mdm norasyikin chapter 2

8/14/2019 Mdm Norasyikin Chapter 2

1/46

Microbial Growth

KineticsChapter 2


2/46

Introduction Growth of microbes are results of:

- replication- change in cell size

Can grow under various physical, chemical andnutritional conditions. Convert nutrient from medium into biological

compounds

substrates + cells product + more cells Rate of growth cell concentration


3/46

FermentationTraditionally-the process for the production

of alcohol or lactic acid from glucose

Broadly-an enzymatically controlled

transformation of organic compound. Fermentation may be carried out as:

Batch process

Continuous process

Fed-batch process


4/46

Types of Processes Batch: (A)

Continuous: (B)

Fed-batch: (C)


5/46

"closed system". Nothing is added exceptoxygen (in the form of air), an antifoam agent,and acid or base (control pH).

- used to produce biomass, primary metabolites

- In order to produce maximum possible biomass atthe end of the process, optimization of cultural

conditions supporting growth should be established.

- For primary metabolite production, conditions toextend the exponential phase (and hence, product

excretion) should be provided

Batch Culture


6/46

Principal advantages of batch cultures are;

low contamination risk;the ability to run different succesive phases in the

same vessel;and close control of the genetic stability of the

microorganism

Batch Culture


7/46

Control systems for batch fermentation arenormally associated with pH, dissolved oxygen

and temperature.

In batch fermentation, if growth is subject tosubstrate inhibition, fermentation has to be

started with low initial substrate

concentration.

This result in lower maximum biomass andhence, probably, lower maximum

concentration of the required product.

Batch Culture


8/46


9/46

Determining Cell Density Number

1. Petroff-Hausser slide/hemocytometer

- 20 grid squares in counted using

microscope-average- Disadvantages:

medium must be free from particles

stain is used to differentiate betweendead/live cells

not suitable for aggregated cultures


10/46

Cont..

2. Plate count:- used for counting viable cell

- unit: colony forming unit (CFU)

- cultures are diluted and pipetted or spread on

agar surfaces

- plates are incubated and viable colonies are

counted

- a good plate count must consist between 30-200 colonies

- suitable foryeast and bacteria

- selection for best medium growth is crucial


11/46

Cont..

3. Electrical resistance of cells

- cells pass the orifice causes resistance

and provide pulses

- number pulses is a measure - number of

cells

- height of pulses- measure cell size


12/46

Cont..

4. Light intensity measurement

- Intensity of light depicts cell concentration- only for diluted suspension


13/46

Determining Cell MassConcentration

Direct method:- Dry weight - biomass determination

- OD spectrophotometer

Indirect method:

- measurement of cellular component

- e.g: enzyme, chlorophyl


14/46

Growth Patterns & Kinetics in Batch*


15/46

The fermenter could be operated in different modes aims at

improving the performance.

BATCH FERMENTATION

Considered to be a closed

system. At time t=0 the

sterilized nutrient solution in

the fermenter is inoculated

with microorganisms and

incubation is allowed to

proceed. In the course of the

entire fermentation, nothing

is added, except oxygen (in

case of aerobic

microorganisms), an antifoam

agent, and acid or base to

control the pH. Thecomposition of the culture

medium, the biomass

concentration, and the

metabolite concentration

generally change constantly

as a result of the metabolismof the cells.


16/46

Cell growth kinetics

Batch culture

Growth rate of bacterial culture (duringexponential phase):

xdt

dx

1,

,

.

=

==

hrategrowthspecific

hrtimet

concbiomassx


17/46

Growth-limiting Nutrient

Nutrient will be exhausted before theothers

Decrease in growth rate due to depletion ofsubstrate may be described using Monod

equation:

sK

s

s+

=max

= specific growth rate coeff.

max = max specific growth ratecoeff.

S = conc. of limiting nutrient

Ks = half saturation coeff.


18/46

Lag Phase

Delay before rapid growth Occurimmediately after inoculation Cell mass increase, number of cells remain

constant

Cells may be damaged Cells may be adapting to media Cells may be old/cold Cells make new ribosomes

Cells make new proteins Cells begin to make cells


19/46

Log/Exponential Phase

Cells divide at or close to maximum

Cell adjusted to new environment

Biomass increases quickly

Nutrients consumed rapidly

Oxygen (if needed) consumed rapidly

Heat produced in some cultures

Changes in pH due to organism

Protein in media may produce foam

Cell mass & numbers multiply rapidly

Balance growthall component growth at same rate

Growth rate independent of nutrient concentration


20/46

Deceleration Phase

Also known as late log phase

Growth decelerates due to:

- depletion of essential nutrient

- accumulation of toxic by product

- unbalanced growth-restructuring of cell


21/46

Stationary Phase

Resting phase Zero growth rate (no cell division) orgrowth rate

equal to the death rate

Nutrients depleted

Oxygen may be limited Release of cellular chemicals e.g. toxins

Cell growth~= cell death

Production ofsecondary metabolites (non growth

related). E.g: antibiotics, hormones Mixed growth and non-growth associated production.

Cell lysis, cryptic growth occur


22/46

Death Phase

Cells decline exponentially

Some may start during stationary phase

Cell autolysis may occur Occur at the end because of nutrient

depletion or waste accumulation

Sometimes death rate change after hours ordays.


23/46

Product Formation

Primary metabolites

- Growth associated

Secondary metabolites

- Stationary growth associated


24/46

Primary Metabolites

Formed during active cell growth

During primary growth phase

Product essential for the metabolic activity &

growth

Produced from growth substrate by the cell

activity

Ex: alcohol, amino acid


25/46

Secondary Metabolites

Formed at the end or during stationary phase

Not essential for growth

Growth conditions crucial to determine thesynthesis rate of secondary metabolites

Over-production often achievable (not growth

related)

Unpredictable - formation is not consistentamong all members of a species

Ex: penicillin, antibiotics.


