ERG 201 Biochemical Engineering (2+1)

Lecture 1

Biotechnology and Bioprocess Engineering – Bioprocess

development – Story of Penicillin – Role of Biologists and Engineers
– Bioprocesses: Regulatory Constraints
TB-1: 1-10
• Dolly (5 July 1996 – 14 February 2003)

was a female domestic sheep, and the

first mammal cloned from an adult

somatic cell, using the process of

nuclear transfer
 Introduction

• Genetic engineering with a purpose

• Genetic manipulation
• New medicines
• Semisynthetic organs grown in large vats
• Abundant and nutritious foods
• Computers based on biological molecules rather than silicon chips
• Superorganisms to degrade pollutants
• Wide array of consumer products and industrial processes
 What is a Bio Engineer?
Biotechnology
Use or development of methods of direct genetic manipulation for a
socially desirable goal
 production of a particular chemical
 production of better plants or seeds
 gene therapy
 use of specially designed organisms to degrade wastes
Bioengineering

Include work on medical and agricultural systems;

(agricultural, electrical, mechanical, industrial, environmental and
chemical engineers, and others)
Biological engineering

Similar but emphasizes applications to plants and animals

Biochemical engineering

Extension of chemical engineering principles to systems using a

biological catalyst to bring about desired chemical
transformations

Bioprocess engineering

Work of mechanical, electrical, and industrial engineers to apply

the principles of their disciplines to processes based on using
living cells or subcomponents of such cells
Biomedical engineering

Separate from biochemical engineering, although the boundary

between the two is increasingly vague, particularly in the areas of
Biomolecular engineering cell surface receptors and animal cell culture

Research at the interface of biology and chemical engineering and

is focused at the molecular level
 Biologists Engineers

• Life sciences • Physical Sciences

• Mathematical theories and quantitative • Mathematical sciences
methods (except statistics) have played a
secondary role

+
Story of Penicillin : How Biologists and Engineers
Work Together
• 1928, Alexander Fleming, St. Mary’s Hospital, London  isolate
the bacterium, Staphylococcus aureus, which causes boils

• Grew bacterium on the surface of a nutrient solution

Staphylococcus aureus

• Contamination of one of the dishes  no bacteria grew near

the invading substance

• Fleming recognized  common mold of the Penicillium genus

(later identified as Penicillium notatum) Contamination

• Fleming using crude extraction methods obtain a tiny quantity

of antimicrobial material named as penicillin
Penicillium notatum
• World War II  antibiotic with minimal side effects and broader applicability was
desperately needed

• Howard Florey and Ernst Chain of Oxford  treat a London bobby for a blood
infection; penicillin worked brought the patient to recovery; penicillin was
exhausted the man relapsed and died

• To make large amounts of penicillin would require a process, and for such a
process development, engineers would be needed, in addition to microbial
physiologists and other life scientists

• In 1939, penicillin final concentration  1 ppm

• Life scientists at the Northern Regional Research Laboratory made corn steep
liquor–lactose based medium increased productivity about tenfold

• Soon processes using tanks of about 10,000 gal were built

• Pfizer first commercial production 14 tanks each of 7000-gal capacity

• United States had the capacity by the end of World War II to produce enough
penicillin for almost 100,000 patients per year

• Progress with penicillin fermentation has continued, as has the need for the
interaction of biologists and engineers

• From 1939 to now, the yield of penicillin has gone from 0.001 to over 50 g/l of
fermentation broth

• Progress has involved better understanding of mold physiology, metabolic

pathways, penicillin structure, methods of mutation and selection of mold
genetics, process control, and reactor design
Schematic of penicillin production process
 Bioprocesses: Regulatory Constraints
• Difficulty of bioprocess engineer regulatory climate
• U.S. FDA (Food and Drug Administration) and its equivalents in other countries must
insure the safety and efficacy of medicines
• Pharmaceutical or biotechnology industry
• X reduction of manufacturing cost
• production of a product of consistently high quality
• FDA approval process
 Drug 6.5 years from discovery stage through preclinical testing in animals
• Human clinical trials
 Phase 1 clinical trials (about 1 year) are used to test safety; typically 20 to 80
volunteers are used
 Phase II clinical trials (about 2 years) use 100 to 300 patients and the emphasis is on
efficacy (i.e., does it help the patient) as well as further determining which side
effects exist
• Compounds that are still promising enter phase III clinical trials (about 3 years) with 1000
to 3000 patients
• FDA data review (about 18 months)
• If clinical trials statistically significant improvements in health with acceptable
side effects - likely to be approved
• Continued monitoring for adverse effects
• Whole drug discovery-through-approval process takes 15 years on the average and
costs about $400 million (in 1996)
• Only one in ten drugs that enter human clinical trials receives approval
• Drugs sold on the market or used in clinical trials must come from good
manufacturing practice (GMP) facilities
• Equipment and procedures must be validated
• Off-line assays done in laboratories must satisfy good laboratory practices (GLP)
• Procedures are documented by SOPs (standard operating procedures)
Thank You
 Lecture 2

Enzyme Kinetics (Single-substrate Reactions) - How Enzymes Work -

Enzyme Kinetics
TB-1: 57-60
 Enzymes
• Proteins of high molecular weight (15,000 < MW < several million daltons) - catalysts

• RNA molecules that have catalytic properties  ribozymes

• Specific, versatile, and very effective biological catalysts  higher reaction rates than
chemically catalyzed reactions

• Named by adding the suffix -ase to the end of the substrate or reaction catalyzed

• Many have more than one subunit

• Some protein enzymes (holoenzyme) require a nonprotein group for their activity
(a cofactor, such as metal ions, Mg, Zn, Mn, Fe, or a coenzyme, such as a complex
organic molecule, NAD (nicotinamide adenine dinucleotide), FAD (flavin adenine
dinucleotide), CoA (coenzyme A), or some vitamins)

• Protein part is apoenzyme (holoenzyme = apoenzyme + cofactor)

• Enzymes that occur in several different molecular forms, but catalyze the
same reaction  isozymes

Five lactate dehydrogenase isozymes

International Classification of Enzymes: Class Names, Code Numbers, and Types of Reactions Catalyzed
 How Enzymes Work
• Enzymes lower the activation energy of the reaction catalyzed by binding the
substrate and forming an enzyme–substrate complex

• Ex. Ea for decomposition (20ºC) of hydrogen peroxide :

• uncatalyzed : 18 kCal/mol
• chemically catalyzed (by colloidal platinum) : 13 kCal/mol
• enzymatically catalyzed (catalase) : 13 kCal/mol  catalase accelerates
108 times
• Ratio of the rates is exp(-7000/2 * 293) / exp(-18,000/2 *293)
Activation energies of enzymatically catalyzed and uncatalyzed reactions
|DG°A2|<|DG°A1|
• Interaction between the enzyme and its substrate is usually by weak forces
(van der Waals and hydrogen bonding)
• Substrate binds to a specific site on the enzyme  active site
 lock (enzyme) -and-key (substrate) model
• Multi-substrate enzyme-catalyzed • Enzymes hold the substrates at
reactions  enzymes hold certain positions and angles to
substrates close to each other improve the reaction rate
 orientation effect
 proximity effect
• Formation of an enzyme–substrate complex  slight changes in 3D shape
 induced fit
Cofactors (Metal Ions) and Coenzymes of Some Enzymes
Thank You
 Lecture 3

Reaction rate and Yield – Mechanistic Models for Simple Enzyme

Kinetics
TB-1: 61 - 63
 Enzyme Kinetics
• Single substrate enzyme catalyzed reactions
• Henri (1902)
• Michaelis-Menten (1913)  saturation kinetics

----------- 1

• S – substrate
• P – product
• ES – enzyme substrate complex
• k1, k-1 , k2 – rate constants
• Main objective of the kinetic study is to develop suitable
mathematical expressions for the rates of enzyme-catalysed
reactions
• What is reaction rate?
• Substrate (S) and Product (P)
S  P
• Reaction rate is v
- ds dp
v = ---- = ---
dt dt
Effect of substrate concentration on the rate of an
enzyme – catalyzed reaction
 Rate expressions

• Rapid equilibrium approach

• Quasi-steady state approach

 Mechanistic Models

• Both the rate expressions  same initial steps

• Rate of product formation ----- 2

 v - rate of product formation or substrate consumption in moles/l-s

• k2 = kcat
• Rate of variation of the ES complex ------- 3

• Enzyme is not consumed, the conservation equation on the enzyme yields

------ 4
 Rapid equilibrium assumptions
• Henri and Michaelis & Menten =
• Equilibrium constant
---------- 5

• [E] = [Eo] – [ES] if enzyme is conserved, then

------------ 6
• K ’m = k-1/k1, -dissociation constant

------------- 7
• Sub. (7) in (2)
------------ 8

where Vm = k2[E0]
 Quasi Steady state assumptions
• G. E. Briggs and J. B. S. Haldane
• Experimental systems - closed system (batch reactor)  [So] > [Eo]
• Since [E0] was small  d[ES]/dt a 0  logic is flawed
In a closed system the quasi-steady-state hypothesis
• if [S0] > > [E0] (for example, 100X)
• applying the quasi-steady-state assumption to rate of variation of ES complex 0
------------- 9

• Sub. enzyme concentration in (4) & (9)

--------------- 10
Time course of the formation of an enzyme/substrate complex and initiation of the steady state
 Solving (10) for [ES]

------------- 11

Sub. (11) into (2), yields

------------- 12

where Km is (k-1 + k2)/k1 and Vm is k2[E0]

Under simple experiments impossible to determine whether Km or K ’m is more suitable

Thank You
 Lecture 4

Experimentally Determining Rate Parameters for Michaelis -

Menten Type Kinetics – Line weaver- Burk and Eadie plots
TB-1: 64 - 66
 Experimentally Determining Rate Parameters for MM Kinetics
• From initial rate experiments, high precision determination of Km and Vm difficult 
more other predictions
• Experimental data - initial-rate experiments
• Batch reactor - known amount of substrate [S0] and enzyme [E0]
• Product or substrate concentration Vs time

• Initial slope of this curve is estimated

v = d[P]/dt |t = 0 = -d [S] / dt |t=0

• Value of v  values of [E0] and [S0]

