Principles of Biochemical

Engineering

Dr. Muhammad Shafiq

Faculty of Engineering & Technology
University of the Punjab, Lahore
[email protected]
 Course contents (2 credit hours)

 Introduction to biochemical engineering

 Introduction to microorganism and biological processes
 Introduction to reaction engineering in general
 Principles of enzyme catalysis
 Introduction to enzyme kinetics
 Bio reactors
 Continuous stirred tank reactor
 Plug flow reactor
 Packed bed reactor
 Heat transfer in bioreactors
 Introduction to bio-product recovery techniques
 Unit operations for bio-systems

2
 Bioreactors

 Bioreactors are the apparatus in which practical biochemical

reactions are performed often with use of enzymes and/or
living cells.

3
 Bioreactors

 Bioreactors which use living cells are usually called

fermenters.
 The apparatus applied to waste water treatment using
biochemical reactions is also an example of a bioreactor.
 Blood oxygenators, that is, artificial lungs can also be regarded
as bioreactors.
Most biochemical reactions occur in the liquid phase, bioreactors
usually handle liquids. Some processes in bioreactors often involve a
gas phase, as in cases of aerobic fermenters. Some bioreactors must
handle particles, such as immobilized enzymes or cells, either
suspended or fixed in a liquid phase (with regards to mass transfer,
microbial or biological cells may be regarded as small particles).

4
 Bioreactors: Types
 On the basis of mode of operation, these are classified into
three classes
 Batch reactors
 Semi-batch reactors
 Continuous reactors
 On the basis of design and working mechanism, these are
classified into following major classes
 Mechanically stirred (agitated) tanks (vessels).
 Bubble columns – that is, cylindrical vessels without
mechanical agitation, in which gas is bubbled through a
liquid.
 Packed-bed reactors (tubular reactors).
 Membrane reactors, using semi-permeable membranes,
usually of sheet or hollow fiber-type.
 . Micro-reactors. 5
 Bioreactors: Types

 On the basis of mode of operation, these are classified into

three classes
 Batch reactors
In a batch reactor, the reactants are initially charged and, after a
certain reaction time, the product(s) are recovered batch wise.
 Semi-batch reactors
In the semi-batch (or fed-batch) reactor, the reactants are fed
continuously, and the product(s) are recovered batch wise.
In these batch and semi-batch reactors, the concentrations of
reactants and products change with time.
 Continuous reactors
In a continuous reactor, the reactants and products are charged
and recovered continuously.

6
 Bioreactors: Types

Classification of bioreactors based on mode of operation

7
Bioreactors: Effects of Mixing on Reactor Performance

Uniformly Mixed Batch Reactor

As there is no entering or leaving flow in the batch reactor, the
material balance equation for a reactant A in a liquid of constant
density is given as:
Rate of products formed = rate of reactants consumed= Accumulation

where rA is the reaction rate (kmolm3/s), V is the liquid

volume (m3), and CA is the reactant concentration (kmol/m3).

8
Bioreactors: Effects of Mixing on Reactor Performance

Continuously Stirred tank reactors

The liquid composition in the CSTR is uniform and equal to that of the
exit stream, and the accumulation term is zero at steady state. Thus,
the material balance for a reactant A is given as:
Reactants in- products out +Generation= accumulation

+ =0

where rA is the reaction rate (kmolm3/s), where F is the volumetric

feed rate (m3/s) and V is the volume of the reactor (m3), and CA is
the reactant concentration (kmol/m3)).

9
 Bioreactors: Effects of Mixing on Reactor Performance

Plug Flow reactor

Type of flow in which each fluid element has the same residence time
and the fluid velocity is assumed to be uniform over the entire cross-
section of the pipe or vessel is termed as plug flow.

10
 CSTR( Continuous stirred tank reactor)

A continuous stirred-tank reactor (CSTR) is an ideal reactor

which is based on the assumption that the reactor contents
are well mixed. Therefore, the concentrations of the various
components of the outlet stream are assumed to be the
same as the concentrations of these components in the
reactor.

11
 CSTR: Working and Applications

CSTR( Continuous stirred tank reactor)

An ideal continuous stirred tank reactor needs the following
criteria to be fulfilled:
 An ideal CSTR is a 100% back-mixed reactor.
 The reaction system is perfectly mixed and there is no
composition change with respect to the position in the reactor.
 Temperature is the same at every point in the reactor.
 The compositions of the exit stream and the reaction mixture
within the reactor are the same.
 The residence time for the individual fluid packets is not the
same. The average of all these residence times is the space
time for the given reaction system

12
 CSTR: Construction

CSTRs consist of a tank, usually of constant volume, a stirring system

to mix reactants together and heating or cooling jacket. Also, feed and
exit pipes are present to introduce reactants and remove products.

