rx = rate of growth
Week 4 – Fundamentals of Microbial Cell Culture & Nutrition Math of bacterial growth & Biomass -
rx = rate of growth X = cell concentration or Biomass (dry weight per volume) μ = growth rate constant (μ = rx / X)
When bacteria divide, they multiply – producing 2 daughter cells by binary fission resulting in exponential increase in cell numbers Doubling time (td) = generation time (ie, time taken for population or organisms to double) Binary fission -
-
asexual reproduction & cell division used by prokaryotes don’t exchange DNA, just doubling and producing identical daughter cells consequences all cells are genetically identical (if no mutations occur) Process o DNA in bacteria is tightly coiled o DNA uncoils & replicates o DNA is pulled to separate poles (size increases to prepare for splitting) o Growth of new cell wall splits bacterium o New cell wall completely splits bacterium o New daughter cells have – tightly coiled DNA, ribosomes, plasmids Different microorganisms have different generation time – depends on type of microorganisms, environment & nutrients available
Generation time
What do cells need to multiply? -
Suitable temperature range (eg E. coli 37*C) Suitable pH range (eg E. coli pH 7.2) Basic elements of life – carbon dioxide, water Nutrients – for growth (eg. Nitrogen, glucose – C,H,O)
Maximal growth rates = exponential growth (unrestricted growth) -
Growth rates will not be maximal if physical conditions are not optimal – max growth rate can be limited by different factors For each set of conditions there are maximal growth rates, μmax
Some issues can arise with cell cultures ie cells did not grow at wanted rate -
Did not provide right environment – unsuitable pH, Temperature Defect in cells – cell growth kinetics retarded Cells are dying cells multiply but die due to over population (competition), cells do not have enough nutrients/build-up of waste products are toxic
Measuring biomass (measure how many bacterial cells) -
Biomass (X) = total amount of microbial cell matter in a system Amount of biomass being formed in a culture depends on the amount of biomass already present dX/dt = μX Change in biomass over time depends on – how many cells are present(X), how fast they can grow and multiply (μ) Measure cells (estimate biomass) by: Method 1) Microscopic count
-
2) Spectrophotometry
-
-
Take sample (1ml out of 100ml), centrifuge, dye, look under microscope, count number of dead cells, repeat N = no. of dead cells/ no. of living cells x100 (since 100ml) Accurate but laborious Measure biomass by measuring turbidity (cloudiness of sample) Turbidity = relationship between biomass & amount of light absorbed or light scattered(optical density) Note: different cell shapes and size behave differently, cells in different growth stage behave differently, each type of spectrophotometer behave differently
3) Electronic particle counting
-
Flow cytometer – difficult when a lot of particles in the medium, expensive
4) Total Viable counts (TVC)
-
Plate microorganisms, grow, count colonies Suitable for colonies that are easy to grow, ie short generation time
5) Study chemical components of cells
-
Eg. DNA, RNA, protein, cell wall May vary in different growth phases
Complexity of cell cultures -
Bacterial growth involves anabolic(build up) and catabolic(break down – energy released from breaking down can be used for anabolic reactions) reactions Two approaches to study growth of bacteria under controlled conditions o Batch culture & Continuous culture
Batch Culture -
Large scale closed system where cells are grown in a fixed volume of nutrient culture medium under specific environmental conditions limited nutrient available Certain density of cells achieved before nutrients used up Growth curve contains 4 phases o Lag phase – very little to no growth; bacterial adapt to environment (begin synthesis of RNA, DNA molecules) o Log phase/Exponential phase – cells doubling at a constant exponential rate; growth not limited by nutrient availability or by toxic products but rather temperature and pH; there will be a μmax o Stationary phase – rate of cell death = rate of cell division; essential nutrients will be used up/ waste products build up inhibiting growth; dX/dt = 0 o Death phase – steady decline in number of cells from starvation/increase in toxic wastes; rate of cell death is generally slower than exponential growth rate; dX/dt= KdX
Fed-batch culture -
-
