Microbial Biotechnology

BT431

Fermentation
 Fermentation: Multiple definitions

• Any process involving the mass culture of microorganisms,

aerobically or anaerobically

• Any biological process that occurs in the absence of oxygen

• Spoilage of food

• The production of alcoholic beverages

 Microbial growth kinetics
of fermentative production process
the # ofbacteria
of
that
are
dying
bacteria that
are
dividing
=

i the media (
ends so bacteria
·rate stops
dividing
M
5 the phase
might extend
for a
year

Adaptation phase

Sanjai Saxena, Applied Microbiology

 Lag phase

• Cells are adapting to environment

• Intense metabolic activity (DNA and enzyme

synthesis)

• Cell growth is minimal in this phase.

 Log phase
• Cell division (logarithmic increase or exponential
increase)

• Generation time is constant = log plot of growth is a

straight line.

• Most metabolic activity (catabolism and anabolism)

• Cell is sensitive to adverse conditions (antibiotics,

changes in environmental factors)
 Stationary phase
plateau
• The number of cells multiplying equals the number of
cells dying

• Lack of nutrients, accumulation of waste products, pH

changes.

• Net growth rate is zero

• Cells still active and produces the secondary metabolites..

 Death phase
• Number of dying cells > number of dividing cells

• Can be a logarithmic decrease

• Depending on the species, some cells can survive long

periods of time.

• Extended death phase has been discovered

Phases of fermentative production process

Upstream
process

Fermentation

Downstream
process
 Phases of fermentative production process
Batch Fermentation

g. 3.2 Different phases of fermentative production process

 After completion of the fermentation time, the amino acids, proteins, nucleotides, nucleic acids,
spent medium is removed and subjected to differ- lipids, carbohydrates, etc., or produce ethanol,
ent processes for product recovery. Meanwhile, acetone and butanol which are by-products of
Inoculum preparation
the production fermenter is cleaned, sterilised
and prepared for the next batch of fermentation.
energy-yielding metabolism. They are also
referred to as primary metabolites being charac-
Hence, during batch fermentation, there is a teristically associated with the growth phase.
downtime between two batches. Mathematically, microbial growth in exponential
to make sure that
we have a pure culture
↓

Fig. 3.1 The process of inoculum development for submerged batch fermentation processes
 Phases of fermentative production process
Batch Fermentation

g. 3.2 Different phases of fermentative production process

 Phases of fermentative production process
Batch Fermentation

g. 3.2 Different phases of fermentative production process

Sanjai Saxena, Applied Microbiology

 Fermenters (Bioreactors)

[Link]
[Link]
 Fermenters (Bioreactors)
24 3 Fermentation Technology

Anaerobic E Aerobic bacteria

bacteria

Sanjai Saxena, Applied Microbiology

Fig. 3.4 Components of a typical bioreactor
 Fermenter control and monitoring
102 102 Chapter
Chapter 6 6
a bacteria, I
Table 6.3 Table 6.3 The
The sensors sensors
used used in fermenter
in fermenter monitoringmonitoring and control
and control
↑turbidity, light
Measurement
Measurement

Sensor Sensor Physical Physical Chemical Chemical BiologicalBiological

ElectrodesElectrodes Temperature
Temperature Dissolved Dissolved
oxygen oxygen BiosensorsBiosensors
for for
(thermistor, resistance resistance Dissolved Dissolved
(thermistor, carbon dioxide
carbon dioxide biologically active
biologically active
thermometer, thermometer)
thermometer, Nutrients Nutrients
thermometer) products products
(biosensors, e.g. for glucose)
(biosensors, e.g. for glucose)
pH pH
Metal ionsMetal ions
Foam levelFoam
detection
level detection
Meters Meters Air flow rate
Airinflow
andrate
outin and out Acid/alkaliAcid/alkali
addition addition
Agitation shaft power
Agitation shaft power
Speed of agitation,
Speed of agitation,
e.g. impeller
[Link]
impeller tachometer
Transducers
Transducers Pressure Pressure
Liquid flow
Liquid flow
Mass spectra Directly on-line or off-line Products
Mass spectra Directly on-line or off-line Products
nutrients and in flow and
nutrients and in flow and
exhaust gases
exhaust gases
Spectrophotometers Biomass
Spectrophotometers Biomass
(determination on-line
(determination on-line
and off-line)
and off-line)

Industrial Microbiology: An Introduction

 Agitation in a fermenter

Agitation mixes the three phases within a fermenter:

1. The liquid phase contains dissolved nutrients and

metabolites

2. The gaseous phase is predominantly oxygen and

carbon dioxide

3. The solid phase is made up of the cells and any solid

substrates that may be present
 Agitation in a fermenter

• Efficient mixing is important for oxygen transfer in aerobic fermentations,

as microorganisms can take up oxygen only from the liquid phase.

