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Citric acid
Citric acid (2-hydroxy-propane-1.2.3-
tricarboxylic acid)
Microbes : [Link], [Link]
Applications of citric acid:
Flavoring agent
Antofoam
Preservation of fats, oils
Detergent
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Factors affecting CA production :
Carbohydrate sources : Molasses, Starch
Ideally, sugar concentration should be 12-
25%.
Trace metal ion: Mn+2
pH
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Nitrogen source
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Production : Submerged versus Solid
state
Recovery of CA
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Ethanol
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It can be produced from i) Hydration of
Ethylene or ii) Fermentation
Gasohol : 20% Ethanol, 80% Petrol
Microbes : [Link], Zymomonas
mobilis, [Link]
Raw materials
Pretreatment of raw materials : Molasses
is diluted to 10-18% sugar. Starch is
broken down to simple sugars. Cellulosic
feed are delignified.
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Glucose 2 Ethanol + CO2
Therotical yield : 1 gm glucose 0.511 g
ethanol
Regulation of synthesis
Fermentation conditions : Anaerobic, pH-
4.5, Temp-20-26 deg C
DSP : Refer flow sheet
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Composition of Biomass
Anwar Z, Gulfraz M, Irshad M. Agro-industrial lignocellulosic biomass a key to unlock the future bio-energy: a brief review. J Radiat Res Appl Sci 2014;7: 163–73
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Penicillin
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Penicillins are a group of antibiotics
which have Beta-lactum ring.
Action : Are effective many gram
positive bacteria
But ineffective against microbes which
produce beta-lactamase
Organism : Penicillium Chrosogenum,
Floxacillin
Regulation of biosynthesis : L-alpha-
aminiadipic acid is common intermediate
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Production process : Aerobic, aeration
rate-0.5 VVM, pH-6.5, Temperature -25-
27 deg C
Medium : Corn steep liquor, Yeast extract
Precursor : Phenyl acetic acid
Recovery : Refer flow sheet
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Low volume , high value product-Insulin
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Insulin is essential peptide harmone for
diabetics
Traditionally , it has been extracted from
animal pancreas and purified by Crystallization
The onset of r-DNA technology offered the
prospectus of producing large quantities of
human insulin employing microbes
Process description : Microbe : E-coli-K12
Mode : Batch
Overall yield : 28%
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Alternate method
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Instead of Lac operaon, genes from tryptophan
operon are used. The tryptophan operaon of
[Link] contains five structural genes (trpE, trpD,
trpC,trpB and trpA), regulatory elements-primary
promoter (trpP), operator (trpO) and attenuator
(trpa).
Beside tryptophan promoter, trpE gene is
inserted and beside that, gene responsible for
proinsulin is inserted.
Tryptophan-trpE gene-gene responsible for
proinsulin-- Transcription Fusion protein (trpE-
met-proinsulin)
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The fusion protein is accumulated in
inclusion bodies.
Fusion bodies should be disrupted, to
release fusion protein
Fusion protein should be cleaved to get
proinsulin and trpE fragments
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Medium : Corn steep liquor, glucose
Sterilization : Continuous, heated to
145oC, held for 20 sec, cooled to 37 deg
C
Fermentation : Aerobic, 37 deg C
DSP : Refer flowsheet
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Cell disruption methods
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Muerien layer -10-80 nm, Peptidoglycan
Periplasmic space- 8 nm- contains enzymes,
Cytoplasmic membrane-made of phospholipids+
proteins+metal ions
Gram negative-Outer membrane- 8 nm
Protein+lipopolysaccharide
Yeats cell wall-70 nm thick, mannan and glucan-
polysaccharide+protein, plasma membrane-
phospholipids and lipoproteins
Fungi cell wall- Cellulose +chitin
Plant cell wall- Cellulose, Mammalian- No cell wall
Animal cytoplasam is stiffened by sterols
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Pressure required for cell
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disruption
Animal cells -2000 psi
insect blood cells require 15,000 psi
yeast cells require up to 20,000 psi
plant tissue -40,000 psi
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Bead Mill
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Bead milling can generate enormous
amounts of heat. While processing
thermolabile material, the milling can be
carried out at low temperatures, i.e. by
adding a little liquid nitrogen into the
vessel
A bead mill can be operated in a batch
mode or
in a continuous mode and is commonly
used for
disrupting yeast cells and for grinding
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Using a small scale unit operated in a
continuous mode, a few kilograms of
yeast cells can be disrupted per hour.
Larger unit can handle hundreds of
kilograms of cells per hour.
Kicks law
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Kinetics of release
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High pressure homogenizer
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Cell wall permeabilization
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Organic solvents in the concentration of
1-3%permieabilize the cell wall without
disrupting it.
