Microbial growth kinetics and fermentation

Measurement of Microbial Growth

3. Measurement of Microbial Growth

A. Measurement of cell numbers

1.Direct microscopic observation on specially etched slides (hemacytometers)

2.Viable cell counts involve plating diluted samples (using a pour plate or spread plate)

B. Measurement of cell mass-may be used to approximate the number of

microorganisms

(Suitable parameters may be dry weight, light scattering in liquid solutions, or

biochemical determinations of specific cellular constituents such as protein, DNA)

Bacterial colonies growing on a

plate of nutrient agar.

1 OD600 nm = 109 cells/ml (E. coli

Saccharomyces cerevisiae 1 OD600 nm = 4-9 x 10^7 cells/ml

If plated on a suitable medium, each viable unit grows and forms a colony.Each colony that can be counted is called a colony forming unit (cfu) and thenumber of cfu's is related to the viable number of bacteria in the sample

DILUTION PLATING

Beer-Lambert Law

Beers Law states that absorbance is proportional to concentrationover a certain concentration range

A = log(IO/I) = cl

A = cl

A = absorbance; = molar extinction coefficient (M-1 cm-1 or mol-1 L cm-1); c =concentration (M or mol L-1); l = path length (cm) (width of cuvette)

A = Absorbance (optical density) IO = Intensity of light on the sample cell I = Intensity of light leaving the sample cell c = molar concentration of solute l = length of sample cell (cm) = molar absorptivity (molar extinction coefficient)

Intensity of spectral lines

Transmittance0II

T 0I Isample

Beer-Lambert law ][010 JII

[J] Molar concentrationLength

Molar absorption coefficient (extinction coefficient) : L mol-1 cm-1

Absorbance AII

A 0log JA

Note that ~ (cm2 mol-1) ~ cross-section for absorption

Plating methods

Measure number of viable cells

Population size is expressed as colony forming units (CFU)

Simple and sensitive

Widely used for viable counts of microorganisms in food, water, and soil

Inaccurate results obtained if cells clump together

plate dilutions of population on suitable solid medium

count number of colonies

calculate number of cells in population

Especially useful for analyzing aquatic samples

Membrane filtration methods

Measurement of Cell Mass

• Dry weight

– time consuming and not very sensitive

• Quantity of a particular cell constituent

– protein, DNA, ATP, or chlorophyll

– useful if amount of substance in each cell is constant

• Turbidometric measures (light scattering)

– quick, easy, and sensitive

Effects of Physical and chemical parameterson cell Physiology

Effect of pH• Acidophiles

– growth optimum between pH 0 and pH 5.5

• Neutrophiles

– growth optimum between pH 5.5 and pH 7

• Alkalophiles

– growth optimum between pH 8.5 and pH 11.5

• most acidophiles and alkalophiles maintain an internal pH near neutrality– The plasma membrane is impermeable to protons

• some synthesize proteins that provide protection– e.g., acid-shock proteins

• many microorganisms change pH of their habitat by producing acidic or basic waste products– most media contain buffers to prevent growth inhibition

Temperature

• organisms exhibit distinct cardinal growth temperatures

– minimal

– maximal

– optimal

Effect of factors: aerobic growth is more efficient.

- aerobic fermentation requires oxygen

- oxygen gas is sparingly soluble in water

- specific growth rate may be limited by DO if DO is below acritical oxygen concentration.growth rate becomes independent of DO concentration.

bacteria and yeast: 5%-10% of the saturated DOmold: 10%-50% of the saturated DO

The saturated DO in aqueous solution is 7 ppm at 25oC and 1atm.

-Dissolved oxygen (DO)

Media Optimization:

Diauxic Growth

The biphasic response of a culture of micro-organisms based on a phenotypic adaptationto the addition of a second substrate; characterized by a growth phase followed by a lagafter which growth is resumed.

Growth of microbial cells

Batch Growth Characteristics

Growth Stages, Effects of Environmental Conditions, Product Formation,Mathematical Models

Fed-batch:No substrate inhibition, desired growth rate, minimization of inhibitory

product formation, High cell density

Continuous Growth Characteristics

Dilution Rate, Optimum Operation

Introduction:

Microbial Growth in Batch

• Region 1:Lag phase– microbes are

adjusting to the new substrate (food source)

• Region 2Exponential growth phase, – microbes have

acclimated to the conditions

• Region 3Stationary phase, – limiting substrate or

electron acceptor limits the growth rate

• Region 4Decay phase, – substrate supply has

been exhausted

Time

log [X]32 41

Lag Phase

Cell growth is the primary response of viable cells tosubstrates and nutrients.

Substrates/nutrients + cells → products + more cells

Product formation is a secondary response.

The second major phase of microbial growth in a batch fermentation ferment processwhich is also known as the logarithmic growth phase.