13
Image taken from http://encyclopedia.che.engin.umich.edu/Pages/Reactors/CSTR/CSTR.html Stirrers
 CSTR: Advantages and disadvantages

Advantages
 Continuous operation of the bio-reactor can increase the
productivity of the reactor significantly.
 It is also easy to automate in order to reduce labor costs.
 Good temperature control
 Easily adapts to two phase runs
 Simplicity of construction
 Easy to clean
Disadvantages:
 low conversion per unit volume
 Channeling is possible with poor agitation.

14
 CSTR: Applications

 Continuous stirred-tank reactors are most commonly used in

industrial processing, primarily in homogeneous liquid-phase
flow reactions, where constant agitation is required.
 CSTR are also mainly used in the Pharmaceutical Industry.
 Fermentation industry (fermentor reactors are mostly CSTRs).

15
CSTR: Working Equations

(1)

XA = conversion at time t
16
 CSTR: Working Equations

Introducing these three terms into (1)

𝐹
  𝐴 0 =𝐹 𝐴 0 − 𝐹 𝐴 0 𝑋 𝐴 +(− 𝑟 ¿¿ 𝐴).𝑉 ¿

  𝑋𝐴
𝑉 =𝐹 𝐴 0 (2)
(−𝑟¿¿ 𝐴)¿
So for a required conversion and known rate of reaction, volume of reactor
can be determined using eq (2).

17
 Class activity

A feed solution containing a reactant A (CA0 =1 kmol/m3) is fed to a

CSTR at a volumetric flow rate of 0.6m3/min, and converted to product
(P) in the reactor. The reaction rate is 1.2 kmol/(min.m3). Determine
the reactor volumes of the CSTR required to attain a fractional
conversion (XA) of 0.95.
  𝑋𝐴
𝑉 =𝐹 𝐴 0
(−𝑟¿¿ 𝐴)¿
  0.95
𝑉 =0.6
1.2

𝑽
  =𝟎 . 𝟒𝟕𝟓 𝐦𝟑

18
Plug Flow Reactors

19
 PFR (Plug Flow Reactor)

Plug flow is an ideal flow reactor in which no back mixing

occurs. The concentrations of both reactants and products in
the plug flow reactor varies continuously along the flow
direction, but are uniform in the direction perpendicular to
flow.

 In an ideal plug flow reactor, it is assumed that there is no

mixing of the medium along the axial direction of the
reactor however, there may be lateral mixing in the
medium at any point along the radial direction.

20
 PFR (Plug Flow Reactor)
In a plug flow reactor the composition of the fluid varies from
point to point along a flow path; consequently, the material
balance for a reaction component must be made for a
differential element of volume dV.

21
PFR (Plug Flow Reactor)

(1)

22
 PFR (Plug Flow Reactor)

Introducing these three terms in eq. 1, we obtain

 
dV =

23
 Advantages and Disadvantage of PFR

Advantages:
 High Conversion per Unit Volume
 Low operating cost
 Continuous Operation
 Good heat transfer
Disadvantage:
 Undesired thermal gradients may exist.
 Poor temperature control
 Shutdown and cleaning may be expensive

24
 Comparison of PFR and CSTR

 A larger volume is always required for the CSTR than for the PFR
in order to attain an equal specific conversion.
 In the CSTR, the reactants in the feed are instantaneously diluted
to the concentrations in the reactor, whereas in the PFR there is
no mixing in the axial direction. Thus, the concentrations of the
reactants in the PFR are generally higher than those in the CSTR,
and reactions proceed under favorable conditions.

25
 Miscellaneous Bioreactors

 Bioreactors, or Fermenters, are the core of any biotechnology-

based production process for vaccines, proteins, organic acids,
amino acids, antibiotics, enzymatic or microbial bio-transformations,
biomedical application.
 A production facility typically has a train of bioreactors ranging from
20 Liters to 250 m3.
 Other than CSTR and PFR, the reactors used in industry includes
 Bubble columns bioreactors
 Air lift column bioreactors
 Fluidized bed bioreactors
 Packed bed bioreactors

26
 Bubble Column reactors

 Bubble columns (BCs) belong to a family of

pneumatic bioreactors. These bioreactors do
not have any mechanical or other moving parts.
 BC bioreactor usually consists of cylinder (with
height to diameter ratio of 4-6). Gas is sparged or
distributed at the base of column through perforated
pipes or plates etc.
 Gas transfer, mixing and reactor performance
factors are mainly influenced by the flow rate of gas
and physical properties of liquid (viscosity, density).
 Mass transfer can be improved by using internal
devices such as perforated plates and vertical
baffles etc.
 Mass and heat transfer increase with increase in
gas flow rates.
27
Bubble Column reactors

28
 Bubble Column reactors

Advantages:
 BCs require very little maintenance
 Less floor space
 low operating costs

Disadvantages:
 Short gas residence time
 Less conversion as compared to other
reactors.