Double reciprocal plot (Lineweaver-Burk plot)

 linearized in double-reciprocal form

1/v versus 1/[S]

 Eadie-Hofstee plot

v (Km + [S]) = Vm [S]

v Km + v [S] = Vm [S]

• Large errors since both coordinates contain 

• Lesser bias on points at low [S]
 Hanes Woolf plot


Multiply by [S] on both sides  • [S]/v versus [S]

• To determine Vm more accurately
 Batch kinetics
• Time course of variation of [S] in a batch enzymatic reaction 

• By integration to yield

[S0] - [S] = Vm t – Km ln {[S0]/ [S]}

Vm t = [S0] - [S] + Km ln {[S0]/ [S]}

 { }

/ by Km
1/t ln[S0]/[S] versus {[S0] - [S]}/t  a line of slope -1/Km & intercept of Vm/Km
 Interpretation of Km and Vm
• Km (or Km’) - intrinsic parameter, Vm is not

• Km expected to change with T or pH

• Vm = f (k2, [E0])

• Highly purified enzyme preparations  [E0]  mol/l or g/l

• Crude preparation  Enzyme concentration  units

• Unit - amount of enzyme that gives a predetermined amount of catalytic activity

under specific conditions
 Interpretation of Km and Vm
• 1 unit  formation of one mmol product/min at a specified pH and T ; [S] >>Km

• Specific activity – No. of units of activity / amount of total protein

• Eg. a crude cell lysate might have a specific activity of 0.2 units/mg protein which
upon purification may increase to 10 units/mg protein

• Remaining catalytically active enzyme only be measured

• Enzyme - denatured (unfolds or 3 D shape altered) by pH or T during purification

 no activity
 Example
To measure the amount of glucoamylase in a crude enzyme preparation, 1 ml of the crude
enzyme preparation containing 8 mg protein is added to 9 ml of a 4.44% starch solution.
One unit of activity of glucoamylase is defined as the amount of enzyme which produces 1
mmol of glucose per min in a 4% solution of Lintner starch at pH 4.5 and at 60°C. Initial
rate experiments show that the reaction produces 0.6 mmol of glucose/ml-min. What is the
specific activity of the crude enzyme preparation?
• Total amount of glucose, Vm = 10 ml / 0.6 mmol glucose/ml-min or 6 mmol glucose / min
• Specific activity , K2
= 6 units / (1 ml protein solution * 8 mg/ml)
= 6 units/8 mg protein
= 0.75 units/ mg protein
• Vm - units such as mmol product/ml-min
• Since Vm = k2E0, the dimensions of k2 must reflect the definition of units in E0
• If, for example, Vm = 1 mmol/ml-min, then k2 = ?

• k2 = 1 mmol/ml-min  0.6 units/ml or k2 = 1.67 mmol/unit-min

Thank You
 Lecture 5 & 6
Enzyme Inhibition - Models for More Complex Enzyme Kinetics –
Effect of pH and Temperature – Theoretical and overall yields

TB-1: 65 – 78
 Models for More Complex Enzyme Kinetics
• Enzymes have more than 1 substrate binding site - binding of 1 substrate to
enzyme facilitates binding of other substrate molecules  allostery or
cooperative binding
 Allosteric enzymes

• Rate expression

------ (1)

n = cooperativity coefficient and n > 1

• If n = 1  MM equation

• n  1  positive cooperativity (rate of reaction )

• n  1  negative cooperativity (rate of reaction )

 Sigmoidal
shape

Comparison of MM & allosteric enzyme kinetics

• Cooperativity coefficient (by rearranging the eqn.)

• Determination of cooperativity coefficient

• Hill equation
K″m= Kh 

Kh – hill constant
 Inhibited enzyme kinetics
• Compounds may bind to enzymes and reduce their activity  enzyme
inhibitors

• Enzyme inhibitions  irreversible or reversible

• Irreversible inhibitors : Heavy metals (lead, cadium, mercury, and others) +

enzyme stable complex - reduce enzyme activity

• Enzyme inhibition - reversed – chelating agents : EDTA & citrate

• Reversible inhibitors -dissociate more easily from the enzyme after

binding
Classes of Inhibited enzyme kinetics

.
 Reversible - Competitive inhibition kinetics
Compete with S for active site of enzyme

Assuming rapid equilibrium

Rate of enzymatic conversion

or

↑ of K’ m, app  ↓ v
 Reversible - Non-Competitive inhibition kinetics
• Bind on sites other than the active site and reduce enzyme affinity to the substrate

I ↑  ↓ Vm & v
 Double reciprocal plot
Rate of enzymatic conversion

Assume same KI & K’m

↑ K’ m & ↓ Vm
 Reversible - Uncompetitive inhibition kinetics
• Bind to the ES complex only and have no affinity for enzyme
Rate of enzymatic conversion

or

↓ Vm & K’ m  v ↓
 Substrate inhibition
• High substrate concentrations may cause inhibition in some enzymatic
reactions

Comparison of substrate inhibited and uninhibited enzymatic

reactions
Reaction Scheme
L-B plot
• At low substrate concentrations, [S]2/KS1 << 1 & no inhibition effect
Rate of enzymatic conversion

or

1/v versus 1/[S]  slope K'm/Vm and intercept of 1/Vm

• At high substrate concentrations, Km / [S] << 1 & inhibition - dominant
Rate of enzymatic conversion

or

1/v versus 1/[S]  slope 1/KS1 . Vm and intercept of 1/Vm

Substrate concentration - maximum reaction rate  dv/d[S] = 0
Thank You
 Enzyme Immobilization
 The restriction of enzyme mobility in a fixed space is known as enzyme
immobilization.
 Immobilization of enzymes provides important advantages- enzyme reutilization,
enzyme recovery, purification processes and may provide a better environment.
 Some of the intracellular enzymes are membrane bound, immobilized enzymes
provide a model system to mimic and understand the action of some membrane-
bound intracellular enzymes.
 Product purity is usually improved

Methods of Immobilization
Entrapment
Entrapment is the physical enclosure of enzymes in a small space.
Matrix entrapment and membrane entrapment (microencapsulation) are the
two major methods of entrapment.
 Matrices used for enzyme immobilization are usually polymeric materials
- Ca-alginate, agar, k-carrageenin, polyacrylamide, and collagen.
 Solid matrices such as activated carbon, porous ceramic, and
diatomaceous earth can also be used.
 The matrix - particle, a membrane, or a fiber
 When immobilizing in a polymer matrix, enzyme solution is mixed with
polymer solution before polymerization.
 Polymerized gel-containing enzyme is either extruded or a template is
used to shape the particles from a liquid polymer-enzyme mixture.
 Membrane entrapment of enzymes is done to entrap an enzyme solution
between thin, semipermeable membranes.
 Membranes - nylon, cellulose, polysulfone and polyacrylate.
 Semipermeable membrane is used to retain high-molecular-weight
compounds (enzyme), while allowing small molecular- weight
compounds (substrate or products) access to the enzyme.
 Microencapsulation - microscopic hollow spheres contains the enzyme
solution, enclosed within a porous membrane.
 Enzyme entrapment problems - enzyme leakage into solution, significant
diffusional limitations, reduced enzyme activity and stability and lack of
control of micro environmental conditions.
 Enzyme leakage - reducing the MW cutoff of membranes or the pore
size of solid matrices.
 Diffusion limitations - reducing the particle size of matrices and/or
capsules.
 Reduced enzyme activity and stability - unfavorable micro environmental
conditions (difficult to control).
 By using different matrices and chemical ingredients, by changing
processing conditions, and by reducing particle or capsule size, more
favorable micro environmental conditions can be obtained.
 Diffusion barrier is usually less significant in microcapsules as compared
to gel beads.
Surface immobilization.
Adsorption and covalent binding.
Adsorption - Attachment of enzymes on the surfaces of support particles by
weak physical forces (van der Waals or dispersion forces).
 The active site of the adsorbed enzyme is usually unaffected and it is
stabilized by cross-linking with glutaraldehyde.
 Support materials - inorganic materials (Alumina, silica, porous glass,
ceramics, diatomaceous earth, clay and bentonite) or organic materials
(Cellulose like CMC, DEAE-cellulose, starch and activated carbon) and
ion-exchange resins (Amberlite, Sephadex and Dowex).
 The surfaces of the support materials - pretreated for effective
immobilization.
Covalent binding – Attachment of enzymes on support surfaces by covalent
bond formation, via certain functional groups (Amino, carboxyl, hydroxyl, and
sulfhydryl).
 These functional groups must not be in the active site.
 Functional groups on support material are usually activated by using
chemical reagents (cyanogen bromide, carbodiimide, and glutaraldehyde).
 The cross-linking agents such as glutaraldehyde, bis-diazobenzidine and 2,2-
disulfonic acid.
 Cross-linking can be achieved in several different ways: enzymes can be
cross-linked with glutaraldehyde to form an insoluble aggregate, adsorbed
enzymes may be cross-linked or cross-linking may take place following the
impregnation of porous support material with enzyme solution.
 Cross-linking may cause significant changes in the active site of enzymes
and also severe diffusion limitations may result.
 Support material and immobilization method vary depending on the enzyme
and particular application.
 Two major criteria used in the selection of support material - the binding
capacity of the support material, which is a function of charge density,
functional groups, porosity, and hydrophobicity of the support surface.
 stability and retention of enzymatic activity, which is a function of functional
groups on support material and microenvironmental conditions.
 If immobilization causes some conformational changes on the enzyme, or if
reactive groups on the active site of the enzyme are involved in binding
leads to a loss in enzyme activity can take place upon immobilization.
Effect of Immobilization Methods on the Retention of Enzymatic Activity
of Aminoacylase
 Diffusional Limitations in
Immobilized Enzyme Systems
• Diffusional resistance is seen in immobilised enzymes
• These resistances vary depends
 Nature of the support material (porous, nonporous)
 Hydrodynamical conditions surrounding the support material
 Distribution of the enzyme inside or on the surface of the support
material.
• Diffusion resistance has a significant effect on the rate of enzymatic
reaction rate depends on the relative rate of the reaction rate and
diffusion rate, which is characterized by the Damköhler number (Da).

where [Sb] is substrate concentration in bulk liquid (g/cm3) and kL is the

mass-transfer coefficient (cm/s)
depending on the value of the Damköhler number. If Da >> 1, the diffusion rate
is
limiting. For Da << 1, the reaction rate is limiting, and for Da ≈ 1, the diffusion
and reaction resistances are comparable. Diffusion and enzymatic reactions
may be simultaneous, with enzymes entrapped in a solid matrix, or may be
two consecutive phenomena for adsorbed enzymes.
Diffusion effects in surface-bound enzymes on nonporous support materials.
When enzymes are bound and evenly distributed on the surface of a
nonporous support material, all enzyme molecules are equally active, and
substrate diffuses through a thin liquid film surrounding the support
surface to reach the reactive surfaces.

The process of immobilization has not

altered the protein structure, and the
intrinsic kinetic parameters (Vm, Km) are
unaltered.
At steady state, the reaction rate is equal to the mass-transfer rate

where V’m is the maximum reaction rate per unit of external surface area and
kL is the liquid mass-transfer coefficient. This equation is quadratic in [Ss], the
substrate concentration at the surface and it can be solved analytically

When enzymes are immobilized on internal pore

surfaces of a porous matrix, substrate diffuses
through the tortuous pathway among pores and
reacts with enzyme immobilized on pore surfaces.
Diffusion and reaction are simultaneous in this case,
as depicted in figure.

Assume that enzyme is uniformly distributed in a

spherical support particle; the reaction kinetics are
expressed by Michaelis–Menten kinetics, and there
is no partitioning of the substrate between the
exterior and interior of the support.
The effectiveness factor is defined as the ratio of the reaction rate with
diffusion
limitation (or diffusion rate) to the reaction rate with no diffusion limitation. The
value of the effectiveness factor is a measure of the extent of diffusion
limitation. For ƞ < 1, the conversion is diffusion limited, whereas for ƞ ≈ 1 values,
conversion is limited by the reaction rate and diffusion limitations are negligible.

When designing immobilized enzyme systems using a particular support, the

main
variables are Vm and R, since the substrate concentration, Km, and De are fixed.
The particle size (R) should be as small as possible within the constraints of
particle integrity, resistance to compression, and the nature of the particle
recovery systems.

The maximum reaction rate is determined by enzyme activity and

concentration in the support. High enzyme content will result in high enzyme
activity per unit of reaction volume but low effectiveness factor.
On the other hand, low enzyme content will result in lower enzyme activity per
unit volume but a high effectiveness factor.
For maximum conversion rates, particle size should be small (Dp ≤ 10 mm) and
enzyme loading should be optimized. Dp ≤ 10 mm and enzyme loadings of less
Electrostatic and Steric Effects in Immobilized Enzyme Systems
• When enzymes are immobilized in a charged matrix as a result of a
change in the microenvironment of the enzyme, the apparent bulk pH
optimum of the immobilized enzyme will shift from that of soluble enzyme.
• The charged matrix will repel or attract substrates, product, cofactors, and
H+ depending on the type and quantity of surface charge.
• For an enzyme immobilized onto a charged support, the shift in the pH-
activity profile is given by

where pHi and pHe are internal and external pH values, respectively; z is
the charge (valence) on the substrate; F is the Faraday constant (96,500
coulomb/eq. g); y is the electrostatic potential; and R is the gas constant.
Expressions similar to other nonreactive charged medium components.
The intrinsic activity of the enzyme is altered by the local changes in pH
and ionic constituents.
Further alterations in the apparent kinetics are due to the repulsion or
attraction of substrates or inhibitors.
• The activity of an enzyme toward a high-molecular-weight
substrate is usually reduced upon immobilization to a much
greater extent than for a low-molecular-weight substrate.

• This is mainly because of steric hindrance by the support. Certain

substrates, such as starch, have molecular weights comparable to
those of enzymes and may therefore not be able to penetrate to
the active sites of immobilized enzymes.

• Immobilization also affects the thermal stability of enzymes.

• Thermal stability often increases upon immobilization due to the

presence of thermal diffusion barriers and the constraints on
protein unfolding.

• However, decreases in thermal stability have been and the pH

stability of enzymes usually increases upon immobilization.
 LARGE-SCALE PRODUCTION OF ENZYMES

• Various enzymes produced at large scale are proteases (subtilisin,

rennet), hydrolases (pectinase, lipase, lactase), isomerases (glucose
isomerase), and oxidases (glucose oxidase).
• These enzymes are produced using overproducing strains of certain
organisms.
• Separation and purification of an enzyme from an organism require
disruption of cells, removal of cell debris and nucleic acids, precipitation
of proteins, ultrafiltration of the desired enzyme, chromatographic
separations (optional), crystallization, and drying.
• The process scheme varies depending on whether the enzyme is
intracellular or extracellular.
• In some cases, it may be more advantageous to use inactive (dead or
resting) cells with the desired enzyme activity in immobilized form. This
approach eliminates costly enzyme separation and purification steps
and is therefore economically more feasible.
• The first step in the large-scale production of enzymes is to cultivate
the organisms producing the desired enzyme. Enzyme production can
be regulated and fermentation conditions can be optimized for
overproduction of the enzyme.
• Proteases -Bacillus, Aspergillus, Rhizopus, and Mucor; pectinases -
Aspergillus niger; lactases - yeast and Aspergillus; lipases - certain
strains of yeasts and fungi; glucose isomerase - Flavobacterium
arborescens or Bacillus coagulans.
• After the cultivation step, cells are separated from the media usually by
filtration or sometimes by centrifugation. Depending on the intracellular
or extracellular nature of the enzyme, either the cells or the fermentation
broth is further processed to separate and purify the enzyme.
• The recovery of intracellular enzymes is more complicated and involves
the disruption of cells and removal of cell debris and nucleic acids.
• In some cases, enzyme may be both intracellular and extracellular, which
requires processing of both broth and cells.
• Intracellular enzymes may be released by increasing the permeability of
cell membrane. Certain salts such as CaCl2 and other chemicals such as
dimethylsulfoxide (DMSO) and pH shift may be used for this purpose.
• If enzyme release is not complete, then cell disruption may be essential.
• The processes used to produce these industrial enzymes have much in
common with our later discussions on processes to make proteins from
recombinant DNA.
MEDICAL AND INDUSTRIAL UTILIZATION OF ENZYMES
 Lecture 9
Microbial Growth - Introduction - Batch
Growth - Quantifying Cell Concentration,
- Growth Patterns and Kinetics in Batch
Culture
•For microbes, growth is their most essential response to their
physiochemical environment.
•Growth →replication and change in cell size.
•Microorganisms can grow under a variety of physical, chemical, and
nutritional conditions.
•In a suitable nutrient medium → organisms extract nutrients from the
medium → convert them into biological compounds.
•Nutrients → Energy production and biosynthesis and product formation.
•Nutrient utilization, microbial mass increases with time and can be
described simply by

•Microbial growth → autocatalytic reaction.

•The rate of growth is directly related to cell concentration and cellular
reproduction is the normal outcome of this reaction.
The rate of microbial growth is characterized by the net specific growth rate,
defined as

•X is cell mass concentration (g/l), t is time (h), and µnet is net specific
growth rate (h-1).
•The net specific growth is the difference between a gross specific growth
rate, mg (h-1), and the rate of loss of cell mass due to cell death or
endogenous metabolism, kd (h-1).
•Microbial growth can also be described in terms of cell number
concentration, N, as well as X.

•where µR is the net specific replication rate (h-1). If we ignore cell death, kd,
then we use the symbol µ’R; and in cases where cell death is unimportant, µ
R will equal µ’R.
BATCH GROWTH
Culturing cells in a vessel with an initial charge of medium that is not altered
by further nutrient addition or removal.
Cultivation is simple and used both in the laboratory and industrially
Quantifying Cell Concentration
•Essential for the determination of the kinetics and stoichiometry of microbial
growth.
•Classified in two categories: direct and indirect
•Direct methods are not feasible →presence of suspended solids or
interfering compounds in the medium
•Either cell number or cell mass can be quantified
Determining cell number density
•Petroff–Hausser slide or a hemocytometer direct cell counting.
•A calibrated grid is placed over the culture chamber, the number of cells per
grid square is counted using a microscope at least 20 grid squares must be
counted and averaged.
•The culture medium should be clear and free of particles.
•Plates containing appropriate growth medium gelled with agar (Petri
dishes) are used for counting viable cells.
•Culture samples are diluted and spread on the agar surface and the
plates are incubated.
•Colonies are counted on the agar surface following the incubation period
and expressed in terms of colony-forming units (CFU).
•Another method is based on the relatively high electrical resistance of
cells