Cells have adjusted to their new environment

The cells are dividing at a constant rate resulting in an exponential increase in thenumber of cells present. This is known as the specific

Growth rate and is represented mathematically by first order kinetics as the following:

where X is the cell concentration, μ is the cell growth rate, and kd is the cell death rate.The term can be referred to as net . μ – kd (μnet) The cell death rate is sometimesneglected if it is considerably low (smaller than the cell growth rate)

Exponential Phase

• Fermentation is a biological reaction in which both electron-donor and electron-acceptor are organic molecules

• In industrial microbiology, fermentation refers to any largescale microbial process ,whether or not, it is biochemically afermentation

• Fermentation processes

Batch fermentation Continuous fermentation Fed-batch fermentation

Fermentation

Batch Fermentation

Reasons for Batch Popularity

• Equations were for cell mass (or other growth-associatedproduct). Many industrial applications are for non-growthassociated products.

• Selective pressure of a chemostat is detrimental toengineered organisms

• Batch is more mechanically reliable

• Batch system is more more flexible

OPEN SYSTEM• All components that compose

the system may enter and leave• Eg. Continuous flow cultures• Biomass balances the output

rate.• Establishment of steady state

may occur

CLOSED SYSTEM• All components that

compose the system may not enter and leave.

• Batch culture which consists of an essential limited amount of nutrient medium.

• Growth rate tends to zero• System is always in

transient state.

Batch fermentation

Growth Kinetics of batch fermentation

Introduction

- Autocatalytic reaction: The rate of growth is directly related to cell concentration

substrates + cells → extracellular products + more cells

∑S + X → ∑P + nX

S: substrate concentration (g/L); X: cell mass concentration (g/L);P: product concentration (g/L); n: increased number of biomass.

Net specific growth rate (1/time):

t: the timedt

dX

X

1net

(1/time) rategrowth specific maximum theis R

mμ

mμnet

Batch Growth Kinetics

Exponential growth phase

Doubling time of cell mass: the time required to double the microbial mass:

netnetnet

693.02ln0/ln

XXd

Fed-Batch Fermentation

Fed-Batch Fermentation• Initially starts in a batch mode and is then

fed according to one of the following feed strategies:

i) Medium used to establish the batch culture is added

ii) Solution of limiting substrate of same concentration as in the initial medium is used

iii) Concentrated solution of the limiting substrate is used

Fed-Batch Nutrients are continuously or semi-continuously fed, while effluent is removed

discontinuously.

Overcome substrate inhibition or catabolite repression by intermittent feeding ofsubstrate by maintaining low substrate concentration.

Used for production of secondary metabolites e.g. antibiotics, lactic acid, E. Colimaking proteins from recombinant DNA technology.

• Before starting a fed-batch process, a batch fermentation should be implementedto "get to know" the fermentation of the microorganism. From a batchfermentation, the operator should have a knowledge of:

• Best abiotic conditions such as temperature, light, agitation, pH, growthmedium, etc.

• Specific needs of precursors, inducers or other enrichment factors

• The different growth phases and the consumed (substrate) and producedcomponents (product of interest and by-product)

• The relationship between the biomass and product formation (growth or non-growth associated product) and the oxygen uptake rates

• Limiting substrate for growth and the relationship between the specific growthrate and the limiting substrate concentration

• Eventual inhibitions from the substrate and/or product

Preliminary knowledge required to implement fed-batch

Though batch fermentation is considered simple, fed-batch fermentation offersthe convenience of better control over substrate concentration variations anddifferentiation of growth leading to improved overall productivity withessentially the same equipment used for batch fermentation.

The productivity of a fermentation process is better in the fed-batch modecompared to a batch mode of operation.

In batch fermentation of S. cerevisiae a dry weight of 10g/L was obtainedwhereas in fed batch mode , with respiratory quotient (RQ) as the controlparameter for glucose feed , a final dry cell weight of 31-56g/L was obtained.

Other technique is High cell density cultivation

Productivity of Fed-Batch fermentation

Growth rate = number of generation/time(k = n/t)

Effect of nutrient concentration on growth

Two basic approaches can be used:

• The constant volume fed-batch culture - Fixed Volume Fed-Batch- The limiting substrate is fed without diluting the culture.

• The Variable Volume Fed-Batch- As the name implies, a variable volume fed-batch is one in which the volume

changes with the fermentation time due to the substrate feed. The way thisvolume changes it is dependent on the requirements, limitations andobjectives of the operator.

Fed-batch fermentation

Feed Rate in Fed-batch culture

F = Fo et

Production of high cell densities due to extension of working time(particularly important in the production of growth-associated products)

Controlled conditions for the provision of substrates during thefermentation

Control over the production of by-products, or catabolite repressioneffects, due to limited provision of only those substrates solely required forproduct formation Allows the replacement of water lost via evaporation

Alternative mode of operation for fermentations involving bioremediationof toxic substrates (cells can only metabolize a certain quantity at a time), orlow solubility Compounds

No additional special pieces of equipment are required to convert frombatch to fed-batch operation

Advantages of Fed-Batch Culture

Requires previous analysis of the microorganism, its requirements, and anunderstanding of its physiology with respect to productivity

Requires a substantial amount of operator skill for set-up, definition anddevelopment of the process

In a cyclic fed-batch culture, care must be taken in the design of the processto ensure that toxins do not accumulate to inhibitory levels, and that nutrientsother than those incorporated into the feed medium do not become limiting

Also, if many cycles are run, the accumulation of non-producing or low-producing mutants may result

Disadvantages of Fed-Batch Culture

• In fed-batch cultivation, feeding strategy is the most important factor in success of theprocess . Different feeding strategies include-

• Constant feeding: Concentrated nutrients are fed into the bioreactor at apredetermined (constant) rate. Because of the increase in culture volume andcell concentration in the bioreactor, the specific growth rate continuouslydecreases, and the increase in cell concentration slows down over time(Jensen and Carlsen, 1990)

• Increased feeding: Nutrient is fed at an increasing (gradual, stepwise, orlinear) rate.