29
 Bubble Column reactors

Applications:
 fermentation (production of ethanol and
mammalian cells).
 biological waste water treatment (due to
aeration).
 Production of a Single-Cell Protein from
Cheese Whey.
 Absorption of oxygen/air in various aqueous
solutions.

30
 Air lift column bioreactor

 Airlift bioreactor is modified form of bubble

reactor.
 In airlift bioreactors, the fluid volume of the
vessel is divided into two interconnected zones
by means of baffle pf draft tube. Only one of
the two zones is sparged with gas.
 The sparged zone is known as riser and other
zone is called as downcomer.
 The bulk density in the riser zone is lower than
the bulk density in downcomer zone.
 Various configurations are possible for air lift
bioreactor such as inner draft tube, outer draft
tube, split loop, external loop etc.

31
Air lift column bioreactor (common configurations)

32
 Air lift column bioreactor

All Air lift bioreactors, regardless of the basic configuration (external loop or
baffled vessel), consist of four main sections.
Riser: The gas is injected at the bottom of this section, and the flow of gas and
liquid is upward.
Downcomer: This section, which is parallel to the riser, is connected to the
riser at the bottom and at the top. The flow of gas and liquid is downward. The
driving force for recirculation is the difference in mean density between the
downcomer and the riser; this difference generates the pressure gradient
necessary for liquid recirculation.
Base: In the vast majority of airlift designs, the bottom connection zone
between the riser and downcomer is very simple. It is usually believed that the
base does not significantly affect the overall behavior of the reactor, but the
design of this section can influence gas holdup, liquid velocity, and solid phase
flow.
Gas separator: This section at the top of the reactor connects the riser to the
downcomer, facilitating liquid recirculation and gas disengagement/ separation.

33
 Air lift column bioreactor construction

34
http://image.slidesharecdn.com/airliftbioreactorppt-130111210405-phpapp02/95/airlift-bioreactor-ppt-8-638.jpg?cb=1357938893
Difference between bubble bioreactor and air lift column
bioreactor

35
 Air lift column bioreactor

Advantages:
 Simple design
 Highly energy efficient compared to CSTRs
 Less floor space.
 low operating cost.
 Better heat transfer.
 Easier sterilization (no agitator/moving part involved)

Disadvantages:
 Higher gas throughput is required.
 Higher pressure drop
 Inefficient for the systems prone to foam formation.

36
 Air lift column bioreactor

Applications:
 Air lift reactors are commonly employed
to shear sensitive cultures.
 These devices are employed in large
scale manufacture of biopharmaceutical
proteins obtained from fragile animal
cells.

37
 Packed bed bioreactor

Also called fixed bed reactor. Packed bed

reactor consists of packing bed(bed of solid
particles, glass beads etc.)
 Biocatalyst is supported on, or within, the
matrix of solids.
 A fluid containing nutrients flows
continuously through the bed to provide
the needs if the immobilized biocatalyst.
 Products are released into the fluid and
are removed in the outflow.
 The flow may be upward or downward.

38
Packed bed bioreactor construction

 Shell (vertical or
horizontal)
 Liquid distributor
 Packing support
 Packing hold down grid
 Gas inlet
 Gas outlet
 Liquid inlet
 Liquid outlet

39
Packed bed bioreactor construction

Packing
 Random packing
 Structured packing

Random packing

Structured packing
40
 Packed bed bioreactor

Advantages:
 Higher conversion per unit mass of catalyst than other
catalytic reactors
 Low operating cost
 Continuous operation
 No moving parts to wear out
 Catalyst stays in the reactor
 Reaction mixture/catalyst separation is easy
 Effective at high temperatures and pressures

Disadvantages:
 Undesired heat gradients
 Poor temperature control
 Difficult to clean
 Undesirable side reactions 41
 Packed bed bioreactors

Applications:
 Mainly used as enzyme immobilized
reactors
 Bio catalytic reactions
 Fermentation

42
 Fluidized bed bioreactors

Fluidization:
A process in which solid particles are set Fluid

suspended in a gas or liquid and the fluid-solid

system behaves like a liquid.