While cells are growing exponentially but nutrients become depleted, concentrated feed medium is added either continuously or intermittently to supply additional nutrients, allowing for a further increase in cell concentration and the length of production phase. To accommodate addition of medium, fed batch culture is started in a volume of much lower than full capacity Would still have the same 4 phases as batch culture but longer exponential time Fed-batch culture is effective for a few types of bioprocesses o Substrate inhibition – some compounds (eg ethanol) can inhibit the growth of microorganisms adding substrates/nutrients appropriately reduces lag time and inhibition of cell growth is reduced o High cell concentration – to achieve high cell concentration, high initial concentration of nutrients are required. But this can be inhibitory therefore use fedbatch culture o Glucose effect (Crabtree effect) – eg in baker’s yeast, aerobic ethanol fermentation occurs in presence of glucose therefore use fed-batch to reduce effect o Catabolite repression – when too high glucose concentration available, increase in ATP concentration leads to repression of enzyme biosynthesis therefore slowly feed glucose o Extension of operation time, supplement water loss due to evaporation, decrease viscosity of nutrient broth
Continuous culture -
-
-
Fresh nutrients continually supplied to a well stirred culture Products and cells are simultaneously withdrawn Continuous culture also known as chemostat At steady state, concentration of cells, products and substrates are constant o Steady state all state variables are constabt o In batch culture: the environment changes continually & terminate after a certain duration, continuous culture can continuously grow Laboratory setup more complex, but can control everything A way of prolonging the balanced growth of early exponential phase Growth container for continuous culture = chemostat/bioreactor Produces microbial product more efficiently than batch fermentation because the chemostat can hold a culture in the exponential growth phase Graph of log cell density vs no of generations
Ideal chemostat balance everything to keep things constant o Control elements – pH, T, dissolved oxygen o Fresh sterile medium is continuously fed o Suspension is removed at the same rate o Liquid volume in the reactor is kept constant
Microbial growth kinetics -
Monod equation is a mathematical model for the growth of microorganisms
-
μ = specific growth rate μmax = maximum specific growth rate of the organism S = concentration of the limiting substrate for growth (ie nutrients) Ks = half velocity constant ie substate concentration when μ/ μmax = ½ Μmax & Ks differs for type of microorganism and environmental conditions
Continuous culture is important for biotechnology -
Cells are generally investigated in steady state analyse metabolic process accurately Provides precise, effective & well controlled way of investigating microbial growth Growth rates and metabolic rates can be experimentally controlled physiological processes related to rates of metabolism can be dissected Some industrial processed are continuous
Using continuous culture in R&D
-
Use microcosms(artificial ecosystems) to predict and simulate the behaviour of natural ecosystems under controlled conditions Antibiotic research – antibiotic resistance can be due to mutations, use chemostats to enrich for specific type of mutants
Type of culture Batch culture
Advantages -
-
Initial capital expenditure is lower only need to supply nutrients once Simple to terminate a contaminated cycle & restart Batch-to-batch variability (can also be disadvantageous)
Disadvantages -
-
-
Fed-batch culture
-
-
-
Continuous culture
-
Increase production of biomass compared to batch culture Get products which are growth associated products – ie get more products from specific phase Production of secondary metabolites Overcome problems in continuous culture such as contamination and mutations
-
More productive then batch cultures (theoretically) Cells can be produced under optimal conditions Lower operating costs
-
-
-
Less effective production of biomass & primary metabolites lag period, non-productive death period, cleaning, refilling, sterilizing Batch-to-batch variability (can also be advantageous) Greater stress in instruments due to increased frequency of usage Greater running costs to prepare and maintain stock cultures More personnel required Additional instruments for feedback control Requires operator skill Time courses might not always follow expected profiles
Higher initial expenditure Few large scale industrial examples have been successful Can get random mutations – since perfect environment cell line keep continuing (no death/removal) longer time to mutate