• Oxygen transfer into liquid from the gaseous phase is enhanced by

agitation because it:
1. Prolongs retention of air bubbles in suspension
2. Reduces bubble size to increase the surface area for oxygen transfer
3. Prevents bubble coalescence
4. Decreases the film thickness at the gas-liquid interface
 Aeration in a fermenter
Oxygen transfer is influenced by the following factors:

1. The prevailing physical conditions; temperature, pressure and surface

area of air/oxygen bubbles

2. The chemical composition of the medium

3. The volume of gas introduced per unit reactor volume per unit time

4. The type of sparger system used to introduce air into the fermenter

5. The speed of agitation

 Aeration in a fermenter
In order for oxygen to transfer from the gaseous phase to an individual cell or site
of reaction, it must pass through several points of resistance :
1. Resistance within the gas film to the phase boundary
2. Penetration of the phase boundary between the gas bubble and bulk liquid
3. Transfer from the phase boundary to the bulk liquid
4. Movement within the liquid
5. Transfer to the surface of the cell
6. Entry into cell
7. Transport to the site of reaction within the cell
liquid film
air bubble

7 O
O2 1 4 6
2 3
5
microorganism
 Fermentation media
1. Water
2. Carbon source (energy source)
• Molasses, malt extract, whey, sulphite waste liquor, alkanes & alcohols,
cellulose, starch & dextrins, fats & oils.
3. Nitrogen Source
• Yeast extract, peptones, corn steep liquor and soya been meal.
4. Vitamins
5. Minerals
6. Precursors
7. Inducer & Elicitors (extrinsic to the system and work as enhancers)
8. Inhibitors
9. Oxygen
10. Cell permeability modifiers
11. Anti-foaming agents -> to stopthe foam from taking oxygen space
 -

protein cannot

Anti-foaming
be sterilized
-

Antifoams used in industrial fermentation should ideally have the following

properties. They should:

1. Be non-toxic to microorganisms
2. Have no effect on taste and odor; a change in the usual organoleptic
properties of the finished goods.
3. Be autoclavable.
4. Not be metabolized by the microorganisms
5. Does not impair oxygen transfer.
6. Be active in small concentrations, cheap, and persistent.
by adding antibiotics
, antimicrobials -
Sterilization
Contamination by a foreign organism may result in:

1. Contamination of the final product

2. Medium would be consumed unnecessarily to support
growth of contaminating organism
3. Contaminated product will overweigh the desire product
4. Contaminated product may interfere with the recovery of the
desired product
5. Unsterile air in aerobic fermentation may result in the
spoilage of the fermentation product
 Sterilization

1. Sterilization of medium
2. Employing as pure inoculum as possible
3. Sterilization of fermenter
4. Sterilization of pipes, valves, etc. Which come in
contact with the fermentation process
5. Sterilization of all materials to be added to fermenter
6. Sterilization of air
 Sterilization of medium
Medium contamination can lead to:

1. Other type of organism to use the nutrients

2. This can lead to changes in chemical structure of the nutrients
3. Change in pH
4. Foam formation
5. Formation of metabolic products–leads to growth of
fermentation
6. Alter the oxidation/reduction potential of the medium
7. Destroy/alter/degrade fermentation product
 Fermentation modes of operation

1. Batch fermentation hard to use for production of

stops after 6 hundreds ofliteres
1-2 days

2. Fed-batch fermentation adding media but not

Lasts for d removing old media
more than Batch fermentation I

3. continuous fermentation
Lasts
d
for months
into a sterile medium held within a sterile fermenter vessel. After medium sterilisation,
the organism is inoculated into the vessel and allowed to grow. The fermentation is ter-
Batch Fermentation
minated when one or more of the following has been reached: (i) microbial growth has
stopped due to the depletion of the nutrients or the build of toxic compounds; (ii) after a
A closed system
fixed predetermined period of time; (iii) the concentration of desired product has been
achieved.