•As cells pass through the orifice, the electrical

resistance increases and causes pulses in
electrical voltage.
•The number of pulses is a measure of the number
of particles.
•The height of the pulse is a measure of cell size.
Probes with various orifice sizes are used for
different cell sizes.
Determining cell mass concentration (Direct methods)
•Determination of cellular dry weight →cell mass concentration →
applicable only for cells grown in solids-free medium.
• If noncellular solids →molasses solids, cellulose, or corn steep liquor →
dry weight measurement will be inaccurate.
•Samples of culture broth are centrifuged or filtered and washed with a
buffer solution or water → dried at 80°C for 24 hours → dry cell weight is
measured.
•Packed cell volume is rapid but roughly estimate the cell concentration in
a fermentation broth → centrifuged in a tapered graduated tube under
standard conditions and the volume of cells is measured.
•Another rapid method → is based on the absorption of light by suspended
cells in sample culture media. (600-700 nm)
•The intensity of the transmitted light is measured using a spectrometer
(turbidity or optical density measurement) → fast, inexpensive, and simple
method.
•The extent of light transmission in a sample chamber is a function of cell
density and the thickness of the chamber.
•Light transmission is modulated by both absorption and scattering.
•The calibration curve relates optical density (OD) to dry-weight
measurements. Such calibration curves can become nonlinear at high OD
Indirect methods
•In many fermentation processes → direct methods cannot be used. In
such cases indirect methods →based mainly on the measurement of
substrate consumption and/or product formation during the course of
growth.
•Intracellular components → RNA, DNA, and protein → measures of cell
growth.
•The ATP concentration in a fermentation broth → measure of biomass
concentration → based on luciferase activity, catalyzes oxidation of
luciferin at the expense of oxygen and ATP with the emission of light
•Nutrients used for cellular mass production → follow microbial growth
(Nitrate, phosphate, or sulfate).
•The utilization of a carbon source or oxygen uptake rate → monitor
cellular growth.
•The products of cell metabolism can be used to monitor and quantify
cellular
•Growth (Ethanol and lactic acid).
•Changes in the viscosity of the fermentation broth → extent of microbial
growth.
Growth Patterns and Kinetics in Batch Culture
When a liquid nutrient medium is inoculated with a seed culture, the
organisms selectively take up dissolved nutrients from the medium and
convert them into biomass. A typical batch growth curve includes the
following phases:
(1) lag phase, (2) logarithmic or exponential growth phase, (3) deceleration
phase,
The lag phase occurs immediately after
(4) stationary phase, and (5) death phase.
inoculation and is a period of adaptation
of
cells to a new environment.
Microorganisms reorganize their
molecular constituents when they are
transferred to a new medium.
Depending on the composition of
nutrients, new enzymes are synthesized,
the synthesis of some other enzymes is
repressed, and the internal machinery of
cells is adapted to the new environmental
conditions.
•During this phase, cell mass may increase a little, without an increase in
cell number density. When the inoculum is small and has a low fraction
of cells that are viable, there may be a pseudolag phase, which is a result,
not of adaptation, but of small inoculum size or poor condition of the
inoculum.
•Low concentration of some nutrients and growth factors may also cause
a long lag Phase. The lag period increases with the age of the inoculum.
•To minimize the duration of the lag phase, cells should be adapted to
the growth medium and conditions before inoculation, and cells should
be young and active, and the inoculum size should be large (5% to 10% by
volume).
•Many commercial fermentation plants → rely on batch culture (high
productivity from a fixed plant size, the lag phase must be as short).
•Multiple lag phases → the medium contains more than one carbon
source (diauxic growth) → shift in metabolic pathways in the middle of a
growth cycle.
•After one carbon source is exhausted, the cells adapt their metabolic
activities to utilize the second carbon source.
•The first carbon source → readily utilizable than the second → presence
of
•more readily available carbon source → represses the synthesis of the
•The exponential growth phase (logarithmic growth phase) → In this
phase, the cells have adjusted to their new environment → cells can
multiply rapidly, and cell mass and cell number density increase
exponentially with time.
•This is a period of balanced growth → All components of a cell grow at
the same rate (Average composition of a single cell remains
approximately constant during this phase of growth).
•During balanced growth, the net specific growth rate determined from
either cell number or cell mass would be the same.
•Nutrient concentrations are large in this phase → the growth rate is
independent of nutrient concentration and the growth rate is first order:

•where X and X0 are cell concentrations at time t and t = 0.

•The time required to double the microbial mass is given by above eqn.
•The exponential growth is characterized by a straight line on a
semilogarithm plot of ln X versus time
τd → doubling time of cell mass.
τ'd → doubling time based on the
replication rate
•The deceleration growth phase follows the exponential phase.
•In this phase → growth decelerates due to either depletion of one or
more essential nutrients or the accumulation of toxic by-products of
growth.
•For a typical bacterial culture, these changes occur over a very short
period of time.
•The rapidly changing environment results in unbalanced growth.
•During unbalanced growth, cell composition and size will change (τd ≠ τ’d )
.
•In the exponential phase, the cellular metabolic control system is set to
achieve maximum rates of reproduction.
•In the deceleration phase, the stresses induced by nutrient depletion or
waste accumulation cause a restructuring of the cell to increase the
prospects of cellular survival in a hostile environment.
•These observable changes are the result of the molecular mechanisms of
repression and induction.
•Because of the rapidity of these changes, cell physiology under conditions
of
•nutrient limitation is more easily studied in continuous culture.
•The stationary phase starts at the end of the deceleration phase, when
the net
growth rate is zero or when the growth rate is equal to the death rate.
•Even though the net growth rate is zero during the stationary phase,
cells are still metabolically active and produce secondary metabolites.
•Primary metabolites are growth-related products and secondary
metabolites are non growth-related. The production of certain
metabolites is enhanced during the stationary phase (e.g., antibiotics,
some hormones) due to metabolite deregulation. During the course of
the stationary phase, one or more of the following phenomena may take
place:
1. Total cell mass concentration → constant, number of viable cells may
decrease.
2. Cell lysis → occur and viable cell mass decrease. A second growth
phase may
Occur (lysis products of lysed cells).
3. Cells may not be growing but may have active metabolism to produce
secondary metabolites (metabolite deregulation). Cellular regulation
changes when certain metabolites are low.
•During the stationary phase, the cell catabolizes cellular reserves for new
building blocksand for energy-producing monomers → Endogenous
metabolism.
•The cell must always expend energy to maintain an energized
membrane (pmf) and transport of nutrients and for essential metabolic
functions such as motility and repair of damage to cellular structures →
energy expenditure → maintenance energy.
•The appropriate equation to describe the conversion of cell mass into
maintenance energy or the loss of cell mass due to cell lysis during the
stationary phase is

•where kd is a first-order rate constant for endogeneous metabolism, and

Xso is the cell mass concentration at the beginning of the stationary
phase. Because S is zero, mg is zero in the stationary phase.
•The reason for termination of growth → exhaustion of an essential
nutrient or accumulation of toxic products.
•If an inhibitory product is produced → accumulates →the growth rate will
slow down and at a certain level of inhibitor concentration, growth will
stop.
•Ethanol production by yeast is an example of a fermentation in which the
product is inhibitory to growth.
•Dilution of toxified medium, addition of an unmetabolizable chemical
compound complexing with the toxin, or simultaneous removal of the
toxin → alleviate the adverse effects of the toxin and yield further growth.

The death phase (decline phase) follows the stationary phase.

•Some cell death may start during the stationary phase, and a clear
demarcation between these two phases is not always possible.
•Dead cells lyse, and intracellular nutrients released into the medium are
used by the living organisms during stationary phase.
•At the end of the stationary phase, because of either nutrient depletion
or toxic product accumulation, the death phase begins.
•The rate of death usually follows first-order kinetics:

•where Ns is the concentration of cells at the end of the stationary phase

and k’d is the first order death-rate constant. A plot of ln N versus t yields
a line of slope
–k’d.
•During the death phase, cells may or may not lyse, and the
reestablishment of the culture may be possible in the early death phase if
cells are transferred into a nutrient-rich medium.
•In both the death and stationary phases, it is important to recognize that
there is a distribution of properties among individuals in a population.
•To better describe growth kinetics, we define some stoichiometrically
related parameters.
•Yield coefficients are defined based on the amount of consumption of
another
material. For example, the growth yield in a fermentation is

•At the end of the batch growth period → apparent growth yield (observed
growth). Because culture conditions can alter patterns of substrate
utilization, the apparent growth yield is not a true constant. For example,
with a compound (such as glucose) that is both a carbon and energy
source, substrate may be consumed as:
Microbial products can be classified in three major categories:
1. Growth-associated products are produced simultaneously with microbial
growth. The specific rate of product formation is proportional to the specific
rate of growth, mg. Note that mg differs from mnet, the net specific growth
rate, when endogeneous metabolism is nonzero. The production of a
constitutive enzyme is an example of a growth-associated product.

2. Non-growth-associated product formation takes place during the

stationary phase when the growth rate is zero. The specific rate of product
formation is constant. Many secondary metabolites, such as antibiotics (for
example, penicillin), are non growth- associated products.

3. Mixed-growth-associated product formation takes place during the slow

growth and stationary phases. In this case, the specific rate of product
formation is given by the following equation:

Lactic acid fermentation, xanthan gum, and some secondary metabolites

from cell
culture are examples of mixed-growth-associated products
 Lecture 9
Factors affecting the growth of microorganisms
The patterns of microbial growth and product formation are influenced by environmental
conditions such as temperature, pH, and dissolved-oxygen concentration.
Temperature is an important factor affecting the performance of cells. According to their
temperature optima, organisms can be classified in three groups:
 Psychrophiles (Topt < 20°C)
 Mesophiles (Topt = from 20° to 50°C), and
 Thermophiles (Topt > 50°C)
 The growth rate approximately doubles for every 10°C increase in temperature.
 Above the optimal temperature range thermal death may occur.
 The net specific replication rate can be expressed by the following equation for temperature
above optimal level:

 At high temperatures, the thermal death rate exceeds the growth rate, which causes a net
decrease in the concentration of viable cells.
 Both µ’R and k’d vary with temperature according to the Arrhenius equation:

 Ea and Ed are activation energies for growth and thermal death.

 The activation energy for growth and thermal death are 10 to 20 kcal /mol and 60 to 80
kcal/mol respectively.
 Thermal death is more sensitive to temperature changes than microbial growth
and temperature also affects product formation and yield coefficient.
 When temperature is increased above the optimum temperature, the
maintenance requirements of cells increase. Maintenance coefficient increases
with increasing temperature with an activation energy of 15 to 20 kcal/mol,
resulting in a decrease in the yield coefficient.
 Temperature also may affect the rate-limiting step in a fermentation process →
the rate of bioreaction might become higher than the diffusion rate, and diffusion
become the rate-limiting step.
 The activation energy of molecular diffusion is about 6 kcal/mol. The activation
energy for most bioreactions is more than 10 kcal/mol, so diffusional limitations
must be carefully considered at high temperatures.
 The graph below depicts Arrhenius plot of growth rate of E. coli B/r which shows
the typical variation of growth rate with temperature.
 Hydrogen-ion concentration (pH) affects the activity of enzymes → microbial
growth rate.
 The optimal pH for growth may be different from that for product formation.
 The acceptable pH range varies about the optimum by ± 1 to 2 pH units.
 Different organisms have different pH optima:( Bacteria pH = 3 to 8; Yeast pH = 3
to 6, Molds pH = 3 to 7, Plant cells pH = 5 to 6 and animal cells pH = 6.5 to 7.5)
 Many organisms have mechanisms to maintain intracellular pH at a relatively
constant level in the presence of fluctuations in environmental pH.
 When pH differs from the optimal value, the maintenance-energy requirements
increase.
 pH optima is that the pH of the medium can be used to select one organism over
another.
 In most fermentations, pH can vary substantially.
 If ammonium is the sole nitrogen source, hydrogen ions are released into the
medium as a result of the microbial utilization of ammonia, resulting in a
decrease in pH.
 If nitrate is the sole nitrogen source, hydrogen ions are removed from the
medium to reduce nitrate to ammonia, resulting in an increase in pH.
 Also pH can change because of the production of organic acids, the utilization of
acids or the production of bases.
 The evolution or supply of CO2 can alter pH greatly in some systems.
 Variation of specific growth rate with pH is depicted in figure above, indicating a
pH optimum.
 Dissolved oxygen (DO) is an important substrate in aerobic fermentations
 It may be a limiting substrate, since oxygen gas is sparingly soluble in water.
 At high cell concentrations, the rate of oxygen consumption may exceed the rate
of oxygen supply, leading to oxygen limitations.
 When oxygen is the rate-limiting factor, specific growth rate varies with dissolved-
oxygen concentration.
 Below a critical concentration, growth or respiration approaches a first-order rate
dependence on the dissolved-oxygen concentration.
 Above a critical oxygen concentration, the growth rate becomes independent of
the dissolved-oxygen concentration.

The figure above depicts the variation of specific growth rate with dissolved-
oxygen concentration.
 Oxygen is a growth-rate-limiting factor when the DO level is below the critical DO
concentration.
 For example, with Azotobacter vinelandii at a DO = 0.05 mg/l, the growth rate is
about 50% of maximum even if a large amount of glucose is present. However,
the maximum amount of cells formed is not determined by the DO, as oxygen is
continually resupplied.
 If glucose were totally consumed, growth would cease even if DO = 0.05 mg/l.
Thus, the extent of growth would depend on glucose, while the growth rate for
most of the culture period would depend on the value of DO.
 The critical oxygen concentration is about 5% to 10% (bacteria and yeast) and
10% to 50% (mold cultures).
 Saturated DO concentration in water at 25°C and 1 atm pressure is about 7 ppm.
 The presence of dissolved salts and organics can alter the saturation value;
increasingly high temperatures decrease the saturation value.
 Oxygen is usually introduced to fermentation broth by sparging air through the
broth.
 Oxygen transfer from gas bubbles to cells is usually limited by oxygen transfer
through the liquid film surrounding the gas bubbles.
 The rate of oxygen transfer from the gas to liquid phase is given by

kL - Oxygen transfer coefficient (cm/h), a - Gas–liquid interfacial area (cm2/cm3),

kLa - Volumetric oxygen transfer coefficient (h-1), C* - Saturated DO
concentration
(mg/l), CL - Actual DO concentration in the broth (mg/l), and
NO2 - Rate of oxygen transfer (mg O2/l.h).

The rate of oxygen uptake is denoted as OUR (oxygen uptake rate):

qO2 - Specific rate of oxygen consumption (mg O2/g dw cells.h)
YX/O2 - Yield coefficient on oxygen (g dw cells/g O2) and
X - Cell concentration (g dw cells/l).

 When oxygen transfer is the rate-limiting step, the rate of oxygen consumption is
equal to the rate of oxygen transfer. If the maintenance requirement of O2 is
negligible compared to growth, then

 Growth rate varies nearly linearly with the oxygen transfer rate under oxygen-
transfer limitations.
 Among the various methods used to overcome DO limitations are the use of
oxygen-enriched air or pure oxygen and operation under high atmospheric
Heat Generation by Microbial Growth
 About 40% to 50% of the energy stored in a carbon and energy source is
converted to biological energy (ATP) during aerobic metabolism, and the rest
of the energy is released as heat.
 For actively growing cells, the maintenance requirement is low, and heat
evolution is directly related to growth.
 The heat generated during microbial growth can be calculated using the heat
of combustion of the substrate and of cellular material.
 A schematic of an enthalpy balance for microbial utilization of substrate is
presented in the figure below:
 The heat of combustion of the substrate is equal to the sum of the metabolic
heat and the heat of combustion of the cellular material.
ΔHs - Heat of combustion of the substrate (kJ/g substrate)
YX/S - Substrate yield coefficient (g cell/g substrate)
ΔHc - Heat of combustion of cells (kJ/g cells)
1/YH - Metabolic heat evolved per gram of cell mass produced (kJ /
g cells)
The above equation can be rearranged to yield:

 ΔHs and ΔHc can be determined from the combustion of substrate and cells.
 Typical ΔHc values for bacterial cells are 20 to 25 kJ/g cells.
 Typical values of YH are glucose, 0.42 g/kcal; malate, 0.30 g/kcal; acetate, 0.21 g/
kcal; ethanol, 0.18 g/kcal; methanol, 0.12 g/kcal; and methane, 0.061 g/kcal.
 Clearly, the degree of oxidation of the substrate has a strong effect on the amount
of heat released.
 Metabolic heat released during fermentation can be removed by circulating
cooling water through a cooling coil or cooling jacket in the fermenter.
 Temperature control (adequate heat removal) is an important limitation on
reactor design.
 The ability to estimate heat-removal requirements is essential to proper reactor
design.
 Lecture 11

Stoichiometry of microbial growth and product formation -

Introduction - Definitions - Stoichiometric Calculations
TB-1: 207-211
Cell growth complex Pseudo
Overall kinetics & stoichiometry of 1000’s of intracellular rxns
Product formation chemical eqns

Chapter  forming eqn. & useful estimates of key yield coefficients

Cell mass yield

Substrates Product yield
Heat evolution

Yield coefficient important ATP consumption

Maintenance coefficient O2

ATP yield coefficient YX/ATP Amount of biomass synthesized per mole of ATP generated
• For many S, YX/ATP = constant
@ 10-11 g dry/mole of ATP heterotrophic growth (anaerobic cdns.)
• For Autotrophic organisms, YX/ATP = 6.5
• Under aerobic cdns., YX/ATP = > 10.5
Maximum theoretical
value
• Maintenance coefficient

- “apparent” yield of biomass

mATP - rate of ATP consumption for maintenance energy

- Maximum energy when there is no maintenance energy is required

• Yields based on oxygen consumption

YX/O2 = 0.17 to 1.5 g biomass/g O2, depending on substrate and organism

 Respiratory quotient

moles of CO2 produced to moles of O2 consumed

 indication of metabolic state

 Example: aerobic growth vs ethanol fermentation in baker’s yeast

 Used in process control

 YP/O

 Ratio of phosphate formed / O2 consumed

indication of conversion efficiency of reducing power into high energy P

bonds (respiratory chain);
• For eucaryotes YP/O  3, Glucose as S

• For procaryotes YP/O - lesser

 YH/O

 Ratio of no. of H+ ions released / unit of O2 consumed

 e- generation  H release

 e- generation/O  normally 4
 e- generation results expulsion of H+  drive the transport of some S or
ATP generation
 Mass and Energy balance

• Becomes simple some cell growth parameters are same

• Irrespective of species or substrates involved Regularities (parameters)
• For example
 = 10. 5 g dry wt/ mol ATP (from growth parameters table)
• Three important regularities (I. G. Minkevich and V. K. Eroshin)
 26.95 kcal/g equivalent of available e- transferred to O2

 4.291 g equi. of available e- per quantity of biomass containing 1 g atom C

 0.462 g C per gram of dry biomass
• YX/e- = 3.14 ± 0.11 g dry wt/g equivalent of electrons

• Observed Avg. values  facilitate estimates of other growth-related parameters

Thank You
 Lecture 12

Elemental Balances - Degree of Reduction - Theoretical

Predictions of Yield Coefficients
TB-1: 212-216
 Stochiometric calculations
• Elemental Balances
 Material balance on biological rxns.  composition of S, P & X

• Determination of X - difficult  varies with organism types

SXP

SX
+
O2
+
NH3
• Balancing - complicated
 Stochiometric coefficient  elemental balances + e-/p+ balances
(based on regularity values)
 Stochiometric calculations
• Typical cellular composition CH1.8O0.5N0.2
• 1 mole of biological material amount containing 1 g atom of C CHαOβNδ
• Simple biological conversion :No extra cellular pdts - only H2O & CO2

• 1 mole of carbohydrate; 1 mole of cellular materials; Elemental balances

m, n, α, β, δ  reaction variables (Known)
a, b, c, d e  unknown variables
(stochiometric coefficients)

• 5 variables + 4 equations  to obtain values for 5 variables, one eqn. - required

• 5th eqn.  Respiratory quotient
Data on Elemental Composition of Several Microorganisms
 Degree of Reduction (γ)
• Complex rxn.  extracellular pdts.  add. stoc. coeff. more info. needed
• γ : no. of equivalents of avg. e-/g atom C
• Used for e-/p+ balances
• Available e- transferred to O2 upon oxidation to CO2, H2O, NH3
• γ  C=4+; H=1; N=-3; O=-2; P=5 and S=6
• γ =valence; outer orbit e-; + donates or - accepts
• Calculation of γ for S
• Methane (CH4)
1(4) + 4(1) = 8; γCH4 =8/1 = 8
• glucose (C6H12O6)
6(4) + 12(1) + 6(-2) = 24; γC6H12O6 =24/6=4
• ethanol (C2H5OH)
2(4) + 5(1) + 1(-2) = 12; γC2H5OH =12/2=6
•
γ & Wt. of 1 C equivalent of 1 mole of some S & X
 Degree of Reduction (γ)
• Aerobic production of single extracellular product

• γs = 1(4) + m(1) + n(-2)  γs = 4 + m - 2n

• γb = (4) + α(2) + β(-2) + δ(-3)  γb = 4 + α - 2 β – 3δ

• γp = (4) + x (1) + y (-2) + z (-3)  γp = 4 + x – 2y – 3z

• CO2 , H2O, NH3 γ =0

• From the above eqn.  c  d  f 1; cα + dz = b; cγb + dγp = γs - 4a

• Partial experimental data, RQ, Y [c = YX/S; d= YP/S] on molar basis

Energy balance for aerobic growth
Q0 = heat evolved / equivalent of avg. e- transferred to O2 (26.95 kcal/g)
Y  c Yb + d Yp = Ys – 4a
Q0 c Yb + Q0 d Yp = Q0 Ys – Q04a

• Rearranging 

• Fractional allocation of av. e- or energy for an organic S

  fraction of av. e- in org. S ie. transferred to O2
b  fraction of av. e- ie. incorporated into X
p  fraction of av. e- ie. incorporated into P
 Theoretical Predictions of Yield Coefficients
• Aerobic fermentation growth in O2