26/46

Microbial Product

Microbial growth, product formation andsubstrate utilization rates are usually

expressed in the form of specific rates

Classified to 3 categories:

growth associated

non growth associated

mixed growth associated


27/46

Growth Associated Product

Produced simultaneously with growth

Specific rate product formation specific

growth rate

Product: primary metabolites

E.g.: Enzyme protease (Bacillus subtilis),

amino acid

h i d d


28/46

Non Growth Associated Product

Production occur during stationary phase

The specific rate of product formation is

constant

Product: secondary metabolite

E. g: hormones, antibiotics (penicillin)

i d G h i d


29/46

Mixed Growth Associated

Production occur during slow growth andstationary phase

E. g: lactic acid & certain secondary

metabolites


30/46

Effect of Environmental FactorsOn Growth

Temperature

pH

Oxygen availability

Osmotic pressure/salt concentration

Nutrient availability


31/46

Feed

medium

reservoir

Outflow

containing

product

Fi

FoProductivity = (F/V) x Product

concentration in outflow

V

CONTINUOUS CULTURE

An open system is set up. Sterile nutrient solution isadded to the bioreactor continuously and anequivalent amount of converted nutrient solution withmicroorganisms is simultaneously taken out of thesystem.

C ti C lt


32/46

Continuous Culture

The importance of continuous culture:- maintenance of a culture in constantenvironmental conditions through continuoussupply of nutrient

- provision of nutrients and removal of wastes.- useful for:

study in a certain growth phase

evolution studies

study the effect of changes in the environmentalon cell physiology


33/46

Continuous Culture

Fresh growth mediumis added continuously

during fermentationand spent medium isremoved.

Fermentation can lastup to 1,000 hours.

ow e row n ont nuous


34/46

ow e row n ont nuousCulture

Fresh medium continually supplied to well-stirredculture

Product (cell/culture medium) continuously

withdrawn

During cultivation, growth & product formation

can be prolonged

At steady state: cell, product and substrate

concentration remain constant An essential nutrient is in limiting quantities

B h C i C l


35/46

Batch vs. Continuous Culture

Batch culture; the culture environment continuouslychanges

Growth, product formation and substrate utilizationall terminate after a certain time interval

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

Growth and product formation can be maintained for

prolonged periods of time At steady state, cell, product and substrate

concentrations remain constant

C t


36/46

Cont

Provides constant environmental

conditions for growth and product formation

and supplies uniform quality product

For growth associated products has higherproductivity than batch culture


37/46

Why is Batch Culture Predominantly Used in

the Biotech Industry

Many secondary products are not growth

associated

Genetic instability

Reliability

Economic considerations

D i f C ti C lt


38/46

Devices for Continuous Culture

Plug flow reactor (PFR)

- Continuous cultivation

- No back mixing-fluid elements containing

active cells cannot inoculate other fluid

elements at different axial positions

- Substrate and cell concentrations varywith axial position in the reactor


39/46

Cont

Chemostat

- also known as a continuous stirred tank reactor(CSTR)- refers to constant chemical environment

- perfectly mixed continuous flow

- equipped with pH, DO, level controller

- feeding of fresh medium and removal of cellsuspension occur at the same rate

- cellular growth is typically limited by one essential

nutrient: other nutrients are in excess

- when operated at steady state: nutrient, product andcell concentrations are constant

- volume of reactor constant

Chemostat


40/46

Chemostat

Effluent

Culture medium

Reservoir: Oneessential nutrientin growth-limitingamount

Flowregulator

Valve

Cont


41/46

Cont

Turbidostat

- Cell concentration in the culture vessel

constant (monitor the OD & feed flow rate)

- Volume is kept constantly by removal of culture

broth- Suitable for microorganisms able to withstand

environmental stress

- Flow rate into the system is adjusted to

maintain preset turbidity (cell density)

- No limiting nutrients

- Operates best at high dilution rates

FED BATCH FERMENTATION


42/46

FED BATCH FERMENTATION

Substrate is added in increments as the

fermentation progresses. In the fed-batch

method the critical elements of the nutrient

solution are added in small concentrations atthe beginning of the fermentation and these

substances continue to be added in small

doses during the production phase.

F d B t h C lt


43/46

Fed-Batch Culture

Batch culture which are fed continuously or sequentiallywith medium without the removal of culture fluid

Established initially in batch mode and is then fedaccordingly to one of the following feed strategies:

the same medium used to establish the batch culture isadded, resulting in an increase in volume

a solution of the limiting substrate at the sameconcentration as that in the initial medium is added,resulting in an increase in volume

a concentrated solution of the limiting substrate is addedat a rate less than in (i) and (ii), resulting in an increasein volume

a very concentrated solution of the limiting substrate isadded at a rate less than in (i), (ii) and (iii), resulting inan insignificant increase in volume

C t


44/46

Cont

strategies (i) and (ii) - variable volume

strategy (iv) - fixed volume

strategy (iii) - culture intermediate between thetwo extremes of variable and fixed volume

Ex:

- production of bakers yeast

- formation of ethanol- production of hepatitis B surface antigen


45/46

Limiting substrate is fed without diluting the culture.

Culture volume can be maintained constant by feedinggrowth-limiting substrate in undiluted form, for example,as a very concentrated liquid or gas (ex. Oxygen).

Alternatively, substrate can be added by dialysis or, in aphotosynthetic culture, radiation can be the growthlimiting factor without affecting culture volume.

1. Fixed volume fed-batch

2. Variable volume fed-batch

Volume changes with fermentation time due tosubstrate feed.


46/46

mdm norasyikin chapter 2

Documents