Rotameter
V

Gas Analysers

O2 CO2
Valve
Air Filter Air Filter
Rotameter
V

Valve
Air Flow Control

Culture Vessel

Figure 4.1 A diagram of a simple batch fermentation. The system is ‘closed’, containing all
Practical Fermentation Technology,
McNeil & Harvey John Wiley & Sons
 Batch Fermentation
Batch fermentation is terminated when one or more of
the following has been reached:

(i) Microbial growth has stopped due to the depletion

of the nutrients or the build of toxic compounds

(ii) After a fixed predetermined period of time

(i) The concentration of desired product has been

achieved.
 h-associated products (primary metabolites) are produced during the exponen-
with their formation decreasing when growth ceases. Typically the rate of
mation directly relates to the rate of growth (Figure 4.3). The fermentation can
they're not
getting utilized
ed at the end of the exponential growth phase before the cell enters stationary
Batch Fermentation
s growth phase is sometimes referred to as the trophophase. Examples of
tabolites are shown in Table 4.1. Nongrowth-associated products (e.g., classic produces
so it
metabolites) have a negligible rate of formation during active cell growth. them not
bacteriaas the cells enter stationary phase (Figure 4.4);
ndary metabolites are produced
produces
metabolites - to benefit
Modes of Fermenter Ope
from them
(regular products) anti cancer
agents
5 it utilizes used
only in
them
stationary phase
so
they've used
when the
e

see
cells are
under stress

so at first use they

Growth associated product formed during the period ofFigure
active 4.4 Nongrowth
culture growth associated product formed during the period of noncul
1then metabolites
2 phase)
(stationary

Primary metabolites or cell mass

Examples of commercially produced primary metabolites Secondary metabolites
ism Growth
Primary associated
metabolite Commercial use Non-growth associated
yces cerevisiae Ethanol Alcoholic beverage Practical Fermentation Technology,
erium glutamacium Amino acids – glutamic Food industry – flavour McNeil & Harvey John Wiley & Sons
 Batch Fermentation
important to I
Advantages: have a batch no.

1. Simplicity of use
2. Operability and reliability: less likely to have instrument failure
on short batch runs
3. Production of secondary metabolites that are not growth-related
4. Fewer possibilities of contamination
5. It is easy to assign a unique batch number to each run,
generating high confidence in the history of each batch of
product
 Batch Fermentation
Disadvantages:

1. Culture ageing, and more importantly differentiation, can be a specific problem,

especially so with growth-related products
2. Build up of toxic metabolites
3. Initial substrate concentrations may have to be limited due to problems with
inhibition and repression effects
4. Batch-to-batch variability
5. The use of batch cultures in industrial systems can lead to an increased
nonproductive periods due to down time required for cleaning, re-sterilization,
filling and cooling of
6. Equipment; If using the organism from one bioprocess to seed another culture,
degeneration or differentiation may occur
7. Cellular autolysis may occur during the decline and stationary phase
8. Complexity of the cultureI since the cell population is heterogeneous and
constantly changing.
 Fed-batch Fermentation
• Similar to batch culture, but do not operate as closed systems.

• At a given point during the fed-batch process one or more substrates,

nutrients, and/or inducers are introduced into the bioreactor.

• Fed-batch cultures can be run in different ways, e.g. at fixed volume or

at a variable volume

• This feeding strategy allows the organism to grow at the desired

specific growth rate, minimizing the production of unwanted by-
products, and allowing the achievement of high cell densities and
product concentrations.
78 Fed-batch Fermentation
Practical Fermentation Technology

Rotameter
V

Gas Analysers

O2 CO2
Valve
Air Filter Air Filter
Rotameter
V

Valve
Air Flow Control

Medium

Pump
Culture Vessel

Feed Tank
Practical Fermentation Technology,
McNeil & Harvey John Wiley & Sons
 [ Sub
Feeding regimes in fed-batch processes (b)

Fermentation Technology Time

intermittent
variable

[ Substrate ]
[ Substrate ]

(a) (c)

Time Time

continuous incremental
[ Substrate ]

[ Substrate ]

(b) (d)

Time Time
Practical Fermentation Technology,

Figure 4.6 Different feeding regimes in fed-batch processes, (a) John

McNeil & Harvey Variable
Wiley & Sonsfee
 Feeding regimes in fed-batch processes
Modes of Fermenter Operation 79

Table 4.4 Advantages and disadvantages of various feeding strategies

Feeding strategy Advantages Disadvantages
Variable By controlling the feed the Controlled by feedback control,
substrate is converted into requires accurate monitoring
either biomass/substrate and operator control
or both, minimising
by-product formation
Continuous Allows bioprocess to run Must have historical process
without operator control trend data
Unexpected changes during the
run will not be accounted for
Intermittent Allows feed to be fully utilised Controlled by feedback control,
before further additions, requires accurate monitoring
reducing catabolite and operator control
repression
Incremental Allows feed to increase with Controlled by feedback control,
biomass formation, requires accurate monitoring
optimizing growth rate and and operator control
product formation

Practical Fermentation Technology,

McNeil & Harvey John Wiley & Sons
 Fed-batch Fermentation
Advantages:

1. Controlling the concentration of the limiting substrate prevents the

repressive effects of high substrate concentration and avoids
catabolite repression.
2. By careful feeding strategy the organism’s growth rate and
subsequent oxygen demand can be controlled.
3. High cell density (up to ten times greater) can be achieved by use of
fed-batch over batch culture.
4. Increased production of non-growth-related secondary metabolites.
5. Reduction of broth viscosity.
 Fed-batch Fermentation
each organism has its
chemical properties
x
own

Disadvantages:

1. Detailed knowledge of the organism’s growth and product formation

pattern is required, especially when no feedback loop is used.
2. Deficiency of reliable online sensors for accurate substrate
determination in near real time.
3. Without feedback control, the feed is predetermined and therefore does
not allow for any fluctuations within the bioprocess.
4. The process operator must be fully trained and highly skilled.
 Continuous Fermentation
bacteria
the keeps dividing
• Continuous cultivation is a method of prolonging the
exponential phase
• Steady state process with less fluctuation in nutrients, cell
number or biomass.
• The organisms are fed with fresh nutrients, and spent medium
and cells are removed from the system at the same rate.
• Several factors remain constant throughout the fermentation,
such as, culture volume, biomass or cell number, product and
substrate concentrations, pH, temperature and dissolved
oxygen.
86 Continuous Fermentation
Practical Fermentation Technology

in 5 out
I
Rotameter
V

Gas Analysers

O2 CO2
Valve
Air Filter Air Filter
Rotameter
V

huge
vessels
Valve
Air Flow Control

Medium

Pump
Culture Vessel

Feed Tank Product Receptacle Practical Fermentation Technology,

McNeil & Harvey John Wiley & Sons
Control Techniques for Continuous Culture – The chemostat

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

• An essential nutrient is in limiting quantities Microbiology McGrow Hill

Control Techniques for Continuous Culture – The chemostat

b A cells are
dying
so much neutrients because we've
the bacteria can't
adding
medic
use it all Figure 7.23
atonce
5
every
minutes removing
old
the bacteria one

decreases has
no
time to
divide so
we
lose
bacteria

Microbiolgogy McGrow Hill

 Control Techniques for Continuous Culture – The turbidstat

The more the absorbed light, the more culture we have, which meansI we need to add media

• Regulates the flow rate of media

through vessel to maintain a
predetermined turbidity or cell
density

• Dilution rate varies

• No limiting nutrient

• Turbidostat operates best at high

dilution rates

[Link]
 Continuous Fermentation
Advantages:

• Productivity and growth rate can be optimized by changing the

feed flow rate during production
• Longer periods of productivity with less down time.
• It can take advantage of cell immobilization, which allows the
maintenance of high cell concentrations in the bioreactor at
low substrate concentrations.
• The effects of environmental or physical factors are more
easily analyzed in a continuous system
• Evolution in these cultures can be readily studied.
 Continuous Fermentation
high chance of
Mutations

Disadvantages:

• The US Food and Drug Administration (FDA) does not accept

continuous culture in the production of therapeutic products as a
Current Good Manufacturing Practice (cGMP). Why?
• Not all products are produced optimally in continuous processes
• Contamination can be a major problem in continuous cultivation
• Culture mutation can easily occur in continuous processes, often
resulting in the micro- organism ‘shedding’ genes required for product
formation, or in the case of genetically engineered organisms, a ‘back
mutation’ can occur, causing the slower-growing recombinant
population to wash out of the vessel.
 Submerged vs. Solid-substrate Fermenters
of Solid Substrate Bioreactors 29

ently, i.e. only once per day; however, Table 3.1 Comparison between submerged and solid
substrate fermentation
is blown forcefully through the bed.
onstitute the group two bioreactors. Submerged fermentation Solid substrate fermentation
Bioreactors: In these bioreactors, the Substrates are soluble Substrate is polymeric and
sugars insoluble
continuously mixed or intermittently
High volume of water Very limited water
with a frequency from minutes to hours consumption and effluent consumption and no
s circulated around the bed. The inter- release release of effluent
mixing of the substrate is carried out In-depth fermentation Shallow fermentation
actors in two modes, viz., stirred drum process process
ors and rotating drum bioreactors. Uniform temperature, pH, Gradient of temperature,
Bioreactors: The characteristic feature C and N source pH, C and N source
distribution distribution
bioreactors is that the bed is agitated
2-phase system (liquid and 3-phase system (solid,
is blown forcefully through the bed. It gas) liquid and gas)
s in two modes, those are mixed Temperature and oxygen Temperature, oxygen
ously and others mixed intermittently transfer are controlled transfer and water content
ervals from minutes to hours between are controlled
xing events. The bioreactors which Inoculum ratio is low Inoculum ratio is large
No intra-particle resistance Intra-particle resistance
his criterion are gas–solid fluidised
Fermenting organism is Fermenting organism
rocking drum and stirred-aerated evenly distributed sticks to the solid surface
ors. throughout the medium and grows
e design of the bioreactor plays a sig- High-energy consuming Low-energy consuming
e in solid substrate fermentation since process process
on of the reactor is based on the mor- High-cost technology Low-cost technology
and growth characteristics of the Low concentration of the High concentration of the
end product end product
nism, aeration of the substrate and the
product being produced. Adapted from Raimbault (1998), Prabhakar et al. (2005)
Sanjai Saxena, Applied Microbiology
penicillin. Thus, stirred tank fermenter (STF) and heat exchange. Conventional fermentation is
became a choice for the production of antibiotics. carried out in a batch mode.

Submerged Fermenters
a Motor b c Gas
Feed Inoculum

Sight Glass

Liquid
Cooling
Jacket

Riser Downcomer

Draft Tube
Impellar Liquid

Sparger Air Inlet Sparger

Sparger
Air
Air Air
Harvest Line (1) Internal Loop (2) External Loop

d UV Lamp Air filter

e
Air
Liquid Disengagement
zone

Catalyst Feed
Light polymer
catalyst particles
Solid matrix packing

Air Product

Gas Feed
Liquid

Sanjai Saxena, Applied Microbiology

Fig. 3.5 Different types of fermenters employed in submerged fermentation processes: (a) stirred tank fermenter, (b)
 Solid-substrate Fermentation

Solid-substrate fermentations are normally multistep processes,

involving:

1. Pretreatment of a substrate that often requires mechanical,

chemical or biological processing

2. Hydrolysis of primarily polymeric substrates, e.g.

Polysaccharides and proteins

3. Utilization of hydrolysis products

4. Separation and purification of end-products

(ii) It controls the key environmental conditions twice per day. The air is circulated around t
such as bed temperature and water activity at bed but not blown forcefully.
Solid-substrate Fermenters
values which are optimal for growth and
product formation by the microorganism.
Packed Bed Bioreactors: In these bioreacto
also the bed is static and mixed ve

Fig. 3.6 Different groups of solid substrate bioreactors: Gr

3, stirred drum bioreactors; Group 4, gas–solid and stirred b
Tray fermenter Packed bed reactor

Sanjai Saxena, Applied Microbiology

 static or mixed very infrequently, i.e. once or
ns twice per day. The air is circulated around the
at
d Solid-substrate Fermenters
bed but not blown forcefully.
Packed Bed Bioreactors: In these bioreactors,
also the bed is static and mixed very

Fig. 3.6 Different groups of solid substrate bioreactors: Group 1, tray fermenter; Group 2, packed bed reactor; Grou
3, stirred drum bioreactors; Group 4, gas–solid and stirred bed reactors
Stirred drum bioreactors Gas–solid and stirred bed reactors

Sanjai Saxena, Applied Microbiology

Solid-substrate Fermentation
106 Chapter 6
Table 6.4 Advantages and disadvantages of solid-substrate midification or
fermentations levels are too lo
does not swell a
Advantages Disadvantages
ever, if the mois
Potentially provide superior Slower microbial growth tion in the poros
productivity diffusion rates
Low-cost media Problems with heat build-up change. Conseq
is reduced and th
Simple technology Bacterial contamination can
be problematic contamination.
Low capital costs Difficulties often encountered
on scale-up T E M P E R AT U R E
Reduced energy requirements Substrate moisture level
difficult to control Heat generation
fermentations a
Low waste-water output
humidity withi
No problems with foaming
largely controll
substrate.
Upstream & Downstream Processing

Upstream processing (USP) involves:

1. Microorganism preparation
2. Fermentation media preparation
3. The fermentation process itself
Upstream & Downstream Processing

Upstream processing (USP) involves:

1. Microorganism preparation
2. Fermentation media preparation
3. The fermentation process

Downstream processing (DSP)

• All processes following the fermentation.