• Yx/e- = 3.14±0.11 g dw/e- (NH3 as N source)

• No. of av. e-/O2 mole 4; O2/S is known YX/S
• e+ in 1 mole of glucose is 6x4=24;
• Cellular yield in av. e- YX/S = 24 X 3.14 = 76 gdw cells / g glucose
• YX/S=76/180=0.4 gdw/mol (Table 6.1 0.38-0.51)
• Fermentation : YX/ATP = 10.2±2 gdw/mol ATP (anaerobic); 6-29 (aerobics)
• mol of ATP/g of S known  YX/S = YX/ATP N
 Example 1

Assume that experimental measurements for a certain organism have

shown that cells can convert two-thirds (wt/wt) of the substrate carbon
(alkane or glucose) to biomass.
a. Calculate the stoichiometric coefficients for the following biological
reactions

b. Calculate the yield coefficients YX/S (g dw cell/g substrate), YX/O2 (g dw

cell/g O2) for both reactions. Comment on the differences.
a. Stoichiometric coefficients

For hexadecane,
amount of carbon in 1 mole of substrate  16(12)  192 g
amount of carbon converted to biomass  192(2/3)  128 g
Then, 128  c(4.4)(12); c  2.42.
amount of carbon converted to CO2  192  128  64 g
64  e (12), e  5.33
The nitrogen balance yields
14b  c(0.86)(14)  b  (2.42)(0.86)  2.085
The hydrogen balance is
34(1)  3b  7.3c  2d  d  12.43
The oxygen balance yields
2a(16)  1.2c(16)  2e(16)  d(16)  a  12.427
For glucose,
amount of carbon in 1 mole of substrate  72 g
amount of carbon converted to biomass  72(2/3)  48 g
Then, 48  4.4c(12); c  0.909.
amount of carbon converted to CO2  72  48  24 g
24  12e; e  2
The nitrogen balance yields
14b  0.86c(14) ; b  0.782
The hydrogen balance is
12  3b  7.3c  2d ; d  3.854
The oxygen balance yields
6(16)  2(16)a  1.2(16)c  2(16)e  16d ; a  1.473
b. yield coefficients

• For hexadecane,

• For glucose,

• Growth yield on more reduced S (hexadecane) > that on partially oxidized S

(glucose), assuming that 2/3 rd all the entering C is incorporated in cellular
structures
• O2 yield on glucose > O2 yield on hexadecane  since glucose is partially oxidized
 Example 2

• Estimate the theoretical growth and product yield coefficients for ethanol
fermentation by S. cerevisiae as described by the following overall
reaction
Since YX/ATP  10.5 gdw/mol ATP and since glycolysis yields 2 ATP/mol of glucose in
yeast,

or
YX/S  0.117 gdw/g glucose
For complete conversion of glucose to ethanol by the yeast pathway, the maximal
yield would be
YP/S = 2(46 )/180 = 0 .51 g ethanol /g glucose
while for CO2 the maximum yield is
YCO2/S = 2(44)/180 = 0.49 g ethanol /g glucose

In practice, maximal yields - not obtained

Product yields - 90 to 95% of the maximal values, because the glucose is converted
into biomass and other metabolic by-products (e.g., glycerol or acetate).
Thank You
 Lecture 13

Models of Microbial Growth - Quantifying Growth Kinetics -

Introduction, - Using Unstructured Nonsegregated Models to
Predict Specific Growth Rate
TB-1: 175-182
 Quantifying Growth Kinetics
• Const. cell comp. & balanced growth  exp phase; other phases
unbalanced growth and varying cell composition
• Recognition of structured nature of cell & segregation of culture into cells
• Models
• Structured & Segregated (realistic & computationally complex)
• Structured & Nonsegregated;
• Unstructured & Segregated;
• Unstructured & Nonsegregated
• Simplicity and initiation  unstructured & nonsegregated models for μ
prediction - Practical
 Using unstructured Non segregated models to
predict specific growth rate
Substrate limited growth
• S – growth rate limiting; other nutrients – no effect
• Langmuir–Hinshelwood or Hougen–Watson  chemical kinetics
• MM  enzyme kinetics
• Monod  Kinetics on cellular systems
μg = (μm S/Ks + S)
• μm – Max. specific growth rate when S>>KS
• Ks – saturation const./half vel. const.  conc. of rate-limiting S (Ks = S ),
when μg = ½ μm
• μg = μm @ S>>KS; μg = μm/KS @ S<<KS
• Monod  semiempirical; suits S limiting, growth and population low
Effect of nutrient concentration on the specific growth rate of E. coli.
• Higher Carbon & Energy consumption.  population rapid, buildup of toxic
metabolic wastes (due to energy spilling rxns.)
• For rapidly growing dense cultures
μg = (μm S) / (Ks0 S0 + S)
μg = (μm S) / (KS1 + Ks0 S0 + S)
• Equations - Alternate to Monod
Better than Monod –
• Blackman eqn. μg = μm if S ≥ 2KS troublesome in
applications
μg = μm S / (2 KS) if S < 2KS
2 constants μm, K
• Tessier eqn.  μg = μm (1 - e-KS)
3 cont. μm, Ks, n
• Moser eqn.  μg = (μm Sn) / (KS+Sn)= μm (1+ KSS-n)-1 n = 1  Monod
μg  X-1
• Contois eqn.  μ = (μ S) / (K X + S)
• Equations  Single differential. eqn.
d/dS = Ka (1-)b
=μg/μm
K, a, b - constants

• More than 1 growth limiting S unresolved  noninteractive S  least μg (Si)

Constants of the Generalized Differential Specific Growth Rate Equation for Different
Models
 Models with growth inhibitors

• At high [S] or [P] & presence of inhibitory substances in the medium 

growth - inhibited, & growth rate -inhibitor concentration
• Microbial growth inhibition pattern  enzyme inhibition
• If a single-substrate enzyme-catalyzed reaction  rate-limiting step in
microbial growth
• Kinetic constants - biologically meaningful
• Underlying mechanism  complicated
• Kinetic constants - do not have biological meanings & curve fitting -
experimental data
1. Substrate inhibition
• Noncompetitive substrate inhibition

or if KI >>> Ks

• Competitive substrate inhibition

• Alleviated by slow, intermittent addition S to growth medium

2. Product inhibition
• Noncompetitive product inhibition

• Competitive product inhibition

• Ethanol inhibition (non competitive PI)

or

• Pm - [P] @ growth stops or

KP – Product Inhibition constant

3. Inhibition by toxic compounds
Competitive inhibition

Noncompetitive inhibition

Uncompetitive inhibition

• In presence of toxic compounds in medium  inactivation of cells or death

• Net specific rate in the presence of death

k’d - death-rate constant (h-1)

Logistic equations
Rel. bet microbial growth yield and [S]
μg = (μm S/Ks + S)
dX/dt = YX/S
No endogenous metabolism
dX/dt  X  dX/dt  μg X
S = {(YX/S So) + Xo – X}/ YX/S

• Sub S in dX/dt 

• Int. form of rate expressions

• Sigmoidal shaped growth curve, X asymptotically reaches YX/S S0 + X0

• Max cell mass X∞ identical to ecological concept of carrying capacity (Max [P])
• Logistic equations are eqn. characterize growth - carrying capacity
• Usual approach μ in terms of unused carrying capacity

• Int. with boundary cond. X(0)=X0  logistic curve

Growth models for filamentous organisms
• Filamentous organisms (molds) – suspension culture
• Filamentous cells grow on moist solid surface  complicated process  growth
kinetics + diffusion of nutrients and toxic metabolic by-products
• Absence of Mass – transfer limitations
• Radius growth in submerged culture or mold colony on agar surface – Increases
with time
dR/dt = kp = constant
• In terms of growth rate of a mold colony

or   kP(36)1/3

• Integration 

• M0  M  M varies with t3
Thank You
 Lecture 14

Models for Transient Behavior - Cybernetic Models

TB-1: 183-189
 Models for Transient Behavior

• Models with time delay

• Ex. pH increases

• Chemically structured models

 Models with time delay

• Time dependent change in cellular composition / biosynthetic capabilities

/ product formation
• Variation in environment  Product formation – time delay
• Ex. atm. T  high at 1 or 2 pm but not at 12 (noon)
• Improved models for dynamic situations  addition of time delay
Comparison of predictions from a model derived from a system-analysis perspective,
predictions from a Monod model, and experiment
 Chemically structured models

• Predictive power of model

• capturing the important kinetic interactions among cellular
subcomponents

• Initially based on two, then three now 20 to 40 subcomponents are

used

• Understanding of detail physical system  two guidelines

First - Intrinsic Concentration
• Intrinsic concentration  (compound / cell mass (or) cell volume)
• Kinetics  not extrinsic concentration  reactor V (complexity)
Second  dilution of intrinsic C by growth

E Coli., growth in glucose Am media one limiting; Rounds DNA and formations
Solid line - material flow; dotted - information; W - waste; A1 NH3, A2 glucose
VR – Reactor total volume
X - extrinsic biomass concentration
Ci - extrinsic concentration of component i
  A

In terms of Intrinsic concentrations ( Ci / X )

• Sub 
 B

•  VR in eqn. A (assume VR – constant) 

• Sub rfi in eqn. B
 Cybernetic models
• Growth under more substrate  Cybernetic models
• Substrate  complementary(C/N source) / substitutable (glucose and
lactose – both supply C/E)
• Cybernetic (goal seeking approach  maximize growth rate)
 Predicting culture growth on a substitutable C source
 Identifying regulatory of a complex biochemical reaction network
structure (such as cellular metabolism)
• Maximization objective oriented mathematical analysis – economic
analysis for resource distribution
• Describes culture growth (components) on a complex medium
• Limitations : objective fn. for any organism – maximize species long-term
survival
• Subobjectives : Maximize growth rate or growth yield - dominate under
some environmental conditions
Thank You
 Lecture 15

Continuous Culture - Introduction, - Some Specific

Devices for Continuous Culture - The Ideal Chemostat
TB-1: 190-196
 How Cells Grow in Continuous Culture