Fluidized bed: A bed of solid particles in a fluidized

state. In a fluidized bed, unlike a fixed bed solid
particles move both with respect to each other and
with respect to the wall of the container.

Fluid

43
Fluidized bed bioreactors, flow regime

Fluid velocity

44
 Fluidized bed bioreactor

Advantages:
 Uniform Particle Mixing
 Better temperature control
 Continuous operation
 Catalyst regeneration is easy
 Effective at high temperatures and pressures

Disadvantages:
 High pressure drop
 Energy intensive operation
 Particle entrainment
 Erosion of Internal Components

45
 Packed bed bioreactors

Applications:
 Mainly used as enzyme immobilized
reactors
 To carry out bio catalytic reactions

46
 Difference between Fluidized bed bioreactors and
packed bed reactor
 In fluidized bed reactor, large fluid-solid contact area is
possible and high rates of heat and mass transfer are
obtained.
 Heat and mass transfer rates are usually higher in a fluidized
bed system as compared to a packed bed system.
 Removal and addition of solid particles are easier therefore a
fluidized bed reactor is preferred over a packed bed reactor
where a catalyst requires frequent regeneration.
 The catalyst particle may be reduced due to attrition. The flow
of solids and fluid are in cocurrent and therefore advantages
of countercurrent are not possible to obtain. Fine particles
may be conveyed which may result in the loss of solids and
there is a need for a separator downstream to recover the
solids.
47
 Photobioreactors

A photobioreactor is a bioreactor that utilizes the light source to

cultivate microorganisms. These organisms use photosynthesis
to generate biomass from light and carbon dioxide and include
plants, macroalgae, microalgae, cyanobacteria etc.
These micro-algae and cynaobacteroa provide important
chemicals such as astaxanthin and β-carotene.

48
Photobioreactors

49
 Photobioreactors

Advantages:
 Cultivation of algae is in controlled circumstances, hence
potential for much higher productivity
 Better control of gas transfer.
 More uniform temperature.
 Better protection from outside contamination.
 Space saving – Can be mounted vertically, horizontally or at
an angle, indoors or outdoors.
Disadvantages:
 Capital cost is very high.
 The technical difficulty in sterilizing these photobioreactors
has hindered their application for algae culture for specific
end-products such as high value pharmaceutical products.

50
 Photobioreactors

Advantages:
 Microalgae are a very diverse group of aquatic
microorganisms which could potentially be used for the
production of a wide variety of compounds.
 Currently these reactors are used for the cultivation of micro-
algae and cynaobacteria which are further used to produce
important chemicals such as astaxanthin and β-carotene

51
Heat Transfer
Introduction

52
 Introduction to heat transfer

 Heat transfer is a science which deals with the energy

transfer between two given locations as a result of
temperature difference.
 Heat transfer deals with rates and predicts how fast or
slow the heat will flow from one point to the other.
Therefore it helps in sizing/designing the heat transfer
equipment.

53
 Introduction to heat transfer

 Knowledge of heat transfer is important in designing

refrigeration systems, power plants, vehicles, major
pieces of equipment for chemical and biochemical
plants.
Think of daily life examples.
What about wearing warm cloths in winter?

54
 Introduction to heat transfer-2
Introduction to heat transfer
(Applications)

Car radiator
Cooling fins in an
electronic device

Boiler

Heat exchanger
55
Figures http://www.lenntech.com/applications/process/boiler/boiler-feed-water.htm
 Units of energy and heat transfer

• SI units of heat is J (Joule), while in English system units are

British thermal unit (Btu).
• SI units of rate of heat transfer = J·s–1, while in English
system units are Btu·h–1.

1.0 calorie = 4.1840 J

1.0 Btu = 1055.06 J = 252.16 cal
1.0 J·s–1 = 1.0 W (Watt)
1.0 Btu·h–1 = 0.29307 W

56
 Modes of heat transfer

heat energy is transferred by three modes:

 Conduction
 Convection
 Radiation

57
 Modes of heat transfer

http://www.beodom.com/en/education/entries/principles-of-thermal-insulation- 58
heat-transfer-via-conduction-convection-and-radiation
 Conduction heat transfer

 Heat conduction is applied to the mechanism of internal

exchange from one body to another in contact, or from one
part of a single body to another part by exchange of activity
at molecular level. This exchange is the kinetic energy
exchange by vibration of the atoms, by movement of free
electrons, or by molecular activity.
Direction of heat

T1 T2

T1 > T2

59
Left figure is taken from http://www.educationalelectronicsusa.com/p/heat-IV.htm
 Convection heat transfer-1
Convection heat transfer

Heat transfer by convection is due to fluid motion by mixing of

one portion of the fluid with another portion. The energy may
be transported from one point to the other by displacement of
fluid itself.