• Exponential feeding: Feeding nutrient at an exponential rate. Cells can growexponentially during the entire culture period if the feed rate of the growth-limiting substrate is increased in proportion to growth. Constant specificgrowth rate can be achieved.

High Cell Density Culture (Nutrient feeding strategy)

Different feeding strategies:

Continuous stirred Tank Reactor CSTR

Continuous stirred Tank Reactor CSTR

In batch culture, the culture environment continuouslychanges

Growth, product formation, and substrate utilization allterminate after a certain time interval

In continuous culture, fresh nutrient medium is continuallysupplied to a well-mixed culture, and products and cells aresimultaneously withdrawn

Growth and product formation can be maintained forprolonged periods of time

At steady state, cell, product, and substrateconcentrations remain constant

Batch vs. Continuous Culture

Importance of continuous culture methods

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

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

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

• food and industrial microbiology

•The Chemostat

•rate of incoming medium = rate of removal of medium from vessel

•an essential nutrient is in limiting quantities

•chemostat operates best at low dilution rate

• dilution rate:- rate at which medium flows through vessel relative to vessel size

• cell density remained unchanged over a wide range of dilution rates

• chemostat operates best at low dilution rate

Dilution rate and microbial growth

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

Most chemostats require some control elements (e.g. pH andDO controllers)

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

Liquid volume in the reactor is kept constant

The Ideal Chemostat

Chemostat:

•Also known as a continuous stirred tank reactor (CSTR)

• Cellular growth is typically limited by one essential nutrient; other nutrients are inexcess

• When operated at steady state, nutrient, product and cell concentrations areconstant

Plug Flow Reactor (PFR):

• Substrate and cell concentrations vary with axial position in the reactor

• An ideal PFR resembles a batch reactor in which distance along the fermenterreplaces incubation time

Reactors for Continuous Culture

Plug Flow Reactor

No longitudinal mixing, all elements of water enter and exit in same order. The time spent in the reactor, the residence time, is equal for all elements:

t = V/Q V = volumeQ = flow rate

Another way to think of the residence time is the time to fill the reactor. This is actually true for any type of reactor.

Plug Flow Reactor Mole Balance

PFR:

The integral form is:

V dF

A

rA

FA 0

FA

This is the volume necessary to reduce the entering molar flow rate (mol/s) from FA0 to the

exit molar flow rate of FA.

Packed Bed Reactor Mole Balance

PBR

The integral form to find the catalyst weight is:

W dF

A

r AFA 0

FA

FA0 FA r AdW dNA

dt

Usage Advantages Disadvantages

1. Large Scale2. Fast Reactions3. Homogeneous Reactions4. Heterogeneous Reactions5. Continuous Production6. High Temperature

1. High Conversion per Unit Volume2. Low operating cost3. Continuous Operation4. Good heat transfer

1. Undesired thermal gradients may exist2. Poor temperature control3. Shutdown and cleaning may be expensive

A turbidostat is a continuous culturing method where the turbidity of the culture is heldconstant by manipulating the rate at which medium is fed. If the turbidity tends to increase,the feed rate is increased to dilute the turbidity back to its set-point. When the turbiditytends to fall, the feed rate is lowered so that growth can restore the turbidity to its set-point.

Regulates the flow rate of media through vessel to maintain a predetermined turbidity orcell density

Dilution rate varies

No limiting nutrient (all nutrients are present in excess)

Regulates the flow rate of media through vessel to maintain a predetermined turbidity orcell density

Use a photocell to measure the absorbance or turbidity

Turbidostat operates best at high dilution rates regulates the flow rate of media throughvessel to maintain a predetermined turbidity or cell density

Turbidostat

Production of high cell densities due to extension of working time

Control over the production of by-products or catabolite repressioneffects due to limited provision of substrates solely required for productformation

Allows the replacement of water loss by evaporation

Maintenance of aerobic conditions within the fermenter

Toxic effects of given metabolites or medium components can be avoided

No additional special piece of equipment is required as compared with thebatch fermentation mode of operation

Precursors and / or inducers can be fed at the right time

Advantages of fed-batch fermentation

• It requires previous analysis of the microorganism, its requirements and the understanding of its physiology with the productivity

• It requires a substantial amount of operator skill for the set-up, definition and development of the process

• In a cyclic fed-batch culture, care should be taken in the design ofthe process to ensure that toxins do not accumulate to inhibitorylevels and that nutrients other than those incorporated into the feedmedium become limiting, Also, if many cycles are run, theaccumulation of non-producing or low-producing variants mayresult.

Disadvantages: