Microbial Microbial Growth Growth KineticKinetic

By By

Khairul Farihan KasimKhairul Farihan Kasim

CO3: CO3:

Ability to define, describe Ability to define, describe and utilize microbial and utilize microbial

growth in fermentation growth in fermentation and biological process and biological process

At the end of the lecture,At the end of the lecture, the students should be able to: the students should be able to:

• name the various phases of growth that occur in closed culture name the various phases of growth that occur in closed culture systems and describe what is occurring in each phasesystems and describe what is occurring in each phase

• determine from experimental data the various parameters (number of determine from experimental data the various parameters (number of generations, specific growth rate constant, mean generation time) that generations, specific growth rate constant, mean generation time) that describe microbial growth in mathematical termsdescribe microbial growth in mathematical terms

• compare and contrast the various methods for measuring microbial compare and contrast the various methods for measuring microbial growthgrowth

• describe the various types of continuous culture systems and explain describe the various types of continuous culture systems and explain the differences in their functionthe differences in their function

• describe the influence of various environmental factors (water describe the influence of various environmental factors (water availability, pH, temperature, oxygen concentration, pressure, availability, pH, temperature, oxygen concentration, pressure, radiation) on the growth of microorganismsradiation) on the growth of microorganisms

• categorize microorganisms according to the environmental factors that categorize microorganisms according to the environmental factors that are conducive to optimal growth of the organismare conducive to optimal growth of the organism

OutlineOutline• Bacterial cell divisionBacterial cell division

– Cell growth and binary fissionCell growth and binary fission– Fts protein and cell divisionFts protein and cell division

• Growth of bacterial populationGrowth of bacterial population– Growth terminology and the concept of Growth terminology and the concept of

exponential growthexponential growth– The mathematics of exponential growthThe mathematics of exponential growth– The growth cycleThe growth cycle

• Continuous culture: The chemostatContinuous culture: The chemostat

• Measuring microbial growthMeasuring microbial growth– Direct measurementDirect measurement– Indirect measurementIndirect measurement

• Growth requirementGrowth requirement– Sources of carbons, energy and Sources of carbons, energy and

electronselectrons

• Environmental effects on microbial Environmental effects on microbial growth growth – TemperatureTemperature– pH, osmolarity and oxygenpH, osmolarity and oxygen

Bacterial cell divisionBacterial cell division

Cell growth and binary fissionCell growth and binary fission• increase in cellular constituents that may increase in cellular constituents that may

result in:result in:– increase in cell numberincrease in cell number

• e.g., when microorganisms reproduce by budding or e.g., when microorganisms reproduce by budding or binary fissionbinary fission

– increase in cell sizeincrease in cell size• e.g., coenocytic microorganisms have nuclear divisions e.g., coenocytic microorganisms have nuclear divisions

that are not accompanied by cell divisionsthat are not accompanied by cell divisions

• microbiologists usually study population microbiologists usually study population growth rather than growth of individual cellsgrowth rather than growth of individual cells

Binary fissionBinary fission

• A process in which a cell grows to A process in which a cell grows to twice its normal size and divides in twice its normal size and divides in half to produce two daughter cells of half to produce two daughter cells of equal size.equal size.

• Binary fission generally involves four Binary fission generally involves four steps as illustrated in next Figure for steps as illustrated in next Figure for a prokaryotic cell. a prokaryotic cell.

One generation

1. The cell replicates its chromosome (DNA 1. The cell replicates its chromosome (DNA molecule). The duplicated chromosomes are molecule). The duplicated chromosomes are attached to the cytoplasmic membrane. (In attached to the cytoplasmic membrane. (In eukaryotic cells chromosomes are attached eukaryotic cells chromosomes are attached to microtubules). to microtubules).

2. The cell elongates and growth between 2. The cell elongates and growth between attachment sites pushes the chromosomes attachment sites pushes the chromosomes apart. (Eukaryotic cells segregate their apart. (Eukaryotic cells segregate their chromosomes by chromosomes by mitosismitosis).).

3. The cell forms a new cytoplasmic 3. The cell forms a new cytoplasmic membrane and wall (septum) across the membrane and wall (septum) across the midline. midline.

• 4. When the septum is completed, 4. When the septum is completed, the daughter cells may remain the daughter cells may remain attached, or they may separate attached, or they may separate completely. When the cells remain completely. When the cells remain attached, further binary fission in attached, further binary fission in parallel planes produces a chain. parallel planes produces a chain. When further divisions are in When further divisions are in different planes, the cells become a different planes, the cells become a cluster. cluster.

• 5. The process repeats 5. The process repeats

Fts protein and cell divisionFts protein and cell division

• process not well understoodprocess not well understood

• protein MreBprotein MreB– similar to eucaryotic actinsimilar to eucaryotic actin– plays a role in determination of cell shape and plays a role in determination of cell shape and

movement of chromosomes to opposite cell movement of chromosomes to opposite cell polespoles

• protein FtsZ,protein FtsZ,– similar to eucaryotic tubulinsimilar to eucaryotic tubulin– plays a role in Z ring formation which is essential plays a role in Z ring formation which is essential

for septationfor septation

• Fts – Fts – 'filamentous temperature sensitive", - 'filamentous temperature sensitive", - the properties of cells harboring mutations in the genes that the properties of cells harboring mutations in the genes that

encode Fts proteins. encode Fts proteins.

• Fts proteins interact to form a Fts proteins interact to form a division apparatus division apparatus in the cell in the cell called the divisome. called the divisome.

• In rod-shaped cells, formation of the divisome begins with the In rod-shaped cells, formation of the divisome begins with the attachment of molecules of FtsZ in a ring around the cell attachment of molecules of FtsZ in a ring around the cell cylinder in the precise center of the cell. cylinder in the precise center of the cell.

• This spot will eventually become the cell division plane. In a This spot will eventually become the cell division plane. In a cell of cell of Escherichia coli Escherichia coli about 10,000 FtsZ molecules about 10,000 FtsZ molecules polymerize to form the ring, and then the ring attracts other polymerize to form the ring, and then the ring attracts other cell division proteins, including cell division proteins, including FtsA FtsA and and ZipAZipA. .

• FtsA is an ATP-hydrolyzing enzyme that provides energy for FtsA is an ATP-hydrolyzing enzyme that provides energy for assembly of the many proteins into the divisome. ZipA is an assembly of the many proteins into the divisome. ZipA is an anchor that attaches the FtsZ ring to the cytoplasmic anchor that attaches the FtsZ ring to the cytoplasmic membrane. membrane.

Concept checkConcept check

• Microbial growth involves an increase in the Microbial growth involves an increase in the number of cells. Growth of most number of cells. Growth of most microorganisms occurs by the process of binary microorganisms occurs by the process of binary fission.fission.

• Cell division and chromosome replication are Cell division and chromosome replication are coordinately regulated, and the Fts proteins are coordinately regulated, and the Fts proteins are they key to these processes. The protein FtsZ they key to these processes. The protein FtsZ defines the division plane in prokaryotes, while defines the division plane in prokaryotes, while Mre proteins help define cell shape.Mre proteins help define cell shape.

Growth of bacterial Growth of bacterial populationpopulation

Growth Terminology and the Growth Terminology and the concept of exponential growthconcept of exponential growth

• The interval for the formation of two cells from The interval for the formation of two cells from one is called a one is called a generationgeneration

• The The time requiredtime required for this to occur is called the for this to occur is called the generation timegeneration time. .

• Generation timeGeneration time is the time required for the cell is the time required for the cell population to double (the cell population to double (the cell mass mass doubles doubles during this period as well). during this period as well).

• Because of this, the generation time is also called Because of this, the generation time is also called the the doubling timedoubling time..

• In nature, microbial doubling times may be In nature, microbial doubling times may be much longer than those obtained in much longer than those obtained in laboratory culture. laboratory culture.

• This is because in nature, ideal growth This is because in nature, ideal growth conditions for a given organism may exist conditions for a given organism may exist only intermittently. only intermittently.

• Depending on resource availability, Depending on resource availability, physiochemical conditions (temperature, physiochemical conditions (temperature, pH, and the like), moisture availability, and pH, and the like), moisture availability, and seasonal changes, bacterial populations in seasonal changes, bacterial populations in nature double only once every few weeks, nature double only once every few weeks, or even longer. or even longer.

A mathematical relationship A mathematical relationship existsexists between the number of cells present in a between the number of cells present in a culture culture iinitiallynitially and the number present and the number present afterafter a period of exponential growth: a period of exponential growth:

N = N = NN0022n n

where where N is the final cell number, N is the final cell number,

NNoo is the initial cell number, is the initial cell number, n n is the number of generations that have is the number of generations that have occurred during the period of exponential occurred during the period of exponential growth. growth.

The Mathematics of The Mathematics of Exponential GrowthExponential Growth

• As one cell divides to become two As one cell divides to become two cells, cells,

2° --'> 22° --'> 211. .

• As two cells become four, As two cells become four,

2211 --'> 2 --'> 222, and so on , and so on

The The generation timegeneration time ( (g)g) of the of the exponentially growing population is (exponentially growing population is (t / t / n)n), ,

where where

tt is the duration of exponential growth is the duration of exponential growth expressed in days, hours, or minutes, expressed in days, hours, or minutes, depending on the organism and the depending on the organism and the growth conditions. growth conditions.

From a knowledge of the initial and final From a knowledge of the initial and final cell numbers in an exponentially growing cell numbers in an exponentially growing cell population, it is possible to calculate cell population, it is possible to calculate nn, , and from and from nn and knowledge of and knowledge of tt, , the the generation time generation time gg..

Relation equation of N and NRelation equation of N and Noo to nto n

The equation The equation N N = = NNoo22nn can be expressed in can be expressed in terms of terms of n n as follows: as follows:

N = N = NNoo22nn

log N = log log N = log NNoo + n log 2 + n log 2

log N – log log N – log NNoo = n log 2 = n log 2

n = log N – log n = log N – log NNoo = log N – log = log N – log NNoo

log 2 0.301log 2 0.301

= 3.3 (log N – log = 3.3 (log N – log NNoo))

exampleexampleN = 10N = 1088, N, Noo = 5 X 10= 5 X 1077, and , and t t = 2= 2

n n = 3.3 (log N – log N= 3.3 (log N – log Noo))

n n = 3.3 [log(10= 3.3 [log(1088) - log(5 X 10) - log(5 X 1077)] )] = 3.3(8 - 7.69) = 3.3(8 - 7.69) = 3.3(0.301) = 3.3(0.301) = 1 = 1

generation time, g = generation time, g = t/n t/n = 2 / 1 = 2 h = 2 / 1 = 2 h

Related growth parameterRelated growth parameter

• The generation time The generation time g g of an exponentially of an exponentially growing culture can also be calculated from the growing culture can also be calculated from the slope of the line slope of the line obtained in the semilogarithmic obtained in the semilogarithmic plot of exponential growth. plot of exponential growth.

• The slope is equal to The slope is equal to 0.301 n/t 0.301 n/t (log (log 2n/t) 2n/t) and in and in the above example would be the above example would be 0.301(1)/2, 0.301(1)/2, or 0.15.or 0.15.

• Since Since g g is equal 0.301/slope, we arrive at the is equal 0.301/slope, we arrive at the same value of 2 for same value of 2 for g. g.

• The term The term 0.301nlt 0.301nlt is called the is called the specific growth specific growth rate, rate, abbreviated abbreviated kk. .

• index of growth is the reciprocal of the generation index of growth is the reciprocal of the generation time, called the time, called the division rate, division rate, abbreviated abbreviated v. v.

• The division rate is equal to 1/The division rate is equal to 1/g g and has units of and has units of reciprocal 1 reciprocal 1 (h-1). (h-1). While the term While the term g g is a measure is a measure of the of the time time it takes for a population to double in it takes for a population to double in cell number, cell number, v v is a measure of the is a measure of the number of number of generations generations that occur per unit time in an that occur per unit time in an exponentially growing culture. exponentially growing culture.

• The slope of the line relating log cell number to The slope of the line relating log cell number to time is equal to time is equal to v /3.3v /3.3

The Growth cycle or phases of The Growth cycle or phases of microbial growthmicrobial growth

• observed when microorganisms are observed when microorganisms are cultivated in cultivated in batchbatch culture culture– culture incubated in a closed vessel with a culture incubated in a closed vessel with a

single batch of mediumsingle batch of medium

• usually plotted as logarithm of cell usually plotted as logarithm of cell number versus timenumber versus time

• usually has four distinct phasesusually has four distinct phases

Lag phaseLag phase• the cells are adjusting to their new environmentthe cells are adjusting to their new environment

• most cells do not reproduce immediately, but most cells do not reproduce immediately, but instead actively synthesize enzymes to utilize instead actively synthesize enzymes to utilize novel nutrients in the medium. novel nutrients in the medium.

• bacteria inoculated from a medium containing bacteria inoculated from a medium containing glucose as a carbon source into a medium glucose as a carbon source into a medium containing lactose must synthesize two types of containing lactose must synthesize two types of proteins: proteins:

– membrane proteins to transport lactose into the membrane proteins to transport lactose into the cellcell

– the enzyme lactase to catabolize the lactose. the enzyme lactase to catabolize the lactose.

Log phaseLog phase• bacteria synthesize the necessary chemicals for conducting metabolism in bacteria synthesize the necessary chemicals for conducting metabolism in

their new environment, and they then enter a phase of rapid chromosome their new environment, and they then enter a phase of rapid chromosome replication, growth, and reproduction. replication, growth, and reproduction.

• population increases logarithmicallypopulation increases logarithmically

• reproductive rate reaches a constant as DNA protein syntheses are reproductive rate reaches a constant as DNA protein syntheses are maximized. maximized.

• more susceptible to antimicrobial drugs that interfere with metabolismmore susceptible to antimicrobial drugs that interfere with metabolism

• preferred for Gram staining because most cells' walls are intact – an preferred for Gram staining because most cells' walls are intact – an important characteristic for correct staining. important characteristic for correct staining.

• the metabolic rate of individual cells is at a maximum during log phasethe metabolic rate of individual cells is at a maximum during log phase

• this phase is sometimes preferred for industrial and laboratory purposes. this phase is sometimes preferred for industrial and laboratory purposes.

Stationary phaseStationary phase• If bacterial growth continued at the exponential rate of the log If bacterial growth continued at the exponential rate of the log

phase, bacteria would soon overwhelm the earth. phase, bacteria would soon overwhelm the earth.

• does not occur because as nutrients are depleted and wastes does not occur because as nutrients are depleted and wastes accumulate, the rate of reproduction decreases. accumulate, the rate of reproduction decreases.

• the number of dying cells equals the number of cells being the number of dying cells equals the number of cells being produced, and the size of the population becomes stationary produced, and the size of the population becomes stationary

• During this phase the metabolic rate of surviving cells declines. During this phase the metabolic rate of surviving cells declines.

• The onset of the stationary phase can be postponed The onset of the stationary phase can be postponed indefinitely by a special apparatus called a indefinitely by a special apparatus called a chemostat, chemostat, which which continually removes wastes (along with old medium and some continually removes wastes (along with old medium and some cells) and adds fresh medium. cells) and adds fresh medium.

• Chemostats are used in industrial fermentation processes. Chemostats are used in industrial fermentation processes.

Stationary PhaseStationary Phase

• total number of viable cells remains total number of viable cells remains constantconstant– may occur because metabolically active may occur because metabolically active

cells stop reproducingcells stop reproducing– may occur because reproductive rate is may occur because reproductive rate is

balanced by death ratebalanced by death rate

Possible reasons for entry Possible reasons for entry into stationary phaseinto stationary phase

• nutrient limitationnutrient limitation

• limited oxygen availabilitylimited oxygen availability

• toxic waste accumulationtoxic waste accumulation

• critical population density reachedcritical population density reached

Starvation responsesStarvation responses

• morphological changesmorphological changes– e.g., endospore formatione.g., endospore formation

• decrease in size, protoplast decrease in size, protoplast shrinkage, and nucleoid shrinkage, and nucleoid condensationcondensation

• production of starvation proteinsproduction of starvation proteins• long-term survivallong-term survival• increased virulenceincreased virulence

Death phaseDeath phase

• If nutrients are not added and wastes are not removed, If nutrients are not added and wastes are not removed, a population reaches a point at which cells die at a a population reaches a point at which cells die at a faster rate than they are produced. faster rate than they are produced.

• Such a culture has entered the death phase (or Such a culture has entered the death phase (or decline decline phase). phase).

• during the death phase, some cells remain alive and during the death phase, some cells remain alive and continue metabolizing and reproducing, but the number continue metabolizing and reproducing, but the number of dying cells exceeds the number of new cells of dying cells exceeds the number of new cells produced, so that eventually the population decreases produced, so that eventually the population decreases to a fraction of its previous abundance. to a fraction of its previous abundance.

• In some cases, all the cells die, while in others a few In some cases, all the cells die, while in others a few survivors may remain indefinitely. The latter case is survivors may remain indefinitely. The latter case is especially true for cultures of bacteria that can develop especially true for cultures of bacteria that can develop resting structures called resting structures called endosporeendospore

Death PhaseDeath Phase

• two alternative hypothesestwo alternative hypotheses– Cells are Cells are VViable iable BBut ut NNot ot CCulturable ulturable

(VBNC)(VBNC)•Cells alive, but dormantCells alive, but dormant

• programmed cell deathprogrammed cell death– Fraction of the population genetically Fraction of the population genetically

programmed to die (commit suicide)programmed to die (commit suicide)

Loss of ViabilityLoss of Viability

Concept checkConcept check

• From knowledge of the initial and From knowledge of the initial and final cell numbers and the time of final cell numbers and the time of exponential growth, the generation exponential growth, the generation time and growth rate constant of a time and growth rate constant of a cell population can be calculated cell population can be calculated directly. Key parameters here are directly. Key parameters here are n, n, g, v, k g, v, k andand t t..

Concept checkConcept check

• If in 8 h an exponentially growing cell If in 8 h an exponentially growing cell population increase from 5x10population increase from 5x1066 cells/ml to 5x10cells/ml to 5x1088 cells/ml, calculate cells/ml, calculate g, g, n, v n, v andand k k..

Concept checkConcept check

• Starting with four bacterial cells per Starting with four bacterial cells per milliliter in a rich nutrient medium, milliliter in a rich nutrient medium, with a 1-h lag phase and a 20-min with a 1-h lag phase and a 20-min generation time, how many cells will generation time, how many cells will there be in 1 liter of this culture after there be in 1 liter of this culture after 1 h? After 2 h? After 2 h if one of the 1 h? After 2 h? After 2 h if one of the initial four cells was dead?initial four cells was dead?

AnswerAnswer

n = 6.6n = 6.6

g = 73 ming = 73 min

v = 0.0136v = 0.0136

k = 0.0041k = 0.0041

answeranswer

All cells are viableAll cells are viable: After one hour, there would : After one hour, there would still be only the four original cells since the still be only the four original cells since the lag time is one hour. lag time is one hour.

After two hours, three divisions would have After two hours, three divisions would have occurred, since the generation time is 20 occurred, since the generation time is 20 minutes; the population would be 4 x 23 = minutes; the population would be 4 x 23 = 32 cells/ml. 32 cells/ml.

One of the initial cells is deadOne of the initial cells is dead: After two hours : After two hours the population would be 3 x 23 = 24 the population would be 3 x 23 = 24 cells/ml.cells/ml.

Concept checkConcept check

• Microorganisms show a characteristic Microorganisms show a characteristic growth pattern when inoculated into a fresh growth pattern when inoculated into a fresh culture medium. There is usually a lag culture medium. There is usually a lag phase, and then growth commences in an phase, and then growth commences in an exponential fashion. As essential nutrients exponential fashion. As essential nutrients are depleted or toxic products build up, are depleted or toxic products build up, growth ceases, and the population enters growth ceases, and the population enters the stationary phase. If incubation the stationary phase. If incubation continues, cells may begin to die.continues, cells may begin to die.

• In what phase of the growth curve are In what phase of the growth curve are cells dividing in a regular and orderly cells dividing in a regular and orderly process?process?

• When does a lag phase usually not occur?When does a lag phase usually not occur?

• Why do cells enter the stationary phase?Why do cells enter the stationary phase?

BATCH CULTUREBATCH CULTURE

• A batch culture is a fixed volume of culture A batch culture is a fixed volume of culture medium that is continually being altered by medium that is continually being altered by the metabolic activities of growing organisms the metabolic activities of growing organisms and is therefore a and is therefore a closed system. closed system.

• In the early stages of exponential growth in In the early stages of exponential growth in batch cultures, conditions may remain batch cultures, conditions may remain relatively constant, but in later stages when relatively constant, but in later stages when cell numbers become quite large, drastic cell numbers become quite large, drastic changes in the chemical composition of the changes in the chemical composition of the culture medium occur. culture medium occur.

• A continuous culture is an A continuous culture is an open system open system of constant volume which fresh medium of constant volume which fresh medium is added continuously and spent culture is added continuously and spent culture medium removed continuously, both at medium removed continuously, both at a constant rate. a constant rate.

• Once such a system is in equilibrium, Once such a system is in equilibrium, the chemostat volume, cell number, and the chemostat volume, cell number, and nutrient status main nutrient status main constant, constant, and the and the system is said to be in steady state.system is said to be in steady state.

CONTINUOUS CULTURE: THE CONTINUOUS CULTURE: THE CHEMOSTATCHEMOSTAT

• The chemostat controls The chemostat controls both the both the growth rate growth rate and and the the population density population density of of the culture simultaneouslythe culture simultaneously

• Two factors are important Two factors are important in such control: in such control: – the the dilution rate dilution rate and the and the – concentration of a limit concentration of a limit

nutrient, nutrient, such as a carbon or such as a carbon or nitrogen source. nitrogen source.

batch culturebatch culture

• nutrient nutrient concentration concentration can affect both can affect both the growth rate the growth rate and the growth and the growth yield of a culture yield of a culture

chemostatchemostat

• growth rate and growth yield can be growth rate and growth yield can be controlled controlled independently of each otherindependently of each other

• Growth rate by adjusting the dilution rate Growth rate by adjusting the dilution rate and the growth yield by varying the and the growth yield by varying the concentration of a nutrient present in a concentration of a nutrient present in a limiting amount. limiting amount.

• wide limits over which the wide limits over which the dilution rate controls growth dilution rate controls growth rate, although at both very rate, although at both very low and very high dilution low and very high dilution rates the steady state rates the steady state breaks down. breaks down.

• At At high high dilution rates, the dilution rates, the organism cannot grow fast organism cannot grow fast enough to keep up with its enough to keep up with its dilution, and the culture is dilution, and the culture is washed out of the washed out of the chemostat. chemostat.

• very very low low dilution rates, a dilution rates, a large fraction of the cells large fraction of the cells may die from starvation may die from starvation

• The The cell density cell density (cells/milliliter) is controlled by (cells/milliliter) is controlled by the level of the limiting nutrientthe level of the limiting nutrient

• If the concentration of this nutrient in the If the concentration of this nutrient in the incoming medium is raised, with the dilution rate incoming medium is raised, with the dilution rate remaining constant, the cell density will increase. remaining constant, the cell density will increase.

• by adjusting dilution rate and nutrient level, the by adjusting dilution rate and nutrient level, the experimenter can obtain dilute, moderate or experimenter can obtain dilute, moderate or dense populations growing at slow, moderate, or dense populations growing at slow, moderate, or rapid growth rates. rapid growth rates.