60
Convection heat transfer-2
Convection heat transfer

In which of the following cases

heat transfer will be higher in
heating a fluid?
a) Conduction or b) Convection

61
 Types of Convective heat transfer

• Free or natural convection heat transfer

• Forced convection heat transfer

62
 Free convection heat heat
Types of Convective transfer
transfer

Free or natural convection heat transfer:

 If the fluid motion is caused by itself due to difference in
densities at two different points such a process is natural
or free convection heat transfer. The density differences
may be caused by temperature differences or
concentration differences at two locations.
 In natural convection, no mechanical means are used to
produce convective currents and convective mixing is a
solely due to natural motion of the fluid. Boiling of milk
and water and heating distant parts of a room in the
presence of a room heater are common daily examples.

63
 ForcedTypes
convection heat
of Convective heattransfer
transfer

Forced convection heat transfer:

If the fluid motion is caused by some external or mechanical
means the heat transfer is due to forced convection. Pumps,
blowers, fans, agitation devices such as impellers are employed
for forced convection heat transfer.

64
Free and forced convection heat
Types of Convective heat transfer
transfer
In which of the following cases do you
expect greater rate of heat transfer?

1. Free convection
2. Forced convection

65
 Radiation heat transfer

Radiation mode is unique as it does not require any

physical medium for heat transfer. Energy transfer by
radiation occurs by means of electromagnetic radiations.

Electromagnetic radiation spectrum

66
Heat transfer coefficient
Heat Transfer Coefficient

Heat transfer coefficient is the ability of the system to transfer

heat.

Units: J/s·m2·K or W/m2·K

67
Individual and overall heat transfer coefficients

Outside film
resistance

Inside film Wall

resistance resistance

68
Individual and overall heat transfer coefficients

q
For inside film resistance:  hi  (T1  T2 )
A

For wall resistance: q (T2  T3 )

 kw 
A x

For outside film: q

 ho  (T3  T4 )
A

69
 Individual and overall heat transfer coefficients

Reciprocal of overall resistance is overall conductance and

frequently called as overall heat transfer coefficient.
1
U
1 x 1
 
hi k ho
The rate of heat transfer is related to area available for heat
transfer and mean temperature difference/

q  U  A  dT

70
Heat Transfer in bioreactor (Fermentor)

71
Heat Transfer in bioreactor (Fermentor)

72
 Importance of heat transfer

 Heat transfer deals with rates and predicts how fast or

slow the heat will flow from one point to the other.
Therefore it helps in sizing/designing the heat transfer
equipment.
 Knowledge of heat transfer helps to find out mass of
cooling agent required to maintain temperature inside
bioreactors etc.

73
Downstream Processing

74
 Downstream Processing
 Downstream processing means the recovery and purification of
bioproducts and the proper treatment and disposal of waste.
 It is an essential step in the manufacture of bioproducts such as
antibiotics, hormones, vaccines, enzymes, natural fragrance and
flavor compounds etc.
 Downstream processing is usually considered a specialized field in
biochemical engineering and chemical engineering.
 Separation and purification of a desired product from the product
mixture e.g. fermentation broth or cell culture supernatant is a
crucial and challenging component of commercial biotechnology.
Product recovery usually accounts for a large portion of the product
cost and in some cases is the major manufacturing cost.
 The ratio of fermentation to product recovery costs is about 60:40
for many antibiotics. For recombinant DNA fermentation products,
the cost of downstream purification is very important and can
amount to 80-90% of the total manufacturing costs.
75
 Downstream Processing

Correlation between concentration in starting material (pre-purification) and approximate

76
or estimated selling price of various biological products.
Downstream Processing

The downstream processing is

usually employed for isolation and
purification of biomolecules and
can be divided into following
stages.
 Solid liquid separation/
clarification
 Cell Disruption
 Concentration/Purification
 Formulation

77
 Solid liquid Separation/ clarification

Many bioreactors contain microorganisms suspended in liquid culture

media as single cells, flocks, or particles formed by mycelia. In such
cases the first separation step is the removal of insoluble biomass by
a solid-liquid separation process such as sedimentation,
centrifugation, flotation, or filtration.
Sedimentation and centrifugation are both based on density
differences between insoluble particles and the surrounding fluid.
 Sedimentation relies on gravity and settling to achieve solid-liquid
separation, and is generally performed in rectangular or circular
flow tanks.
 Centrifugation, on the other hand, involves mechanical application
of a centrifugal force to obtain a solid concentrate and clarified
supernatant.