• It has the primary aim of efficiently, reproducibly and
safely recovering the target product to the required
specifications
• Maximize recovery yield and minimize costs
110 Chapter 7

Upstream Processes

Microorganism
Fermentation raw materials
Initial isolation Sources of carbon, nitrogen, phosphorus and
sulphur, minor elements, trace elements,
growth factors, water, etc. (availability, cost,
Strain improvement stability, and pretreatment and sterilization
requirements)

Production strain
Constraints: nutritional requirements, metabolic Media development
controls, shear sensitivity, temperature optima,
morphology, O2 and CO2 effects and requirements,
Propagation Maintenance
genetic stability, metabolic by-products, viscosity
medium medium
effects

Starter culture Production

propagation medium

+/–
Oxygen
pH control
Supported or suspended growth.
Antifoam
Fermenter type, stirring mechanism, size,
Cooling/heating
geometry, mode of operation,
Fermentation instrumentation and automation

Downstream Processes in situ DSP

Cell separation
ex situ DSP centrifugation
Influenced by product
concentration and stability. or filtration
Other considerations are Biomass waste:
yield at each step, process if product is Harvested cells Spent medium
costs and purity requirements extracellular

Intracellular Extracellular
or product
periplasmic product

Concentration
Cell disruption step
Primary
recovery

Cell Centrifugation
debris or ultrafiltration

Cell-free Inclusion Medium

extract bodies concentrate

Dialysis, precipitation, partition, Product

chromatographic steps, ultrafiltration, distillation, etc. purification

Crystallization, drying, lyophilization, Finishing

sterile filtration, packaging, etc. processes

Effluent Finished product Industrial Microbiology: An Introduction

 USP
110 Chapter 7

Upstream Processes

Microorganism
Fermentation raw materials
Initial isolation Sources of carbon, nitrogen, phosphorus and
sulphur, minor elements, trace elements,
growth factors, water, etc. (availability, cost,
Strain improvement stability, and pretreatment and sterilization
requirements)

Production strain
Constraints: nutritional requirements, metabolic Media development
controls, shear sensitivity, temperature optima,
morphology, O2 and CO2 effects and requirements,
Propagation Maintenance
genetic stability, metabolic by-products, viscosity
medium medium
effects

Starter culture Production

propagation medium

+/–
Oxygen
pH control
Supported or suspended growth.
Antifoam
Fermenter type, stirring mechanism, size,
Cooling/heating
geometry, mode of operation,
Fermentation instrumentation and automation
 +/–
Oxygen

DSP
pH control
Supported or suspended growth.
Antifoam
Fermenter type, stirring mechanism, size,
Cooling/heating
geometry, mode of operation,
Fermentation instrumentation and automation

Downstream Processes in situ DSP

Cell separation
ex situ DSP centrifugation
Influenced by product
concentration and stability. or filtration
Other considerations are Biomass waste:
yield at each step, process if product is Harvested cells Spent medium
costs and purity requirements extracellular

Intracellular Extracellular
or product
periplasmic product

Concentration
Cell disruption step
Primary
recovery

Cell Centrifugation
debris or ultrafiltration

Cell-free Inclusion Medium

extract bodies concentrate

Dialysis, precipitation, partition, Product

chromatographic steps, ultrafiltration, distillation, etc. purification

Crystallization, drying, lyophilization, Finishing

sterile filtration, packaging, etc. processes

Effluent Finished product

 +/–
Oxygen

DSP
pH control
Supported or suspended growth.
Antifoam
Fermenter type, stirring mechanism, size,
Cooling/heating
geometry, mode of operation,
Fermentation instrumentation and automation

Downstream Processes in situ DSP

Cell separation
ex situ DSP centrifugation
Influenced by product
or filtration
-cnm
concentration and stability.
Other considerations are Biomass waste:
yield at each step, process if product is Harvested cells Spent medium
costs and purity requirements extracellular

Intracellular Extracellular
or product
periplasmic product

Concentration
Cell disruption step
Primary
recovery
Centrifugation
obtain
Cell
debris or ultrafiltration
to
the product
Cell-free Inclusion Medium
extract bodies concentrate

Dialysis, precipitation, partition, Product

chromatographic steps, ultrafiltration, distillation, etc. purification

Crystallization, drying, lyophilization, Finishing

sterile filtration, packaging, etc. processes

Effluent Finished product

 +/–
Oxygen

DSP
pH control
Supported or suspended growth.
Antifoam
Fermenter type, stirring mechanism, size,
Cooling/heating
geometry, mode of operation,
Fermentation instrumentation and automation

Downstream Processes in situ DSP

Cell separation
ex situ DSP centrifugation
Influenced by product
concentration and stability. or filtration
Other considerations are Biomass waste:
yield at each step, process if product is Harvested cells Spent medium
costs and purity requirements extracellular

Intracellular Intracellular Extracellular extracellular

or product
collectthe cells periplasmic product 3 collect the media
Concentration
Cell disruption step
Primary
recovery

Cell Centrifugation
debris or ultrafiltration

Cell-free Inclusion Medium

extract bodies concentrate

Dialysis, precipitation, partition, Product

purification
1. We add ammonium sulfate chromatographic steps, ultrafiltration, distillation, etc.

