalall terminate after a certain time interval

Continuous culture: fresh nutrient medium is Continuous culture: fresh nutrient medium is continually supplied to a well-mixed culture, continually supplied to a well-mixed culture, products and cells are simultaneously withdrawnproducts and cells are simultaneously withdrawn

Growth and product formation can be maintained for Growth and product formation can be maintained for prolonged periods of timeprolonged periods of time

At steady state, cell, product and substrate At steady state, cell, product and substrate concentrations remain constantconcentrations remain constant

Cont…Cont…

Provides constant environmental conditions Provides constant environmental conditions for growth and product formation and for growth and product formation and supplies uniform quality productsupplies uniform quality product

For growth associated products has higher For growth associated products has higher productivity than batch cultureproductivity than batch culture

Why is Batch Culture Predominantly Why is Batch Culture Predominantly Used in the Biotech IndustryUsed in the Biotech Industry

Many secondary products are not growth Many secondary products are not growth associatedassociated

Genetic instabilityGenetic instability ReliabilityReliability Economic considerationsEconomic considerations

Devices for Continuous CultureDevices for Continuous Culture

Plug flow reactor (PFR)Plug flow reactor (PFR)

- Continuous cultivation- Continuous cultivation

- No back mixing-fluid elements containing - No back mixing-fluid elements containing

active cells cannot inoculate other fluid active cells cannot inoculate other fluid

elements at different axial positionselements at different axial positions

- Substrate and cell concentrations vary - Substrate and cell concentrations vary

with axial position in the reactorwith axial position in the reactor

Cont…Cont… ChemostatChemostat

- also known as a continuous stirred tank reactor (CSTR)- also known as a continuous stirred tank reactor (CSTR)- refers to constant chemical environment- refers to constant chemical environment- perfectly mixed continuous flow- perfectly mixed continuous flow- equipped with pH, DO, level controller- equipped with pH, DO, level controller- feeding of fresh medium and removal of cell - feeding of fresh medium and removal of cell

suspension occur at the same ratesuspension occur at the same rate- cellular growth is typically limited by one essential - cellular growth is typically limited by one essential

nutrient: other nutrients are in excessnutrient: other nutrients are in excess- when operated at steady state: nutrient, product and - when operated at steady state: nutrient, product and

cell concentrations are constantcell concentrations are constant- volume of reactor constant- volume of reactor constant

Cont…Cont… TurbidostatTurbidostat

- Cell concentration in the culture vessel - Cell concentration in the culture vessel constant (monitor the OD & feed flow rate)constant (monitor the OD & feed flow rate)

- Volume is kept constantly by removal of culture - Volume is kept constantly by removal of culture brothbroth

- Suitable for microorganisms able to withstand - Suitable for microorganisms able to withstand environmental stressenvironmental stress

- Flow rate into the system is adjusted to maintain preset - Flow rate into the system is adjusted to maintain preset turbidity (cell density)turbidity (cell density)

- No limiting nutrients- No limiting nutrients- Operates best at high dilution rates- Operates best at high dilution rates

Ideal ChemostatIdeal Chemostat

An ideal chemostat is a perfectly mixed, An ideal chemostat is a perfectly mixed, continuous-flow, stirred-tank reactorcontinuous-flow, stirred-tank reactor

Most chemostats require some control Most chemostats require some control elements (pH and DO controllers)elements (pH and DO controllers)

Fresh sterile medium is fed to the Fresh sterile medium is fed to the completely mixed and aerated reactor, cell completely mixed and aerated reactor, cell suspension is removed at the same ratesuspension is removed at the same rate

Liquid volume in the reactor is kept Liquid volume in the reactor is kept constantconstant

Cont..Cont.. Feed sterile media into a culture at the same rate in Feed sterile media into a culture at the same rate in

which it is removedwhich it is removed Essential nutrient is limiting so that flow rate Essential nutrient is limiting so that flow rate

determines growth ratedetermines growth rate Dilution rate - rate at which medium flows through Dilution rate - rate at which medium flows through

vessel relative to vessel sizevessel relative to vessel size Cell density maintained at wide range of dilutionCell density maintained at wide range of dilution Rates and chemostat operates best at low dilution Rates and chemostat operates best at low dilution

ratesrates No matter how fast the media goes in the bacteria No matter how fast the media goes in the bacteria

cannot grow faster than they would in batch culture cannot grow faster than they would in batch culture under the conditions employed within the chamberunder the conditions employed within the chamber

Fed-Batch CultureFed-Batch Culture Batch culture which are fed continuously or sequentially Batch culture which are fed continuously or sequentially

with medium without the removal of culture fluid with medium without the removal of culture fluid Established initially in batch mode and is then fed Established initially in batch mode and is then fed

accordingly to one of the following feed strategies:accordingly to one of the following feed strategies:i.i. the same medium used to establish the batch culture is the same medium used to establish the batch culture is

added, resulting in an increase in volumeadded, resulting in an increase in volumeii.ii. a solution of the limiting substrate at the same a solution of the limiting substrate at the same

concentration as that in the initial medium is added, concentration as that in the initial medium is added, resulting in an increase in volumeresulting in an increase in volume

iii.iii. a concentrated solution of the limiting substrate is added a concentrated solution of the limiting substrate is added at a rate less than in (i) and (ii), resulting in an increase at a rate less than in (i) and (ii), resulting in an increase in volumein volume

iv.iv. a very concentrated solution of the limiting substrate is a very concentrated solution of the limiting substrate is added at a rate less than in (i), (ii) andadded at a rate less than in (i), (ii) and ( (iii), resulting in iii), resulting in an insignificant increase in volumean insignificant increase in volume

Cont…Cont…

strategies (i) and (ii) - variable volumestrategies (i) and (ii) - variable volume strategy (iv) - fixed volumestrategy (iv) - fixed volume strategy (iii) - culture intermediate between the strategy (iii) - culture intermediate between the

two extremes of variable and fixed volumetwo extremes of variable and fixed volume Ex: Ex:

- production of bakers’ yeast- production of bakers’ yeast- formation of ethanol- formation of ethanol- production of hepatitis B surface antigen- production of hepatitis B surface antigen