• Batch culture
one time input
change of culture environment

[S]  & [P]  with time

• Continuous culture
μ, P formation maintained; steady state – X, P, S  const env.
Important tool to determine the response of micro - organisms to
their env. and to produce desired products under optimal env. cdns.
 Specific devices

• Chemostat
• Turbidostat

• Plug flow reactors

 Chemostat

• Cellular growth - limited

by one essential nutrient
& other nutrients are in
excess
• @ Steady state 
Nutrient, Product & Cell
concentrations -
constant.  constant
chemical environment
Continuous-culture laboratory setup
 Turbidostat
• Constant cell concentration Monitoring culture OD
• If turbidity exceed limit pump activated & fresh medium addition
• Constant culture volume  removing equal amt. of culture fluid
• Less used than chemostat more elaborate, dynamic environment, study
on env. stress (ex. High etOH conc.) – cell conc. maintained constant

Laboratory setup
 1. Reservoir -sterile medium
2. Valve controlling flow of medium
3. Spent medium - outflow
4. Photocell
5. Light source

Chemostat Turbidostat
 Plug Flow reactor (PFR)
• No back mixing
• Fluid elements - active cells cannot inoculate other fluid elements @ diff. axial positions
• Liquid recycle required  Continuous inoculation of nutrient media
• [S] and [X]  varies with axial positions
• Ideal PFR - batch reactor  distance along the fermenter replaces incubation time
• Waste treatment - PFR behavior  Multistage chemostats  PFR dynamics (No. of
stages  5)
 Ideal Chemostat
• CFSTR – continuous flow, stirred tank reactor
• Control elements  pH, DO
• Feeding sterile medium and cell suspension removal @ same rate
• Constant liquid V
Material balance – cell conc.

F - flow rate of nutrient solution, l/h

VR - culture volume, l (assumed constant)
X - cell concentration, g/l
g - growth rate constant, h-1
kd - endogenous (or death) rate, h-1
• Balances  cell number; Influence of kd  substrate balance equation
• Most experiments  measuring total cell mass rather than No.
• Rearranging eqn based on [X]

D - dilution rate ; D = F/VR. D - reciprocal of residence time

• At steady state dX/dt = 0; initial sterile as X0 = 0; kd<<μg μg = D ( kd = 0)
• μg - limited by at least one substrate in a chemostat  performance Monod eqn.

 identical to MM kinetics - 1/ μg Vs 1/S  μm & Ks values

• if D> μm  culture wash out

• D< μm 
Material balance - limiting S - absence of endogenous metabolism

S0 & S - feed and effluent [S], g/l

qP - specific rate of extracellular product formation, g P/g cells h
YMX/S - max. yield coefficient, g X/ g S & YP/S - yield coefficient, g P/g S

Extracellular product formation - negligible & system @ dS/dt = 0

g = D at steady state if kd = 0 
Sub. 
• dX/dt = 0; X0 = 0
D = g – kd  net or g  D  kd

• Sub g value in

YMX/S – Max. yield coeff. (no endogenous metabolism or maintenance energy

 Rearranged

or

ms - maintenance coefficient based on S

YAPX/S - apparent yield
YMX/S – constant
YAP – varies with growth conditions if k > 0
 Presence of endogenous metabolism, net  m S/(Ks  S)  kd

and

In addition - maintenance effect, consider extracellular S  extracellular P conversion

Unstructured model - conversion as instantaneous
Otherwise - perceptible period of S uptake, prior to P conversion  change in X amt.
Unlike maintenance, period b/w S uptake and use for maintenance fxns. - long,
allow S to become incorporated into X
 X - degraded when needed
Assume instantaneous conversion of S to extracellular P, cell – catalyst
Product formation balance:
For nongrowth-associated P formation, qP - constant ()
For growth-associated P formation, qP – fxn of g
Substrate balance:
• Biomass balance - unchanged - case with endogenous metabolism

• Solving eqn for X 

• Productivity of a chemostat – P & X. (Prx) - DP & DX

• Dilution rate - maximizes productivity  diff. DP or DX w.r.t D & = 0

• Dopt - whether endogenous metabolism and/or P formation

• When kd = 0 and qP = 0,

• Dopt for biomass production (DX) 

• S0 >> Ks, Dopt  D = m or washout pt

• Stable chemostat operation with D  mm - very difficult, unless flow rate & liquid V – maintained Const.
• D < Dopt - good compromise between stability and biomass productivity
Thank You
 Lecture 16

The Chemostat as a Tool - Deviations from Ideality

TB-1: 197 - 200
 Chemostat as a Tool

• Tool  study on mutation, selection of cultures, effect of env. change on cell

physiology
• Natural or induced mutation [d(gene str.) – new char or trait than parental]
• Errors in DNA replication : Avg. F: 10-6 – 10-8 gene per generation
• [cell] : 109 cells/ml culture  probability is high  variety of mutant cells form
• Natural mutation  little sig.  unless alters f(protein) involved in growth
• Chemostat  selection of special organisms
 selection or enrichment nutrient media
• Organisms growing on etOH  etOH + mineral salt medium intro
• Toxic refractory compounds oxidizing organisms, Thermophillic organism
 Deviation from Ideality

• Fermentations  X complete mixing

• Utilization and pdn. of polysaccharides or – viscous broth, Form of film
growth on solid surfaces
• Segregated reactor model is useful
• Ex. A two compartmental model  well mixed region and stagnant region
 Simple 2 compartmental model for an incompletely mixed chemostat

• Imperfectly mixed chemostat - stagnant biotic phase, dilution rate > m without washout
• Washout dilution - setting S0 = S1 = S2
• Washout dilution rate
Thank You
 Lecture 18

Bioreactor Design - Choosing the Cultivation Method -

Modifying Batch and Continuous Reactors - Chemostat
with Recycle
TB-1: 245-249
 Why bioreactor design important?
• Unlike - traditional chemical processes
• reactor section - major component
• Usually > 50% of total capital cost

• Reactor choice
• Biocatalyst selection
• Culture - choice
• Separation
• Purification train
 Choosing cultivation method
• batch or continuous cultivation
• Productivity - production of cell mass or a primary product / time
• Batch reactor - four distinct phases
• lag phase,
• exponential growth phase,
• harvesting,
• preparation for a new batch (e.g., cleaning, sterilizing, and filling)
• tl = (lag time + harvesting time +preparation time)
• Normally - tl = 3 to 10 h
• Total cycle time, tc = t + tl =
Xm - maximal attainable cell concentration

X0 - cell concentration at inoculation

Cell mass production - batch
• Total amount of cell mass produced  total amount of growth-extent-limiting
nutrient present and its yield coefficient

• Rate of cell mass production in one batch cycle

Productivity = (Xm – Xo)/ tc

Cell mass production – continuous (chemostat)
• Maximum productivity  differentiating DX with respect to D and setting dDX/dD
to zero

• Best productivity - Monod kinetics Dopt x Xopt =

Yc =

When S0 >> KS


• Ratio for rates of biomass formation 

Commercial fermentation

• Xm/X0 =10 - 20

For E.coli.

• Xm/X0 = 20

• tl = 5 h,

If μm=1 h-1

rc,opt/rb = ln (20) + 5 = 8
 Why commercial processes are batch?
• Growth associated products

2o pdt. productivity - not made by growing cells  G represses pdt formation

• Productivity (2o pdt.) : batch > chemostat
• Genetic instability – Chemostat – Mutation (less prod. than parental strain)
• Operability and reliability
in batch - Pdt variability - in downstream processing - undesirable
Problems in long terms conti.culture (sterility, pump/control failure)
• Market economics
cont. dedicated processing (single)
batch – flexibility (matches lesser qnty. reqt. i.e. common)
 Modifying batch and continuous reactor
• Higher the cell conc. higher the rate of conversion  cell recycle

Cell recycle
Cell separator – sedimentation
Centrifugation
Micro filtration

Chemostat with recycle

Material balance :

 - recycle ratio based on volumetric flow rates

C - concentration factor or ratio of [cell] in cell recycle stream to [cell] in reactor effluent
F - nutrient flow rate
V - culture volume
X0 and X1 - [cell] in feed and recycle streams
X2 – [cell] in effluent from cell separator
• Steady state dX1/dt=0, X0=0

• Since C >1, α(1-c)<0, μnet < D i.e. chemostat can be operated at D > μnet recycle
• Material balance – growth-limiting substrate

• @ Steady state, dS/dt=0

Sub kd=0 

• Steady state cell conc. increased by factor of (1+α-αC)

• S in effluent, Monod kinetics and endogenous metabolism is neglected

• Sub. S in X1
Thank You
 Lecture 19

Multistage Chemostat Systems - Fed-batch Operation -

Perfusion Systems

TB-1: 250-262
 Multistage chemostat systems
• Fermentation
• Secondary metabolite pdn. - Growth & pdt. formation –
separate – optimal cdns. – different
• Cdns. – T, pH, limiting Nutrients – each stage - varied 
multistage systems
• Structured models – accurate
• growth in 2nd & subsequent stage
• Unstructured models – simplicity
 2 stage chemostat system
• Biomass and substrate balances – 1st stage

• Biomass balance – 2nd stage

@ steady state ( V2 & equate to 0)

1st stage 2nd stage
Production 2o metabolites
D2X1 – D2X2 + 2 X2 = 0 (F/V = D)

2 = D2 (1 – X1/X2) , where X1/X2  1 and 2  D2

Substrate balance - limiting substrate – 2nd stage

@ steady state ( V2 & equate to 0)

D2S1 - D2S2 - 2 X2 /YMX/S = 0

where D2 = F/V2 and

To solve X2 and S2

 Sub in and
• Feed stream - added to 2nd stage
• 2nd feed stream - additional nutrients, inducers, hormones, or inhibitors
Biomass balance – 2nd stage

@ steady stage , when X’ = 0

where &

Substrate balance – 2nd stage

Eqn. 2 & S solved  X2 & S2

 Fed batch operation

• In fed-batch culture
• nutrients are continuously or semi continuously fed
Repeated fed-
• effluent is removed discontinuously batch culture

Substrate inhbition

Schematic of a fed-batch culture

• Batch culture – [biomass] @ time

In batch, S <<< S0  0 and Xo  X

Any particular time

Xt  X V
X  Xt / V V – culture volume at time t

Rate of increase in culture volume

dV/dt  F F – flow rate
F/V  D
• Rate of change in biomass concentration

(Quotient Rule of differentiation)

dXt / dt = net Xt ; dV/dt  F;