for precipitation ofprotein 2. Dialysis: we have

dialysis
Crystallization, drying, lyophilization,
membranes that
has Finishing
pores

cause it alters the proteins's

allows
sterile filtration, that
packaging, etc. the liquid processes
to pass but not the

proteins
solubility Effluent so that's we get
howproduct
Finished
rid ofthe ammoniumsulfate
3 the proteins are in the buffer
 +/–
Oxygen

DSP
pH control
Supported or suspended growth.
Antifoam
Fermenter type, stirring mechanism, size,
Cooling/heating
geometry, mode of operation,
Fermentation instrumentation and automation

Downstream Processes in situ DSP

Cell separation
ex situ DSP centrifugation
Influenced by product
concentration and stability. or filtration
Other considerations are Biomass waste:
yield at each step, process if product is Harvested cells Spent medium
costs and purity requirements extracellular

Intracellular Extracellular
or product
periplasmic product

Concentration
Cell disruption step
Primary
recovery

Cell Centrifugation
debris or ultrafiltration

Cell-free Inclusion Medium

extract bodies concentrate
If my protein is 60KDac
I
buy a dialysis bay that Dialysis, precipitation, partition, Product
has smaller pores size, chromatographic steps, ultrafiltration, distillation, etc. purification

so protein won't
the lowhighte
or
-> freze

pass's only the salts

Crystallization, drying, lyophilization,
sterile filtration, packaging, etc.
dryingthe liquid that
the
Finishing
processes

so I need prior knowledge about

my protein
has the
protein

Effluent Finished product

 DSP Downstream processing 111
Cell separation
Sedimentation Broth conditioning
or flotation Flocculation
Centrifugation Disc stack, tubular, multichamber
Filtration Depth filters and cross-flow systems

Clarified medium
Harvested cells
(extracellular products)
(intracellular products)
Cell disruption
Mechanical Liquid shear homogenization, bead
mills, sonication
Non-mechanical Antibiotics, autolysis, detergents,
enzymes, osmotic shock
Clarification
Centrifugation Disc stack, tubular, multichamber
Filtration Depth filters and membrane systems

Concentration
Precipitation Ammonium sulphate, solvents
Chromatography Gel chromatography
Filtration Cross-flow membrane filtration systems
Partition Two-phase systems
Distillation Pot still, continuous still

High-resolution techniques
Chromatography Adsorption, affinity, gel filtration,
HPLC, hydrophobic, ion exchange, metal
chelate, etc.
Electrophoresis Isoelectric focusing
Dialysis Diafiltration, electrodialysis

Finishing/Packaging
Crystallization Added salts, solvents
Filtration Membrane systems
Gel chromatography Finishing, not concentration
Typical unit processes used in Drying Freeze-drying (lyophilization),
ream processing. spray drying, tray drying
 DSP Downstream processing 111
Cell separation
Sedimentation Broth conditioning
or flotation Flocculation
Centrifugation Disc stack, tubular, multichamber
Filtration Depth filters and cross-flow systems

Clarified medium
Harvested cells
(extracellular products)
(intracellular products)
Cell disruption
Mechanical Liquid shear homogenization, bead
mills, sonication
Non-mechanical Antibiotics, autolysis, detergents,
enzymes, osmotic shock
Clarification
Centrifugation Disc stack, tubular, multichamber
Filtration Depth filters and membrane systems

Concentration affinity chromotography

profeing, protein
* a Precipitation
Chromatography
Filtration
1.
Ammonium sulphate, solvents

adding proteins
the
Gel chromatography
Cross-flow membrane filtration systems
&
!
Partition Two-phase systems
columns
!
Distillation 2 wash Pot still,to
continuous still
remove we have antibodies
the unbound
High-resolution techniques proteins on the column, so
Chromatography Adsorption, affinity, gel filtration,
HPLC, hydrophobic, ion exchange, metal
3. elution to deattach
Electrophoresis
chelate, etc.
Isoelectric focusing
only specific proteins
will be attached
Dialysis
the proteins
Diafiltration,
from the
electrodialysis

Finishing/Packaging
Abs by: 1. pH changing
Crystallization
Filtration
Added salts, solvents
Membrane systems
2. Salt changing
to the Abs, the
Gel chromatography Finishing, not concentration
binding is reversable
Typical unit processes used in Drying Freeze-drying (lyophilization),
ream processing. spray drying, tray drying
 DSP Downstream processing 111
Cell separation
Sedimentation Broth conditioning
or flotation Flocculation
Centrifugation Disc stack, tubular, multichamber
Filtration Depth filters and cross-flow systems