78
 Solid liquid Separation/ clarification

The earliest uses of centrifugation were in sugar manufacture and in

separating cream from milk. In fact, cream separation is thought to
have been the primary application of the world's first continuous
centrifugal separator.

Lab centrifuge Industrial centrifuge

79
Solid liquid Separation/ clarification

Sedimentation Tank
80
 Solid liquid Separation/ clarification

In flotation, particles are absorbed on gas bubbles, get trapped in a

foam layer and can be collected. For example, recovery of Bacillus
sphaericus strain spores from fermented medium is performed by
froth flotation.

Gas 81
 Solid liquid Separation/ clarification
The separation of solids from a liquid by means of porous medium or
screen which retains the solids and allows the liquid to pass is called
filtration. Very small items (such as bacteria) can also be separated
from fluids by filtration process.
 Filtration is probably the most common means for separating solids.
Filtration offers many advantages for separating solids from liquids,
as it separates mainly on the basis of a single physical parameter-
the size of the solute.
 Moreover, filtration can be very cost effective; compared to
centrifugation it consumes less energy and requires considerably
less capital investment.
 Versatility is another advantage.
 Separation of insoluble membranes exclude solutes as small as 10
o
A or so in diameter, whereas other membranes allow passage of
particles in the hundred-micron (million-A) size range.
 In addition, many different biocompatible filter materials are 82
available.
 Solid liquid Separation/ clarification

Modes for Filtration process

Feed
Feed Retentate

Permeate Permeate

Flux
Cake thickness

Cake thickness
Flux

a) Dead-end filtration b) Cross-flow filtration

83
Solid liquid Separation/ clarification

Classification of Filtration process

84
 Classification of Filtration process

Filtration Driving
Pore size Application
process force, ΔP
Removal of suspended particles,
concentration of insoluble material, cells
Microfiltration 0-1 bar 0.02-10 μm
from fermentation broth, clarification of
disrupted cell slurries

Concentration or separation of dissolved

molecules, separation of cells from a
product that has been secreted,
Ultrafiltration 0-10 bar 0.001-0.2 μm
concentration of cells, removal of cell
debris, concentration of protein solutions,
removal of viruses from protein solutions.

 10-3 -10-2 μm
Reverse 30-100
Removal of salts from water
osmosis bar
85
 Filtration Equipment

The most common apparatus for large-scale dead-end filtration is the

rotary vacuum filter. Commonly employed in the fermentation industry.

Working: The rotary vacuum filter consists of a large, perforated,

horizontal drum, the inside of which is under partial vacuum. The drum
is partially immersed in a bath that contains a filter aid, an inert,
microporous material derived from diatomaceous earth or from perlite.
As the drum rotates through the bath, the vacuum draws liquid through
the filter and solids are retained on the cylinder surface (the filter is
thus precoated). The bath is then filled with product-containing broth to
which filter aid has been added. Rotation of the precoated filter and
maintenance of the vacuum results in the build up of biomass on the
precoat, just as a cake would accumulate on a laboratory filter. As the
cake rotates out of the broth, it is washed, dewatered, and removed by
continuous scraping.
86
 Filtration Equipment

Rotary vacuum filter

87
 Filtration Equipment

Second most commonly used filtration equipment is filter press.

Working: The filter press is built up of a sequence of perforated plates

altering with hollow frames mounted on suitable ports. The plates are
covered with a filter medium to create a series of cloth walled
chambers into which slurry can be forced under pressure using
hydraulic press normally. The solids are retained within chambers
while filtrate is discharged from hollow pores on the plate and further
to drain points.

88
Filtration Equipment

Filter Press

89
Filtration Equipment

Filter Press

90
 Cell Disruption

If the desired product is an intracellular substance, such as an

intracellular enzyme, cell disruption is an important early step in
product recovery.
Methods used for disrupting microorganisms can be classified as
physical (mechanical), chemical, or biological.
 Physical methods can employ shear (e.g., passage through a
homogenizer under high pressure), agitation with abrasives
(grinding in a ball mill with glass or metal beads), and repeated
freezing and thawing (freeze-thaw).
 Chemical and biological methods include treatment with alkali,
detergents, or organic solvents, osmotic shock, and enzymatic
digestion. The effectiveness of a particular disruption technique is
usually assessed in terms of the degree of cell breakage and/or the
level of enzyme activity recovered in the disrupted suspension.

