Factors Affecting Fermentation

Mr. Nilesh Gaikar, Asst. Professor

Department: School of Pharmacy

Subject: Pharmaceutical Biotechnology
Semester: V

Teaching Aids Service by

KRRC Information Section
Fermenter - Basic Function

The basic function of a fermenter is to provide a suitable environment in

which an organism can efficiently produce a target product that may be
- cell biomass,
- a metabolite,
- or bioconversion product.
INOCULUM DEVELOPMENT

• The inoculum is the starter culture that is injected into the fermenter
• It must be of sufficient size for optimal growth kinetics
• Since the production fermenter in industrial fermentations is so large, the
inoculum volume has to be quite large
 The preparation of a population on microorganisms from a dormant stock culture to an active
state of growth that is suitable for inoculation in the final production stage is called inoculum
development.
 As a first step in inoculum development, inoculum is taken from a working stock culture
to initiate growth in a suitable liquid medium
 It is usually done in a stepwise manner to increase the volume to the desired level.
 Typically the inoculum volume used for production stage is about 5% of the medium volume.
 Inoculum preparation media are designed for rapid microbial growth.
 production process depend on inducible enzymes.
 Contamination free during inoculum development.
 Temp. control methods
Microbes brings about fermentation by secreting certain enzymes which

have an optimum temperature or a temperature range .

Temperature: Temperature affects the solubility and diffusivity of oxygen in the
fermentation broth.
The solubility of oxygen decreases but diffusivity increase with the rise in
temperature.
Cooling water in cooling jacket or coil
 HEAT TRANSFER CONFIGURATIONS
The primary heat transfer configurations in fermentation vessels are:

i. External jackets

ii. Internal coils

iii. External surface heat exchanger

The internal coils though provide better heat transfer capabilities, but they
cause problems of microbial film growth on coil surfaces, alteration of mixing
patterns and fluid velocities.
The external surface heat exchangers, the media is pumped through an
external heat exchanger where the heat transfer takes place through the surface
of exchanger tubes.
 pH control

 Most microorganisms have narrow pH growth ranges (pH 5 - 7 )

 The buffering in culture media is generally low

 Metabolites which released into the medium can change the pH

 Initial pH of the fermentation medium must be very well defined and optimized
depending on the microorganism, substrate and production technique
 Addition of base ( NaOH ) or acid ( HCl )
 Addition of physiological acid substance ((NH4)2SO4) or physiological alkali
substance (ammonium hydroxide)
 Dissolved oxygen (DO ） Control
 Most industrial fermentations are aerobic processes

 Supplying oxygen to aerobic cells has a problem ： oxygen is poorly soluble in

water

 Oxygen supplying is the rate limiting step in an aerobic fermentation

 The objective is to maintain the optimal dissolved oxygen concentration above a
critical concentration to avoid inhibition of the cell growth rate due to lack of
oxygen.
 Factors affecting dissolved oxygen
concentration

 Temperature

 Elevation

 Salinity

 Turbulence
Increase aeration and agitation rate maintain a constant dissolved oxygen
concentration during the fermentation process
 Important factor in a fermenters

Provision for adequate mixing of its contents

Mixing in fermentation
 to disperse the air bubbles
 to suspend the cells
 to enhance heat and mass transfer in the medium
AERATION AND AGITATION

 Aeration refers to the process of introducing air to increase oxygen

concentration in liquids
 Aeration may be performed by bubbling air through the liquid,
spraying the liquid into the air or agitation of the liquid to increase
surface absorption
 Agitation – uniform suspension of microbial cells in homogeneous
nutrient medium
STRUCTURAL COMPONENTS INVOLVED IN
AERATION AND AGITATION

 Agitator (impeller)

 Baffles

 Aeration system (sparger)

AGITATOR (impeller)
Achieve mixing objectives – bulk fluid and
gas-phase mixing, air dispersion, oxygen
transfer, heat transfer, suspension of solid
particles and maintaining uniform environment
throughout vessel contents.
 Agitator design and operation

Radial flow impellers - Rushton turbine

 The most commonly used agitator in microbial fermentations

 Like all radial flow impellers, the Rushton turbine is designed to provide the
high shear conditions required for breaking bubbles and thus increasing the
oxygen transfer rate.
AERATION SYSTEM (SPARGER)
 Introduces air into liquid of fermenter

 Three basic types – porous sparger

1. Orifice sparger – a perforated pipe
2. Nozzle sparger – an open or partially
closed pipe
3. Combined sparger-agitator
 Sparger structures can affect the overall transfer of oxygen into the
medium, as it “influences the size of the gas bubbles produced.
 Small bubbles are desirable because the smaller the bubble, the larger the
surface area to volume ratio, which provides greater oxygen transfer.
 However, spargers with small pores that are effective in producing small
air bubbles are more prone to blockage and require a higher energy input.
 The availability of the oxygen depends on:
 · Solubility
 · Mass transfer rate of oxygen in the fermentation broth
 · Rate of utilization of DO by microbial biomass.
MEDIA FOR INDUSTRIAL FERMENTATIONS
 The media is the feed solution
 It must contain the essential nutrients needed for the microbe to grow

 Factors of consideration when choosing media

-Quality consistence and availability
-Ensure there are no problems with Media Prep or other aspects of
production process