Clarified medium
Harvested cells
(extracellular products)
(intracellular products)
Cell disruption
Mechanical Liquid shear homogenization, bead
mills, sonication
Non-mechanical Antibiotics, autolysis, detergents,
enzymes, osmotic shock
Clarification
Centrifugation Disc stack, tubular, multichamber
Filtration Depth filters and membrane systems

Concentration
Precipitation Ammonium sulphate, solvents
Chromatography Gel chromatography
Filtration Cross-flow membrane filtration systems
Partition Two-phase systems
Distillation Pot still, continuous still

High-resolution techniques
Chromatography Adsorption, affinity, gel filtration,
HPLC, hydrophobic, ion exchange, metal
chelate, etc.
Electrophoresis Isoelectric focusing
Dialysis Diafiltration, electrodialysis

Finishing/Packaging
Crystallization Added salts, solvents
Filtration Membrane systems
Gel chromatography Finishing, not concentration
Typical unit processes used in Drying Freeze-drying (lyophilization),
ream processing. spray drying, tray drying
 DSP Downstream processing 111
Cell separation
Sedimentation Broth conditioning
or flotation Flocculation
Centrifugation Disc stack, tubular, multichamber
Filtration Depth filters and cross-flow systems

Clarified medium
Harvested cells
(extracellular products)
(intracellular products)
Cell disruption
Mechanical Liquid shear homogenization, bead
mills, sonication
Non-mechanical Antibiotics, autolysis, detergents,
enzymes, osmotic shock
Clarification
Centrifugation Disc stack, tubular, multichamber
Filtration Depth filters and membrane systems

Concentration
Precipitation Ammonium sulphate, solvents
Chromatography Gel chromatography
Filtration Cross-flow membrane filtration systems
Partition Two-phase systems
Distillation Pot still, continuous still

High-resolution techniques
Chromatography Adsorption, affinity, gel filtration,
HPLC, hydrophobic, ion exchange, metal
chelate, etc.
Electrophoresis Isoelectric focusing
Dialysis Diafiltration, electrodialysis

Finishing/Packaging
Crystallization Added salts, solvents
Filtration Membrane systems
Gel chromatography Finishing, not concentration
Typical unit processes used in Drying Freeze-drying (lyophilization),
ream processing. spray drying, tray drying
 DSP Downstream processing 111
Cell separation
Sedimentation Broth conditioning
or flotation Flocculation
Centrifugation Disc stack, tubular, multichamber
Filtration Depth filters and cross-flow systems

Clarified medium
Harvested cells
(extracellular products)
(intracellular products)
Cell disruption
Mechanical Liquid shear homogenization, bead
mills, sonication
Non-mechanical Antibiotics, autolysis, detergents,
enzymes, osmotic shock
Clarification
Centrifugation Disc stack, tubular, multichamber
Filtration Depth filters and membrane systems

Concentration
Precipitation Ammonium sulphate, solvents
Chromatography Gel chromatography
Filtration Cross-flow membrane filtration systems
Partition Two-phase systems
Distillation Pot still, continuous still

High-resolution techniques
Chromatography Adsorption, affinity, gel filtration,
HPLC, hydrophobic, ion exchange, metal
chelate, etc.
Electrophoresis Isoelectric focusing
Dialysis Diafiltration, electrodialysis

Finishing/Packaging
Crystallization Added salts, solvents
Filtration Membrane systems
Gel chromatography Finishing, not concentration
Typical unit processes used in Drying Freeze-drying (lyophilization),
ream processing. spray drying, tray drying
 DSP Downstream processing 111
Cell separation
Sedimentation Broth conditioning
or flotation Flocculation
Centrifugation Disc stack, tubular, multichamber
Filtration Depth filters and cross-flow systems

Clarified medium
Harvested cells
(extracellular products)
(intracellular products)
Cell disruption
Mechanical Liquid shear homogenization, bead
mills, sonication
Non-mechanical Antibiotics, autolysis, detergents,
enzymes, osmotic shock
Clarification
Centrifugation Disc stack, tubular, multichamber
Filtration Depth filters and membrane systems

Concentration
Precipitation Ammonium sulphate, solvents
Chromatography Gel chromatography
Filtration Cross-flow membrane filtration systems
Partition Two-phase systems
Distillation Pot still, continuous still

High-resolution techniques
Chromatography Adsorption, affinity, gel filtration,
HPLC, hydrophobic, ion exchange, metal
chelate, etc.
Electrophoresis Isoelectric focusing
Dialysis Diafiltration, electrodialysis

Finishing/Packaging
Crystallization Added salts, solvents
Filtration Membrane systems
Gel chromatography Finishing, not concentration
Typical unit processes used in Drying Freeze-drying (lyophilization), Industrial Microbiology: An Introduction
ream processing. spray drying, tray drying