EXPERIMENTAL USES OF EXPERIMENTAL USES OF THE CHEMOSTATTHE CHEMOSTATapplications such as applications such as • the study of a particular enzymethe study of a particular enzyme

– enzyme activities may be quite lower in stationary phase cells than in enzyme activities may be quite lower in stationary phase cells than in exponential phase cells and thus chemostat-grown cultures are ideal.exponential phase cells and thus chemostat-grown cultures are ideal.

• in microbial ecologyin microbial ecology

• enrichment and isolation of bacteria enrichment and isolation of bacteria

• constant supply of cells in exponential phase growing at a known rateconstant supply of cells in exponential phase growing at a known rate

• study of microbial growth at very low nutrient concentrations, close to study of microbial growth at very low nutrient concentrations, close to those present in natural environmentthose present in natural environment

• study of interactions of microbes under conditions resembling those in study of interactions of microbes under conditions resembling those in aquatic environmentsaquatic environments

• food and industrial microbiologyfood and industrial microbiology

Concept checkConcept check

• Continuous culture devices Continuous culture devices (chemostats) are a means of (chemostats) are a means of maintaining cell populations in maintaining cell populations in exponential growth for long periods. exponential growth for long periods. In a chemostat, the rate, and the In a chemostat, the rate, and the population size is governed by the population size is governed by the concentration of the growth-limiting concentration of the growth-limiting nutrient entering the vesselnutrient entering the vessel

• Differentiate the definition of batch Differentiate the definition of batch cultures and continuous culture cultures and continuous culture

• What are the advantages of What are the advantages of chemostat compare to batch culture. chemostat compare to batch culture.

• A A batch culturebatch culture is a fixed volume of culture is a fixed volume of culture medium that is medium that is continuallycontinually being altered by the being altered by the metabolic activitiesmetabolic activities of growing organism and is of growing organism and is therefore a therefore a closed systemclosed system. In the early stage of . In the early stage of exponential growth in batch culture, conditions exponential growth in batch culture, conditions may remain may remain relatively constantrelatively constant, but in later , but in later stages when cell numbers become quite stages when cell numbers become quite large, large, drastic changes in the chemical composition drastic changes in the chemical composition of of the culture medium occur.the culture medium occur.

• A continuous culture is an A continuous culture is an open systemopen system of of contrast volume to which contrast volume to which fresh mediumfresh medium added added continuously and continuously and spent culture mediumspent culture medium is added is added continuouslycontinuously both at a both at a constant rateconstant rate . Once such . Once such a system is in equilibrium, the chemostate a system is in equilibrium, the chemostate volume, cell number, and nutrient status remain volume, cell number, and nutrient status remain constant, and the system is said to be in constant, and the system is said to be in steady steady statestate..

• The chemostat controls both the The chemostat controls both the growth rategrowth rate and the and the population population densitydensity of the culture of the culture simultaneously simultaneously . Two factors are important in . Two factors are important in such control: the such control: the dilution ratedilution rate and the and the concentration of a limiting concentration of a limiting nutrientnutrient, such as a carbon or nitrogen source. , such as a carbon or nitrogen source.

• In a batch culture, In a batch culture, nutrient concentration can affect both rate and nutrient concentration can affect both rate and the growth yield of a culturethe growth yield of a culture. At . At very low concentrationsvery low concentrations of a given of a given nutrient, the nutrient, the growth rate is reducedgrowth rate is reduced, probably because the nutrient , probably because the nutrient cannot be transported into the cell fast enough to satisfy metabolic cannot be transported into the cell fast enough to satisfy metabolic demand. At demand. At moderate or higher nutrient levelsmoderate or higher nutrient levels, the , the growth rate growth rate may not be affectedmay not be affected while the while the cell yield continues to increasecell yield continues to increase. .

• In contrast to a batch culture, in a In contrast to a batch culture, in a chemostatchemostat, growth rate and , growth rate and growth yield can be controlled growth yield can be controlled independently of each otherindependently of each other, the , the former by former by adjustingadjusting the the dilution ratedilution rate and the latter by and the latter by varying the varying the concentrationconcentration of a nutrient present in a limiting amount. A of a nutrient present in a limiting amount. A practical advantage to the chemostat is that a practical advantage to the chemostat is that a population population may bemay be maintained maintained in thein the exponential growth phase exponential growth phase for long periods, thus, for long periods, thus, experiments can be planned in detail and then performed experiments can be planned in detail and then performed whenever most convenient.whenever most convenient.

Measuring microbialMeasuring microbial growthgrowth

Direct methodsDirect methods

• viable plate countsviable plate counts

• membrane filtrationmembrane filtration

• microscopic countsmicroscopic counts

• the use of electronic countersthe use of electronic counters

• the most probable number the most probable number method. method.

Viable plate countViable plate count

• spread and pour plate techniquesspread and pour plate techniques– diluted sample of bacteria is spread over solid diluted sample of bacteria is spread over solid

agar surface or mixed with agar and poured agar surface or mixed with agar and poured into Petri plateinto Petri plate

– after incubation the numbers of organisms are after incubation the numbers of organisms are determined by counting the number of colonies determined by counting the number of colonies multiplied by the dilution factormultiplied by the dilution factor

– results expressed as colony forming units results expressed as colony forming units (CFU)(CFU)

Viable plate countViable plate count• What if the number of cells in even a very small sample is What if the number of cells in even a very small sample is

still too great to count? still too great to count?

• for example, a 1-milliliter sample of milk containing 20,000 for example, a 1-milliliter sample of milk containing 20,000 bacterial cells per ml were plated on a Petri plate, there bacterial cells per ml were plated on a Petri plate, there would be too many colonies to count. would be too many colonies to count.

• In such cases, we make a series of dilutions and count the In such cases, we make a series of dilutions and count the number of colonies resulting on a spread or pour plate number of colonies resulting on a spread or pour plate from each dilution. from each dilution.

• We count the colonies on plates with 25-250 colonies and We count the colonies on plates with 25-250 colonies and multiply the number by the reciprocal of the dilution to multiply the number by the reciprocal of the dilution to estimate the number of bacteria per ml of the original estimate the number of bacteria per ml of the original culture. culture.

• This method is called a viable plate count This method is called a viable plate count

• The accuracy of a viable plate count is The accuracy of a viable plate count is also dependent on also dependent on – the homogeneity of the dilutions, the homogeneity of the dilutions, – the ability of the bacteria to grow on the the ability of the bacteria to grow on the

medium used, medium used, – the number of cell deaths, and the number of cell deaths, and – the growth phase of the sample population. the growth phase of the sample population.

• Thoroughly mixing each dilution, Thoroughly mixing each dilution, inoculating multiple plates per dilution, inoculating multiple plates per dilution, and using log-phase cultures minimize and using log-phase cultures minimize errors. errors.

Membrane filtrationMembrane filtration

• In this method, a large sample (perhaps as In this method, a large sample (perhaps as large as several liters) is poured (or drawn large as several liters) is poured (or drawn under a vacuum) through a membrane filter under a vacuum) through a membrane filter with pores small enough to trap the cells. with pores small enough to trap the cells.

• The membrane is then transferred onto a The membrane is then transferred onto a solid medium, and the colonies present solid medium, and the colonies present after incubation are counted. In this case, after incubation are counted. In this case, the number of colonies is equal to the the number of colonies is equal to the number of CFUs in the original large sample. number of CFUs in the original large sample.

Membrane filtrationMembrane filtration

Plating methods…Plating methods…

• simple and sensitivesimple and sensitive

• widely used for viable counts of widely used for viable counts of microorganisms in food, water, and microorganisms in food, water, and soilsoil

• inaccurate results obtained if cells inaccurate results obtained if cells clump togetherclump together

Microscopic countMicroscopic count

• easy, easy, inexpensive, and inexpensive, and quickquick

• useful for useful for counting both counting both eucaryotes and eucaryotes and procaryotesprocaryotes

• cannot cannot distinguish living distinguish living from dead cellsfrom dead cells

Electronic countersElectronic counters• useful for large microorganisms and blood cells, useful for large microorganisms and blood cells,

but not procaryotesbut not procaryotes

• microbial suspension forced through small microbial suspension forced through small orificeorifice

• movement of microbe through orifice impacts movement of microbe through orifice impacts electric current that flows through orificeelectric current that flows through orifice

• instances of disruption of current are countedinstances of disruption of current are counted

• it is less useful for bacterial counts because of it is less useful for bacterial counts because of debris in the media and the presence of debris in the media and the presence of filaments and clumps of cells.filaments and clumps of cells.

Flow cytometryFlow cytometry

• is a variation of counting with a is a variation of counting with a Coulter counter. Coulter counter.

• A cytometer uses a light-sensitive A cytometer uses a light-sensitive detector to record changes in light detector to record changes in light transmission through the tube as transmission through the tube as cells pass. cells pass.

Most Probable NumberMost Probable Number

• a statistical estimation technique a statistical estimation technique based on the fact that the more based on the fact that the more bacteria in a sample, the more bacteria in a sample, the more dilutions are required to reduce their dilutions are required to reduce their number to zero. number to zero.

Indirect methodsIndirect methods

• Metabolic ActivityMetabolic Activity

• Dry WeightDry Weight

• TurbidityTurbidity

Metabolic ActivityMetabolic Activity

• Under standard temperature conditions, the Under standard temperature conditions, the rate at which a population of cells utilizes rate at which a population of cells utilizes nutrients and produces wastes is dependent nutrients and produces wastes is dependent on their number. on their number.

• Once they establish the metabolic rate of a Once they establish the metabolic rate of a microorganism, scientists can indirectly microorganism, scientists can indirectly estimate the number of cells in a culture by estimate the number of cells in a culture by measuring changes in such things as measuring changes in such things as nutrient utilization, waste production, or pH. nutrient utilization, waste production, or pH.

Dry WeightDry Weight

• The abundance of some microorganisms, The abundance of some microorganisms, particularly filamentous microorganisms, is particularly filamentous microorganisms, is difficult to measure by direct methods. difficult to measure by direct methods.

• Instead, these organisms are filtered from their Instead, these organisms are filtered from their culture medium, dried, and weighed. culture medium, dried, and weighed.

• The The dry weight method dry weight method is suitable for broth is suitable for broth cultures, but growth can not be followed over time cultures, but growth can not be followed over time because the organisms are killed during the because the organisms are killed during the processprocess

TurbidityTurbidity

• As bacteria reproduce in a broth culture, As bacteria reproduce in a broth culture, the broth often becomes the broth often becomes turbid turbid (cloudy)(cloudy)

• An indirect method for estimating the An indirect method for estimating the growth of a microbial population growth of a microbial population involves measuring changes in turbidity involves measuring changes in turbidity using a device called a using a device called a spectrophotometer spectrophotometer

more cells

more lightscattered

less lightdetected

• dry weightdry weight– time consuming and not very sensitivetime consuming and not very sensitive

• quantity of a particular cell constituentquantity of a particular cell constituent– e.g., protein, DNA, ATP, or chlorophylle.g., protein, DNA, ATP, or chlorophyll– useful if amount of substance in each useful if amount of substance in each

cell is constantcell is constant

• turbidometric measures (light scattering)turbidometric measures (light scattering)– quick, easy, and sensitivequick, easy, and sensitive

Growth requirementGrowth requirement

Sources of carbon, energy Sources of carbon, energy and electronsand electrons

- From inorganic source (i.e COFrom inorganic source (i.e CO22) that ) that called called autotrophsautotrophs..

- From organic molecules (proteins, From organic molecules (proteins, carbohydrates) they acquire from carbohydrates) they acquire from other organisms.We called as other organisms.We called as heterotrophsheterotrophs..

Carbon (C) requirementsCarbon (C) requirements

Source of energySource of energy- From redox reactions involving inorganic and From redox reactions involving inorganic and

organic chemicals that called organic chemicals that called chemotrophschemotrophs..

- From light source that called as From light source that called as phototrophs.phototrophs.

-Therefore based on carbon and energy -Therefore based on carbon and energy sources, microbes an be categorized into one sources, microbes an be categorized into one of four basic groups:of four basic groups:

i. Photoautotrophsi. Photoautotrophsii. Chemoautotrophsii. Chemoautotrophsiii. Photoheterotrophsiii. Photoheterotrophsiv. Chemoheterotrophsiv. Chemoheterotrophs

Electron requirements or H Electron requirements or H atoms for hydrogen bonding atoms for hydrogen bonding and electron transferand electron transfer

- From same organic molecules that From same organic molecules that provide C and energy are called provide C and energy are called organotrophsorganotrophs..

- From inorganic sources (H- From inorganic sources (H22, NO, NO2-2-, H, H22S) S) are called are called lithotrophslithotrophs..

Environmental effects on Environmental effects on microbial growthmicrobial growth

TemperatureTemperature

pHpH

water water availabilityavailability

oxygenoxygen

TemperatureTemperature• minimum minimum temperature below temperature below

which growth no longer occurs,which growth no longer occurs,

• an an optimal optimal temperature at temperature at which growth is most rapid, which growth is most rapid, and and

• maximum maximum temperature above temperature above which growth is not possible which growth is not possible

• cardinal temperaturescardinal temperatures

• an characteristic of each an characteristic of each organism but are not organism but are not completely: fixed entities, as completely: fixed entities, as they can be modified slightly they can be modified slightly by other factors of the by other factors of the environment, in particular, by environment, in particular, by the composition of the growth the composition of the growth medium. medium.

Temperature classes of Temperature classes of organismorganism

• Optimum growth temperature classificationsOptimum growth temperature classifications– Close to upper range of growth temperaturesClose to upper range of growth temperatures– Too high and enzymes cease to function Too high and enzymes cease to function

(denaturation)(denaturation)

• 4 groups:4 groups:

– Psychrophiles (-5°C to 15°C)Psychrophiles (-5°C to 15°C)• Arctic and Antarctic bacteriaArctic and Antarctic bacteria• Spoilage of refrigerated goodsSpoilage of refrigerated goods• Some Some Pseudomonas Pseudomonas sp.sp.

– Mesophiles (25°C to 45°C)Mesophiles (25°C to 45°C)• E. coli E. coli and many pathogensand many pathogens• Core body temp is 37°C and extremities coolerCore body temp is 37°C and extremities cooler

– Mycobacterium leprae Mycobacterium leprae (leprosy) attacks extremites but (leprosy) attacks extremites but not main trunknot main trunk

– Syphilis treatment was heat shock- induce fever or hot Syphilis treatment was heat shock- induce fever or hot spasspas

– Thermophiles (45°C to 70°C)Thermophiles (45°C to 70°C)•Hot springsHot springs•Compost heapsCompost heaps•Water heatersWater heaters•Lactobacillus delbrueckii (bulgaricus)- Lactobacillus delbrueckii (bulgaricus)-

yogurt productionyogurt production

– Hyperthermophiles (70°C to 110°C)Hyperthermophiles (70°C to 110°C)•Many ArchaeaMany Archaea•Hydrothermal vents of ocean floorsHydrothermal vents of ocean floors•Pyrolobus fumarimii Pyrolobus fumarimii up to 113°Cup to 113°C

Water availabilityWater availability

• Solute concentrations in the cell’s exterior Solute concentrations in the cell’s exterior environment can cause water to diffuse out of environment can cause water to diffuse out of the cell- plasmolysis and cytoplasmic membrane the cell- plasmolysis and cytoplasmic membrane shrinks away from cell wallsshrinks away from cell walls

• Some cells avoid plasmolysis by increasing Some cells avoid plasmolysis by increasing solute concentration insidecellssolute concentration insidecells

• Osmotolerant organisms-(to about 10% NaCl)Osmotolerant organisms-(to about 10% NaCl)– Staphylococcus Staphylococcus spsp

• Halophiles – require NaCl ions (> 20% NaCl)Halophiles – require NaCl ions (> 20% NaCl)– Many ArchaeaMany Archaea

pHpH

• Develop specialized processes to transport H+ across Develop specialized processes to transport H+ across the cytoplasmic membrane to maintain a nearly neutral the cytoplasmic membrane to maintain a nearly neutral internal pHinternal pH

• Like temperature, there are optimum growth conditionsLike temperature, there are optimum growth conditions

• 3 major groups:3 major groups:– Neutralophiles (optimum pH 7)Neutralophiles (optimum pH 7)

• Most bacteriaMost bacteria– Acidophiles (optimum pH below 5.5)Acidophiles (optimum pH below 5.5)– Alkalophiles (optimum pH above 8.5)Alkalophiles (optimum pH above 8.5)

• Acido- or alkalotolerant organisms can survive in the pH Acido- or alkalotolerant organisms can survive in the pH environments, however do not grow (multiply) under environments, however do not grow (multiply) under those conditionsthose conditions

Oxygen availabilityOxygen availability

OO22 concentration varies concentration varies– 20% of earth’s atmosphere20% of earth’s atmosphere– Subsurfaces of soils and some aquatic habitats Subsurfaces of soils and some aquatic habitats

have much lesshave much less– Stomach and intestines are devoid of OStomach and intestines are devoid of O22

Determining requirement: absolutes and tolerancesDetermining requirement: absolutes and tolerances• Shake tubes:Shake tubes:

– solid medium boiled to drive off Osolid medium boiled to drive off O22

– cooled but molten agar is inoculated with bacteria cooled but molten agar is inoculated with bacteria and bacteria distributed throughout mediumand bacteria distributed throughout medium

– agar solidifies impeding diffusion of Oagar solidifies impeding diffusion of O22 into bottom into bottom of tube (stratification of Oof tube (stratification of O22))

– watch for region of bacterial growthwatch for region of bacterial growth

Obligate aerobesObligate aerobes– Need ONeed O22 for aerobic respiration to produce for aerobic respiration to produce

cellular energycellular energy– Pseudomonas Pseudomonas sp.sp.