Ex. Cane molasses, beet molasses, cereal grains

 Where biomass or primary metabolites are the target product, the objective is to
provide a production medium that allows optimal growth of the m/o
 Secondary metabolite production is not growth related. Consequently, media are
designed to provide an initial period of cell growth, followed by conditions optimized
for secondary metabolite production. At this point the supply of one or more nutrients
(carbon, phosphorus or nitrogen source) may be limited and rapid growth ceases
 Compounds that are rapidly metabolize may repress product formation.
 Certain media nutrients or environmental conditions may affect the
physiology, biochemistry, and morphology of the microorganism.
 In some yeasts the single cells may develop into pseudo-mycelium or
flocculate, and filamentous fungi may form pellets. This is not desirable as
it affects the product yield
 Carbon Sources
Factors influencing the choice of C-source
The rate at which C-sources metabolized
Price & availability
Media sterilization

Carbohydrates: Starch from cereal, grains and maize; malt from barley; sucrose from sugar cane;

impure form: cane molasses, corn steep liquor, whey from diary industry

Oil & Fats: Fatty acid contents (also antifoaming properties))

Hyrdocarbons and their derivatives: n-alkanes for production of organic acids, aminoacids,

vitamins

Molasses, a byproduct of sugar production, is one of the cheapest sources of carbohydrate.

Malt extract, an aqueous extract of malted barley, is an excellent substrate for many fungi, yeasts,

and actinomycetes.

Nitrogrn sources: yeast extract, soyabean meal, corn steep liquor, peptone etc.
FOAMING
Foams consist of liquid lamellas filled with gas

How is foam formed?

 Surface-active media (peptides) and foaming components (proteins)
 High aeration and agitation rates

Foaming

 Removal of cells from culture

 Physical changes
 Reduction in working volume
 Lower mass & heat ratios
 Decrease in sterility
 FOAM CONTROL METHODS

1. Mechanical defoaming

Foam-breaker （ Defoamers ）
 Defoaming blades in fermentation tank
 Separate rotor in the upper area of the culture tank

2. Chemical defoaming
Addition of chemical antifoam agents in the beginning

（ 1 ） Animal and vegetable oils , 0.1%  0.2%

（ 2 ） Polyether surfactant （ Defoamer GPE-1 ） , 0.02%-0.03%

Ideal antifoam
 Should disperse easily and have fast action on foam
 Should be active at low concentrations
 Should be long acting in preventing new foam formation
 Should not be metabolized by m/o
 Should be nontoxic to m/o
 Should not cause any problem in the extraction step
 Should be cheap
 Should be sterilazable

Examples: Silicones, sulphonates, esters, fatty acids, alcohols

 STRAIN OPTIMIZATION
Criteria in the choice of organism:

1. The nutrional characteristics of the organism

2. The optimum temperature of the organism
3. The toxicity of organism and the product
4. The suitability of the organism
5. The stability of organism
6. The productivity of organism
7. The ease of product recovery from culture
 In selecting strains or mutants for large-scale production, several important factors
need consideration. These include stability of strains without undergoing
physiological or biochemical degeneration upon subculture for mass propagation, non-
utilization of the acid formed and non-formation of the other metabolic acids like
gluconic, oxalic, and malic acid
Isolation: Long + expensive procedure
it is essential to
– keep the desirable characteristics
– eliminate genetic change
– retain viability
– protect against contamination
Avoid subculturing :
Small probability of having mutations at each cell division, repeated sub-
culturing carries the risk of contamination..
STRAIN IMPROVEMENT

Three approaches can be used

 Mutant strain
 Recombination
 Recombination DNA technology

 Many mutation bring about marked changes in a biochemical character of

practical interest called as major mutants.

 A mutant strain of Streptomyces aurofaciens produces 6- dimethyl tetracyline

in prrsence of tetracyline, this demethylated form of tetracyline is the major
commercial tetracyline.
 Most improvements in biochemical production have been due to the step wise
accumulation of Minor genes.
 Penicillium chrysogenum – increased peniciilin production
 Each cycle of selection was preceeded by mutagen (chemical) treatment and gave
only small increase in yield.
 Several cycles (dozen) of selection , a strain (E15-1) was obtained that yielded 55%
more penicillin than the original strain
Recombination
As formation of new gene combinations among those present in different strains

Once several different mutants have been isolated , recombination is use for both
genetic analysis as well as strain Improvement.

Recombination DNA technology

 It involves the isolation and cloning of genes of interest , production of the

necessary gene constructs using appropriate enzymes and then transfer and
expression of these gens into an appropriate host organism.
 Fermentation Basics (Kinetics)

Four Phases of Bacterial Growth Curve

 Lag phase

 Log phase

 Stationary phase

 Death phase
 Lag Phase

 Period of adjustment to new conditions

 Cell growth is minimal

Log Phase

 Cell growth rate and metabolic activity are the highest

Number of cells produced ﹥Number of cells dying

 Cells are most susceptible to adverse environmental factors (e.g.

radiation, antibiotics)
 Stationary Phase

 Population size begins to stabilize

Number of cells produced = Number of cells dying

Factors that slow down microbial growth

• Accumulation of toxic waste materials

• Acidic pH of media

• Limited nutrients

• Insufficient oxygen supply

 Death or Decline Phase

 Population size begins to decrease

Number of cells dying > Number of cells produced

 Most of the nutrients in the medium have been consumed