rx = rate of growth X = cell concentration or Biomass (dry weight per volume) μ = growth rate constant (μ = rx / X)
When bacteria divide, they multiply – producing 2 daughter cells by binary fission resulting in exponential increase in cell numbers Doubling time (td) = generation time (ie, time taken for population or organisms to double) Binary fission -
-
asexual reproduction & cell division used by prokaryotes don’t exchange DNA, just doubling and producing identical daughter cells consequences all cells are genetically identical (if no mutations occur) Process o DNA in bacteria is tightly coiled o DNA uncoils & replicates o DNA is pulled to separate poles (size increases to prepare for splitting) o Growth of new cell wall splits bacterium o New cell wall completely splits bacterium o New daughter cells have – tightly coiled DNA, ribosomes, plasmids Different microorganisms have different generation time – depends on type of microorganisms, environment & nutrients available
Generation time
What do cells need to multiply? -
Suitable temperature range (eg E. coli 37*C) Suitable pH range (eg E. coli pH 7.2) Basic elements of life – carbon dioxide, water Nutrients – for growth (eg. Nitrogen, glucose – C,H,O)
Maximal growth rates = exponential growth (unrestricted growth) -
Growth rates will not be maximal if physical conditions are not optimal – max growth rate can be limited by different factors For each set of conditions there are maximal growth rates, μmax
Some issues can arise with cell cultures ie cells did not grow at wanted rate -
Did not provide right environment – unsuitable pH, Temperature Defect in cells – cell growth kinetics retarded Cells are dying cells multiply but die due to over population (competition), cells do not have enough nutrients/build-up of waste products are toxic
Measuring biomass (measure how many bacterial cells) -
Biomass (X) = total amount of microbial cell matter in a system Amount of biomass being formed in a culture depends on the amount of biomass already present dX/dt = μX Change in biomass over time depends on – how many cells are present(X), how fast they can grow and multiply (μ) Measure cells (estimate biomass) by: Method 1) Microscopic count
-
2) Spectrophotometry
-
-
Take sample (1ml out of 100ml), centrifuge, dye, look under microscope, count number of dead cells, repeat N = no. of dead cells/ no. of living cells x100 (since 100ml) Accurate but laborious Measure biomass by measuring turbidity (cloudiness of sample) Turbidity = relationship between biomass & amount of light absorbed or light scattered(optical density) Note: different cell shapes and size behave differently, cells in different growth stage behave differently, each type of spectrophotometer behave differently
3) Electronic particle counting
-
Flow cytometer – difficult when a lot of particles in the medium, expensive
4) Total Viable counts (TVC)
-
Plate microorganisms, grow, count colonies Suitable for colonies that are easy to grow, ie short generation time
5) Study chemical components of cells
-
Eg. DNA, RNA, protein, cell wall May vary in different growth phases
Complexity of cell cultures -
Bacterial growth involves anabolic(build up) and catabolic(break down – energy released from breaking down can be used for anabolic reactions) reactions Two approaches to study growth of bacteria under controlled conditions o Batch culture & Continuous culture
Batch Culture -
Large scale closed system where cells are grown in a fixed volume of nutrient culture medium under specific environmental conditions limited nutrient available Certain density of cells achieved before nutrients used up Growth curve contains 4 phases o Lag phase – very little to no growth; bacterial adapt to environment (begin synthesis of RNA, DNA molecules) o Log phase/Exponential phase – cells doubling at a constant exponential rate; growth not limited by nutrient availability or by toxic products but rather temperature and pH; there will be a μmax o Stationary phase – rate of cell death = rate of cell division; essential nutrients will be used up/ waste products build up inhibiting growth; dX/dt = 0 o Death phase – steady decline in number of cells from starvation/increase in toxic wastes; rate of cell death is generally slower than exponential growth rate; dX/dt= KdX
Fed-batch culture -
-