Toluene permeabilization of yeast cell
walls to release histidyl –t RNA
synthetase and enolase.
Ethyl acetate has been used to release
periplasmic invertase and alpha
glucosidase in yeast.
DMSO to release Ajmalicine from plant
cell walls of Catharanthus rosens
Enzymes digestion
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Lysozyme to isolate glucose isomerase
from Streptomyces Sp.
Gram negative bacteria requires
detergents such as EDTA or Triton X-100
Glycosidases –break polysaccharide
chains, Acetylmuramyl –L-
alanineamidase cleave junction between
polysaccharide and peptide
Endopeptidase –split polypeptide chains
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Yeast cells-split mixture of enzymes-
protease, mannanase or chitinase.
Sequential enzymatic lysis provides
controlled lysis
Sequential disruption of microbial cells
depends on: type of microbial cells,
products to be harvested and process
conditions
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Applications
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Preparation of protoplasts, cell fusion and
transformation of yeast.
Production of intracellular enzymes
Preparation of soluble glucan polysaccharide
Alkali extraction of yeast protein
Ethanol recovery
Release of human serum albumin
Various bacteriolytic and yeast lytic enzymes are
produced from [Link], Staphylococcus,
streptomyces globisporius, Cytophaga, Arthobacter
Yeast cell wall degrading enzyme Kitalase from fungus
Rhizoctonia Sp. has been used in the liberation of
invertase from [Link]
Mechanical cell disruption
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Lab scale- Ball mill or Waring blender
Waring blender –useful for animal cells,
tissues and mycelial organisms
Industrial scale – ball mill or
homogenizer
Bead mill disruption
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Release of intracellular
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enzymes
Enzymes more difficult than proteins as
they are located in different regions
within cell
Example : glucose-6-phosphate
dehydrogenase is released along with
proteins from bakers yeast while
fumarase present in mitochondria is
released slowly.
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In contrast, alpha-glucosidase and
restriction endoglucanase are located
within periplasmic space and released
before cytoplasmic proteins.
Rate of enzyme release =f (indication of
location)
Rate of enzyme release and enzyme
inactivation need to be balanced.
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Removal of Insoluble, Biomass (and
Particulate Debris):
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Flocculation :
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Filtration
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Filtration is a separation process in which
a solid-liquid mixture called the feed (or
the suspension) is forced through a
porous medium on which the solids are
deposited or in which they are
entrapped.
The porous medium which allows the
liquid to go through while retaining the
solids is called the filter. The retained
solid is called "the residue" or "the cake"
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Filtration can be broadly classified into
three categories
If recovery of solids from high solid content
slurry is desired, the process is called cake
filtration.
The term clarification is applied when the solid
content in the feed does not exceed 1 wt %
In a clarification process the filtrate is the
primary product. The third type of filtration is
called cross-flow filtration in which the liquid
flows parallel to the filtration medium
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Cross-flow filtration is mainly used for
membrane filtration and will be discussed
in details in the chapter on membrane
based bioseparation processes
Some of the applications of filtration in the
bio-industry are:
Recovery of crystalline solids
Recovery of cells from fermentation medium
Clarification of liquids and gases
Sterilization of liquids
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Surface filtration: The particles are retained by a
screening action and held on the external
surface of the filter. The particles are not allowed
to enter the filtration medium. Cake filtration
and cross-flow filtration are based on surface
filtration.
Depth filtration: Particles are allowed to
penetrate pores and pore networks present in
the filtration medium. They are retained within
the filter by three mechanisms: direct
interception, inertial impaction and diffusional
interception
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In direct interception the particles enter
the pores or pore networks within the
filtration medium and get trapped where
the pore diameter becomes equal to the
particle diameter
Particles whose diameters are
significantly smaller than the pore
diameter get trapped where pores are
already constricted by collected particles
Particles being carried by fluids possess
momentum on account of their mass and
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Pores present in most filtration media
are tortuous in nature. The flow of fluids
through these pores is usually laminar in
nature on account of their small
diameters
At tortuous sections of pores the fluid
streamlines follow the curves while
particles due to their inertia continue to
move straight and as a result hit the
filter medium, lose their momentum and
are retained.
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This collection mechanism which is referred to as
inertial impaction is important for particles larger
than 1.0 micron in diameter. Diffusional
interception is more relevant to filtration of gases
The gas molecules, due to their random motion
continually bombard suspended particles,
particularly those smaller than 0.3 micron in
diameter.
The suspended particles therefore deviate from
their streamlines and impact on the filter medium.
Once this happens, the particles lose their
momentum and are retained.