Obligate anaerobesObligate anaerobes– Cannot multiply if OCannot multiply if O22 is present is present

– Cellular energy produced by anaerobic Cellular energy produced by anaerobic respiration or fermentationrespiration or fermentation

– Bacterioides Bacterioides sp. and sp. and Clostridium Clostridium sp.sp.

Facultative anaerobesFacultative anaerobes– Grow best if OGrow best if O22 is present but also grow without it is present but also grow without it– Use aerobic respiration if OUse aerobic respiration if O22 is present, otherwise is present, otherwise

undergo fermentation or anaerobic respirationundergo fermentation or anaerobic respiration– Aerobic respiration provides more ATP = better growthAerobic respiration provides more ATP = better growth– E. coli E. coli and and Saccharomyces cervisiaeSaccharomyces cervisiae

• MicroaerophilesMicroaerophiles– Requires low levels of ORequires low levels of O22 for aerobic respiration for aerobic respiration– High concentrations of OHigh concentrations of O22 are inhibitory are inhibitory– Helicobacter pyloriHelicobacter pylori

• Aeroltolerant anaerobesAeroltolerant anaerobes– Can grow in presence of OCan grow in presence of O22 but do not use it for energy but do not use it for energy

productionproduction– Use only fermentative pathways = obligate fermentersUse only fermentative pathways = obligate fermenters– Streptococcus pyogenes Streptococcus pyogenes (strep throat)(strep throat)

Growth versus oxygen Growth versus oxygen concentrationconcentration

Microbial Growth in Microbial Growth in Natural EnvironmentsNatural Environments

• microbial environments are complex, microbial environments are complex, constantly changing, often contain constantly changing, often contain low nutrient concentrations low nutrient concentrations (oligotrophic environment) and may (oligotrophic environment) and may expose a microorganism to expose a microorganism to overlapping gradients of nutrients overlapping gradients of nutrients and environmental factorsand environmental factors

Growth Limitation by Growth Limitation by Environmental FactorsEnvironmental Factors

• Leibig’s law of the minimumLeibig’s law of the minimum– total biomass of organism determined total biomass of organism determined

by nutrient present at lowest by nutrient present at lowest concentrationconcentration

• Shelford’s law of toleranceShelford’s law of tolerance– above or below certain environmental above or below certain environmental

limits, a microorganism will not grow, limits, a microorganism will not grow, regardless of the nutrient supplyregardless of the nutrient supply

Responses to low nutrient levels Responses to low nutrient levels (oligotrophic environments)(oligotrophic environments)

• organisms become more competitive in organisms become more competitive in nutrient capture and use of available nutrient capture and use of available resources resources

• morphological changes to increase surface morphological changes to increase surface area and ability to absorb nutrientsarea and ability to absorb nutrients

• mechanisms to sequester certain nutrientsmechanisms to sequester certain nutrients

Counting Viable but Counting Viable but Nonculturable Vegetative Nonculturable Vegetative ProcaryotesProcaryotes

• stressed microorganisms can temporarily stressed microorganisms can temporarily lose ability to grow using normal lose ability to grow using normal cultivation methodscultivation methods

• microscopic and isotopic methods for microscopic and isotopic methods for counting viable but nonculturable cells counting viable but nonculturable cells have been developedhave been developed

Counting and Identifying Counting and Identifying Microorganisms in Natural Microorganisms in Natural EnvironmentsEnvironments

– difficult to culture organisms from natural difficult to culture organisms from natural environmentsenvironments

– previously stressed microbes are very previously stressed microbes are very sensitive to secondary stresses and may sensitive to secondary stresses and may not grow on media normally used to not grow on media normally used to cultivate themcultivate them

– Postgate Microviability Assay uses change Postgate Microviability Assay uses change in morphology on agar surface as indication in morphology on agar surface as indication of “life signs”of “life signs”

BiofilmsBiofilms

• ubiquitous in natureubiquitous in nature• complex, slime enclosed colonies complex, slime enclosed colonies

attached to surfaces attached to surfaces • when form on medical devices such when form on medical devices such

as implants often lead to illnessas implants often lead to illness• can be formed on any conditioned can be formed on any conditioned

surface surface

Biofilm formationBiofilm formation

• microbes reversibly attach to microbes reversibly attach to conditioned surface and release conditioned surface and release polysaccharides, proteins, and DNApolysaccharides, proteins, and DNA

• additional polymers are produced as additional polymers are produced as biofilm maturesbiofilm matures

• interactions occur among the interactions occur among the attached organisms attached organisms

Biofilm microorganismsBiofilm microorganisms

• extracellular matrix and change in extracellular matrix and change in attached organisms’ physiology protects attached organisms’ physiology protects them from harmful agents such as UV them from harmful agents such as UV light and antibiotics light and antibiotics

• sloughing off of organisms can result in sloughing off of organisms can result in contamination of water phase above the contamination of water phase above the biofilm such as in a drinking water systembiofilm such as in a drinking water system

Cell to Cell CommunicationCell to Cell Communication

• acylhomoserine lactone (AHL) is an acylhomoserine lactone (AHL) is an autoinducer molecule produced by autoinducer molecule produced by many grammany gram organisms organisms

• concentration present allows cells to concentration present allows cells to access population densityaccess population density

• process is called quorum sensingprocess is called quorum sensing

Quorum SensingQuorum Sensing

• AHL or other signal molecule diffuses AHL or other signal molecule diffuses across plasma membrane across plasma membrane

• at high concentrations it enters cellat high concentrations it enters cell

• once inside the cell it induces once inside the cell it induces expression of target genes that expression of target genes that regulate a variety of functionsregulate a variety of functions

End of chapter End of chapter

Microbial growth kineticMicrobial growth kinetic

Next chapterNext chapter

Fermentation processFermentation process