91
Cell Disruption

92
 Cell Disruption: summary
 To release maximum amount of the product in an active state.
 Factors to consider: inactivating effects such as shear, temperature
and choice of disruption methods.
 Mechanical disruption is the most common means to release
intracellular products in laboratory and in industry.
 Ultrasonication is common in the lab-scale but the removal of heat
is difficult on a larger scale.
Two common mechanical cell disruption industrial processes are
1. High pressure homogenization: the cell suspension is forced at
high pressure through an orifice to emerge at atmospheric
pressure. The sudden release of pressure creates high shear.
2. Vigorous agitation with abrasives: agitation with glass in bead
mills ruptures the cells by high shear and impact with the cells.
(Size of beads: 0.2-0.5 mm for bacteria; 0.4-0.7 mm for yeasts.)

93
 Cell Disruption: summary
Two common non-mechanical cell disruption industrial processes are
1. freeze/thaw: freeze at -80 oC and rapid thaw at 37 oC; disrupt the
cells by causing changes in the structure of the cell wall and
membrane.
2. Organic solvents and detergents: extract cells which are more
permeable and soluble.

94
 Concentration of biological product
After initial separation, the filtrate contains 85-90% of solvent (mostly
water). Removal of solvent can be done in different ways, for example
 Distillation
 Evaporation
 Extraction
 Precipitation

95
 Concentration of biological product: Distillation
Distillation is one of the most important processes for separating the
components of a solution. The solution is heated to form a vapor of the
more volatile components in the system, and the vapor is then cooled,
condensed, and collected as drops of liquid. By repeating vaporization
and condensation, individual components in the solution can be
recovered in a pure state.

Distilled alcohol has been used in non-beverage applications as a

solvent, and for medicinal purposes for many centuries.
96
Concentration of biological product: Distillation

Ethanol distillation from fermentation mixture

97
 Concentration of biological product: Evaporation
Works by evaporating the solvents using heat. It is simple but energy-
consuming, normally using steam as the heat source in a large scale.
Applicable for food and other stable biologics, but seldom suitable for
processing of biologically active proteins.

Falling film evaporators (FFE) are commonly employed evaporator in

Industry. In FFE, the liquid to be concentrated flows down long
tubes/plates and distributes uniformly over the heating surface as a
thin film. Part of the liquid is evaporated and exits as the vapor. Suited
for viscous products and is often used in fermentation industry.

98
Concentration of biological product: Evaporation

Falling film evaporator

99
 Concentration of biological product: Extraction
 Extraction is a process in which two phases come into contact with an
objective of transferring a solute or particle from one phase to the other.
If two phases are poorly miscible liquids, the technique is called liquid-
liquid extraction (or solvent extraction). Liquid-liquid extraction is
commonly used for the separation and purification of biological
products.
 Efficiency of the process depends on the distribution of substances
between two phases.
Distribution Partition Coefficient:
In an extraction process, the compounds are separated due to their
different partitioning between two phases that is a result of the difference
in their relative solubilities in two liquid phases. Distribution of a solute
between the two phases is usually expressed in terms of partition
coefficient.
Ciextract
K
Ciraffinate 100
 Concentration of biological product: Extraction
Types of Extraction employed in industry
Organic solvent extraction: Small lipophilic molecules can be
extracted by organic solvents.
Physical extraction: distribution is based on the physical preference.
This applies to nonionizing compounds and the extraction is optimized
by screening for the solvents that have a high K value.
Dissociative extraction: distribution is based on the difference in the
dissociation constant of the ionizable components, e.g. extraction of
penicillin.
Aqueous two-phase extraction: Solvents used for extraction are
water soluble and mostly used for extraction of protein etc.

101
 Concentration of biological product: Extraction
Example: Extraction of proteins using Aqueous two-phase systems
(ATPS)
Some water based polymer solutions are immiscible with each other or
with salt solution of high ionic strength and are capable of forming two
two phases on mixing.
For industrial processes, polyethylene glycol (PEG)/salt and Dextran
system is often used for their low cost to extract valuable proteins, from
cellular debris etc.

102
 Concentration of biological product: Extraction
Example: Extraction of proteins using Aqueous two-phase systems
(ATPS)
 Some water based polymer solutions are immiscible with each other
or with salt solution of high ionic strength and are capable of forming
two phases on mixing.
 For industrial processes, polyethylene glycol (PEG)/salt and Dextran
system is often used for their low cost to extract valuable proteins,
from cellular debris etc.
 Partitioning of a component is based on its surface characteristics,
nature of phase components and the ionic composition.
 Phase separation is slow (from minutes to hours) but can be
speeded up by centrifugation.