While cells are growing exponentially but nutrients become depleted, concentrated feed medium is added either continuously or intermittently to supply additional nutrients, allowing for a further increase in cell concentration and the length of production phase. To accommodate addition of medium, fed batch culture is started in a volume of much lower than full capacity Would still have the same 4 phases as batch culture but longer exponential time Fed-batch culture is effective for a few types of bioprocesses o Substrate inhibition – some compounds (eg ethanol) can inhibit the growth of microorganisms adding substrates/nutrients appropriately reduces lag time and inhibition of cell growth is reduced o High cell concentration – to achieve high cell concentration, high initial concentration of nutrients are required. But this can be inhibitory therefore use fedbatch culture o Glucose effect (Crabtree effect) – eg in baker’s yeast, aerobic ethanol fermentation occurs in presence of glucose therefore use fed-batch to reduce effect o Catabolite repression – when too high glucose concentration available, increase in ATP concentration leads to repression of enzyme biosynthesis therefore slowly feed glucose o Extension of operation time, supplement water loss due to evaporation, decrease viscosity of nutrient broth
Continuous culture -
-
-
Fresh nutrients continually supplied to a well stirred culture Products and cells are simultaneously withdrawn Continuous culture also known as chemostat At steady state, concentration of cells, products and substrates are constant o Steady state all state variables are constabt o In batch culture: the environment changes continually & terminate after a certain duration, continuous culture can continuously grow Laboratory setup more complex, but can control everything A way of prolonging the balanced growth of early exponential phase Growth container for continuous culture = chemostat/bioreactor Produces microbial product more efficiently than batch fermentation because the chemostat can hold a culture in the exponential growth phase Graph of log cell density vs no of generations
Ideal chemostat balance everything to keep things constant o Control elements – pH, T, dissolved oxygen o Fresh sterile medium is continuously fed o Suspension is removed at the same rate o Liquid volume in the reactor is kept constant
Microbial growth kinetics -
Monod equation is a mathematical model for the growth of microorganisms
-
μ = specific growth rate μmax = maximum specific growth rate of the organism S = concentration of the limiting substrate for growth (ie nutrients) Ks = half velocity constant ie substate concentration when μ/ μmax = ½ Μmax & Ks differs for type of microorganism and environmental conditions
Continuous culture is important for biotechnology -
Cells are generally investigated in steady state analyse metabolic process accurately Provides precise, effective & well controlled way of investigating microbial growth Growth rates and metabolic rates can be experimentally controlled physiological processes related to rates of metabolism can be dissected Some industrial processed are continuous
Using continuous culture in R&D
-
Use microcosms(artificial ecosystems) to predict and simulate the behaviour of natural ecosystems under controlled conditions Antibiotic research – antibiotic resistance can be due to mutations, use chemostats to enrich for specific type of mutants
Type of culture Batch culture
Advantages -
-
Initial capital expenditure is lower only need to supply nutrients once Simple to terminate a contaminated cycle & restart Batch-to-batch variability (can also be disadvantageous)
Disadvantages -
-
-
Fed-batch culture
-
-
-
Continuous culture
-
Increase production of biomass compared to batch culture Get products which are growth associated products – ie get more products from specific phase Production of secondary metabolites Overcome problems in continuous culture such as contamination and mutations
-
More productive then batch cultures (theoretically) Cells can be produced under optimal conditions Lower operating costs
-
-
-
Less effective production of biomass & primary metabolites lag period, non-productive death period, cleaning, refilling, sterilizing Batch-to-batch variability (can also be advantageous) Greater stress in instruments due to increased frequency of usage Greater running costs to prepare and maintain stock cultures More personnel required Additional instruments for feedback control Requires operator skill Time courses might not always follow expected profiles
Higher initial expenditure Few large scale industrial examples have been successful Can get random mutations – since perfect environment cell line keep continuing (no death/removal) longer time to mutate