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Centrifugation
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Centrifugation is used when gravity force
is not significant
Used for separating solids from liquids
( Centrifugal sedimentation)
Separation of Immiscible liquids with
close densities(centrifugal decantation)
Filtration of solids by Centrifugal
filtration
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Centrifugal sedimentation
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Tubular bowl centrifuge
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Method 2
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Selection of centrifuge
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Laboratory centrifuge
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Laboratory centrifuges are used for
small-scale separation and clarification
(i.e. removal of particles from liquids)
Typical liquid volumes handled by such
devices are in the range of 1 - 5000 ml
There are two types of rotors: fixed angle
rotors and swing out rotors. A fixed angle
rotor holds the centrifuge in a fixed
manner at particular angle to the axis of
rotation
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Swing out rotors hold the tubes parallel
to the axis of rotation while the rotor is
stationary but when the rotor is in
motion, the tubes swing out such that
they are aligned perpendicular to the
axis of rotation
When the centrifuge tubes are spun, the
centrifugal action creates an induced
gravitational field in an outward direction
relative to the axis of rotation and this
drives the particles or precipitated
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Typical rotation speeds of laboratory
centrifuges range from 1,000 - 15,000 rpm
The magnitude of the induced gravitational
field is measured in terms of the G value: a G
value of 1000 refers to an induced field that is
thousand time stronger than that due to gravity
The G value which is also referred to as the RCF
(relative centrifugal force) value depends on
the rotation speed as well as the manner in
which the centrifuge tubes are held by the rotor
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Quite clearly the G value in a centrifuge tube
will depend on the location, the highest value
being at the bottom of the tube and the lowest
value being at the top
This implies that a particle will experience
increasing G values while moving towards the
bottom of the centrifuge tube
In most cases the average G value that a
particle is likely to experience i.e. the numerical
mean of the maximum and minimum values is
used for process calculations.
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Nomograms provided by centrifuge
manufacturers correlating the radial distance and
rotation speed with the G value are commonly
used in the laboratory-scale calculations
Typical G values employed in laboratory
centrifuges range from 1,000 - 20,000
It is virtually impossible to make very exact
calculations for a laboratory centrifugation
process. This is due to the fact that it usually
takes a certain amount of time after start-up for
the rotation speed of the centrifuge to reach the
operating value
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Similarly it takes a certain amount of
time for the rotation speed to decrease
from the operating speed to zero at the
end of the process.
Moreover as mentioned previously, the
settling particles go through different G
value zones while moving toward the
bottom of the centrifuge tube
An empirical correlation is commonly
used for estimating the complete
centrifugation time:
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The sedimentation coefficient correlates
with the material properties as shown
below:
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For the same rotation speed, a fixed
angled rotor would have a lower k-factor
on account of the smaller difference
between rmax and rmin
Hence the time required for precipitating
a given sample would be less with the
fixed angled rotor
This is intuitively evident from looking at
Fig. 6.3 which shows that the distance
the particles have to travel for
precipitation is less with the angled rotor
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However, fixed angled rotors are heavier
and require much higher energy to
operate .
Swing out rotors are preferred for
centrifuging substances with high
sedimentation coefficients such as cells
and coarse particles while precipitated
macromolecules and finer particles are
centrifuged using fixed angled rotors.
Preparative centrifuge
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Preparative centrifuges can handle significantly larger
liquid volumes than laboratory centrifuges, typically
ranging from 1 litre to several thousand litres.
Preparative centrifuges come in a range of designs,
the common feature in these being a tubular rotating
chamber
The suspension to be centrifuged is fed into such a
device from one end while the supernatant and
precipitate are collected from the other end of the
device in a continuous or semi-continuous manner.
A simple diagram of the most common type of
preparative centrifuge (the tubular bowl centrifuge) is
shown in Fig. 6.4
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Typical rotating speeds for preparative
centrifuges range from 500 - 2000 rpm
The motion of a particle at any point
within a tubular centrifuge in the radial
direction is governed by the following
force balance equation:
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Ultracentrifugation
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An ultracentrifuge is a special type of
centrifuge in which the rotor rotates at a
much higher speed than a standard
centrifuge. Typical rotation speeds in
ultracentrifuges range from 30000 rpm -
50000 rpm.
An ultracentrifuge is usually used for
separating macromolecules from solvents or
for fractionating mixtures of macromolecules.
Ultracentrifuges are used for analytical as
well as for preparative applications.
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An analytical ultracentrifuge (AUC) is
mainly used for studying the properties
of macromolecules as well as for
analyzing complex mixtures of
macromolecules.
Preparative ultracentrifuges are used to
purify macromolecules such as proteins
and nucleic acids based on their physical
properties such as size, molecular
weight, density and mobility.
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The high rotating speeds used in
ultracentrifuges can generate
considerable amount of heat. Therefore
cooling arrangements are required in
these devices.