103
Concentration of biological product: Extraction

Flow sheet for Aqueous two-phase systems (ATPS) 104

 Concentration of biological product: Precipitation
 Precipitation is a very valuable and relatively simple technique for
recovery of many biological products. It is most commonly used in
the purification of antibiotics, biopolymers, and proteins.
Proteins currently isolated by precipitation include analytical and
industrial enzymes, blood-plasma proteins, and food proteins. These
products include proteins produced by genetic engineering as well
as those produced naturally from microbial, plant, and mammalian
sources.
On industrial scale precipitation is carried out using various methods
such as
 Addition of salt (salting out)
 Addition of solvents
 By varying pH
 By varying temperature
 Addition of polymer etc.

105
Concentration of biological product: precipitation

106
 Purification of biological product: Chromatography

 Chromatography is a technique for separation and

purification the components of a mixture on the basis of the
relative amounts of each solute distributed between the
mobile phase and stationary phase. The mobile phase may
be either a liquid or a gas, while the stationary phase is either
a solid or a liquid.
 The degree of purification in previous steps is limited, usually
need several chromatography steps to yield high purity.
Which chromatography to use depends on the characteristics
of the proteins, such as size and shape, overall charge,
surface hydrophobic groups, and ability to bind various
ligands.

107
 Chromatography

1903: The Russian scientist M. Tswett presented

a lecture “On a new category of adsorption
phenomena and their application to biochemical
analysis”. This event is nowadays considered as
the starting point of liquid chromatography.
The Russian word Tswett means „color“.

The separation mechanism is a distribution between the stationary

and mobile phase. 108
Chromatography: working principle

109
Chromatography: working principle

110
 Chromatography
Various types are available:
 Size Exclusion Chromatography
 Ion Exchange Chromatography
 Reverse phase Chromatography
 Gel permeation Chromatography

111
 Chromatography

Gas Chromatography

112
Image taken from https://en.wikipedia.org/wiki/Gas_chromatography
 Chromatography

Chromatogram

113
Image taken from http://www.4college.co.uk/a/Cd/Glc.php
 Chromatography

Liquid Chromatography

114
Image taken from https://en.wikipedia.org/wiki/Gas_chromatography
 Chromatography

Liquid Chromatography

115
Image taken from https://en.wikipedia.org/wiki/Gas_chromatography
 Formulation of bioproduct

Formulation step is very important and mostly the last step in

downstream processing. It helps to enhance shelf life of
product. Valuable product is formulated mostly by drying and
removal of final moisture contents, shaped and sent to
market.
 Most commonly applied drying techniques are Freeze
Drying, vacuum Drying, spray drying etc.
Freeze Drying is a process which extracts water from foods and
other products so that the foods or products remain stable and are
easier to store at room temperature (ambiant air temperature). It is
also called Lyophilization. It is carried out using a simple principle
of physics called sublimation. Sublimation is the transition of a
substance from the solid to the vapour state, without first passing
through an intermediate liquid phase.
116
Formulation of bioproduct

Freeze Drying

117
 Formulation of bioproduct: Freeze drying
Advantages:
 It has many advantages compared to other drying and preserving
techniques.
 Freeze Drying maintains food/ biochemical and chemical reagent
quality because they remains at a temperature that is below the
freezing-point during the process of sublimation.
 The use of Freeze Drying is particularly important when processing
lactic bacteria, because these products are easily affected by heat.
 Food/biochemicals and chemical reagents which are Freeze Dryed can
usually be stored without refrigeration, which results in a significant
reduction of storage and transportation costs.
 Freeze Drying greatly reduces weight, and this makes the products
easier to transport. For example, many foods contain as much as 90%
water. These foods are 10 times lighter after Freeze Drying.
Disadvantages:
 Long processing time is required.
 Capital cost is more as compared to other dryers.
118
 Formulation of bioproduct: Spray Drying

A spray dryer is a device for drying, utilizing a spray. It mixes a

heated gas with an atomized (sprayed) liquid stream within a
vessel (drying chamber) to accomplish evaporation and
produce a free flowing dry powder with a controlled average
particle size.
 Spray drying is a very widely applied, technical method used
to dry aqueous or organic solutions, emulsions etc., in
industrial chemistry and food industry.
 Dry milk powder, detergents and dyes are just a few spray
dried products currently available. Spray drying can be used
to preserve food or simply as a quick drying method.

119
Formulation of bioproduct

Spray Drying
120
 Downstream Processing Summary
 Downstream processing consists of most important steps
in recovery of valuable of product.
 Major steps include
 Solid liquid separation/ clarification
 Cell Disruption
 Concentration/Purification
 Formulation
 It helps to enhance the shelf life of final product.
 It accounts for the major cost of final product.

121