Sub. dXt / dt ; dV/dt and D in dX/dt

dX/dt  [V net Xt - Xt F ] / V2

Sub Xt  X V
dX/dt  [(V net X V) / V2]- [(X V F) / V2]

Sub F/V  D
• When S – totally consumed, S  0 and X  Xm  YMX/S So

• S in unit V – consumed  dX/dt  0

Quasi steady state  Nutrient feed  nutrient consumption
net  D

If maintenance energy – neglected

Balance on the rate-limiting substrate without maintenance energy

St - total amt of the rate-limiting S in culture

dSt / dt = 0 (no change in [S])

IIIly biomass concentration

dXt/dt = Xm (dV/dt)

= Xm F

= YMX/S So F

Integrating

 dXt/dt  YMX/S So F

 dXt = YMX/S So F dt

Xt  Xot  YMX/S So F t 
Product formation

Xt = V Xm; V = V0 + Ft

dPt = qp Xm (Vo + Ft) dt

dPt = qp Xm Vo  dt + qp Xm Ft  dt

Pt - Pot = qp Xm [Vo  dt + F  t dt]

= qp Xm [Vo t + F t2 /2]
 Fed-batch culture @ quasi-steady state

In terms of [P]

In some fed batch operations – part of

culture V – removed @ certain intervals (b) Variation of [P] with t
(reactor V – limited)  repeated fed
batch culture

(a) Variation of (V ) ,  , [X] & [S] with t

Cycle time, tw = constant @ quasi steady state
[P] @ end of each cycle

Dw = F/Vw,
Vw - culture volume at the end of each cycle
V0 - residual culture volume after removal
 - fraction of culture volume remaining at each cycle = V0/Vw

Sub tw in Pw 
 Perfusion system
• Alternate to fed batch
• Mostly for animal culture
• Constant medium flow, cell retention (membrane (immobilized) / screen /
centrifuge),
• Selective removal of dead cells
 Pros
Cons

• Removal
• Handling large amount of medium
• Cell debris
and nutrients
• Inhibitory by-products
• Expensive
• Enzymes
• Dead cells • Sterilization – maintenance

• High per-unit volumetric productivity

Thank You
 Lecture 20

IMMOBILIZED CELL SYSTEMS

 Immobilization of cells is almost as common as enzyme immobilization.
 Immobilization → restriction of cell mobility within a defined space.
 Immobilized cell cultures have the following potential advantages over suspension
cultures.
 Provides high cell concentrations.
 Provides cell reuse and eliminates the costly processes of cell recovery and cell
recycle.
 Eliminates cell washout problems at high dilution rates.
 The combination of high cell concentrations and high flow rates allows high
volumetric productivities.
 Provide favorable micro-environmental conditions (cell–cell contact, nutrient–
product gradients, pH gradients) for better performance of the biocatalysts.
 Improves genetic stability.
 Protection against shear damage

 The primary advantage of immobilized cells over immobilized enzymes is that

immobilized cells can perform multistep, cofactor-requiring, biosynthetic reactions
that are not practical using purified enzyme preparations.
Active Immobilization of Cells
 Active immobilization is entrapment or binding of cells by physical or chemical
forces.
 The two major methods → entrapment and binding.
 Physical entrapment within porous matrices is the most widely used method of
cell immobilization.
 Various matrices can be used for the immobilization of cells.
 Porous polymers (agar, alginate, κ -carrageenan, polyacrylamide, chitosan, gelatin,
collagen), porous metal screens, polyurethane, silica gel, polystyrene, and
cellulose triacetate.
 Polymer beads should be porous enough to allow the transport of substrates and
products in and out of the bead
They are usually formed in the presence of cells and can be prepared by one of the
following methods:
1. Gelation of polymers
2. Precipitation of polymers
3. Ion-exchange gelation
4. Polycondensation
5. Polymerization

Encapsulation and macroscopic membrane-based reactors is another method of cell

entrapment.
Cell immobilization using entrapment on different support material

Cell Immobilization by Surface Attachment

 Immobilization of cells → surfaces materials→ physical adsorption or covalent
binding.
 Adsorption →advantage → direct contact between nutrient and support materials.
 The control of micro-environmental conditions is a problem in porous support
materials.
 A ratio of pore to cell diameter of 4 to 5 is recommended.
 At small pore sizes→ accessibility of the nutrient into inner surfaces of pores
(limiting factor).
 Optimal pore size →maximum rate of bioconversion.
 Adsorption capacity and strength of binding → two major factors that affect the
selection of a suitable support material.
 The adsorption of cells →covalent bonding, H bonds, or van der Waals forces.
 Adsorption → simple and inexpensive method of cell immobilization.
 Limited cell loadings and rather weak binding forces reduce the attractiveness of
this method.
 Hydrodynamic shear around adsorbed cells should be very mild to avoid the
removal of cells from support surfaces.
 Functional groups on cell and support material surfaces are not usually suitable for
covalent binding.
 Binding surfaces need to be specially treated with coupling agents (glutaraldehyde
or carbodiimide) or reactive groups for covalent binding.
 Covalent binding forces are stronger than adsorption forces, resulting in more
stable binding.
 The direct cross-linking of cells by glutaraldehyde to form an insoluble aggregate is
Passive Immobilization: Biological Films
 Biological films are the multilayer growth of cells on solid support surfaces.
 The support material can be inert or biologically active.
 Biofilm formation is common in natural and industrial fermentation systems,
such as biological waste-water treatment and mold fermentations.
 The interaction among cells and the binding forces between the cell and
support
material may be very complicated.
 In mixed-culture microbial films, the presence of some polymer-producing
organisms
facilitates biofilm formation and enhances the stability of the biofilms. Micro-
environmental conditions inside a thick biofilm vary with position and affect the
physiology of the cells.
 In a stagnant biological film, nutrients diffuse into the biofilm and products
diffuse
out into liquid nutrient medium. Nutrient and product profiles within the biofilm
are
important factors affecting cellular physiology and metabolism.
 The thickness of a biofilm is an important factor affecting the performance of the
biotic
phase. Thin biofilms will have low rates of conversion due to low biomass
concentration,
and thick biofilms may experience diffusionally limited growth, which may or may
not be
beneficial depending on the cellular system and objectives.
 Nutrient-depleted regions may also develop within the biofilm for thick biofilms. In
many cases, an optimal biofilm thickness resulting in the maximum rate of
bioconversion exists and can be determined.
 In some cases, growth under diffusion limitations may result in higher yields of
products as a result of changes in cell physiology and cell–cell interactions.
 In this case, improvement in reaction stoichiometry (e.g., high yield) may overcome
the reduction in reaction rate, and it may be more beneficial to operate the system
under diffusion limitations.
 Usually, the most sparingly soluble nutrient, such as dissolved oxygen, is the rate-
limiting nutrient within the biofilm.
Diffusional Limitations in Immobilized Cell Systems
 Immobilization of cells may cause extra diffusional limitations as compared to
suspension cultures.
 The presence and significance of diffusional limitations depend on the relative
rates of bioconversion and diffusion, which can be described by the Damköhler
number (Da)

rmax - maximum rate of bioconversion (mg S/l h), De - effective diffusivity

of the rate-limiting substrate, δ - thickness of diffusion path (or liquid film), and S0 -
bulk substrate concentration in liquid phase.
 When the film-theory model applies, De/δ - mass transfer coefficient (i.e., kL =
De/ δ).
 If Da >> 1, the rate of bioconversion is diffusion limited;
Da << 1, the rate is limited by the rate of bioconversion; and for Da =1, the
diffusion
and bioreaction rates are comparable.
 It is desirable to keep Da < 1 to eliminate diffusion limitations when the
productivity of a cell population does not improve upon immobilization due to
cell–cell contact and nutrient gradients.
 Diffusional limitations may be external, intra particle or both.
 If the external mass transfer is limiting, an increase in liquid-phase turbulence
should result in an increase in the reaction rate.
 In case of intra particle mass-transfer limitations, a reduction in particle size or an
increase in the porous void fraction of the support material should result in an
increase in the rate of the bioreaction.
 The thickness of a biofilm or the size of microbial floc increases with time during
the growth phase.
 A microbial floc is an aggregation of many cells, and these aggregates can be
more than 1 mm in diameter.
 Since the rate of increase in biofilm thickness is much slower than the rate of
substrate uptake, the system can be assumed to be at quasi-steady state for
relatively short periods.
 The simplest case is to assume that the system is at quasi-steady state and all
the cells inside the biofilm are in the same physiological state.
 In this situation we write a steady-state substrate balance within the biofilm by
using average kinetic constants for the biotic phase (living cells).
 Lecture 21

Mass transfer – Molecular diffusion – theory – role of

diffusion in bioprocess – Film theory – convective mass
transfer
TB-2: 190-194
 Mass transfer

• Transfer of mass from higher conc. to lower:

• Oxygen  Air bubble to bulk liquid (gas-liquid interface)
• Penicillin  aqueous to organic solvents (butyl acetate)

• Liquid - solid  nutrients (liquid) to pellets, flocs or

films of cell or enzyme
 Molecular diffusion
• Movement of compound molecules due to conc. difference
• Direction required to destroy the concentration gradient
• High to low  diffusion -continuous
 Diffusion theory
• Binary mixtures  mixtures or solutions – 2 components
• Closed system – Molecule components A & B
• CA  CA1 to CA2; distance y

 No large scale fluid motion

 Mixing solely by molecular movement
 Fick’s law of diffusion
• Mass flux α Conc. Gradient

• JA - Mass flux
• NA - Mass transfer rate
• D AB - Binary diffusion coefficient- diffusivity of A in mixture of A & B
• dCA/dy - Concentration gradient or change in conc. of A with distance

Mass flux
• Rate of mass transfer/unit area ┴ to the direction of movement
• gmol/s/m2
• D depends on A, B, T. Gases  P; Liquid  approx. linear to conc.
 Analogy b/w Mass, heat & momentum transfer
• Flux α to driving force with prop. const (physical quantity)

• Mass flux α conc. difference 

• Heat transfer α temp. difference 

• Mass flux α velocity. Difference 

- Sign  J, q, T transfer always opposite to the direction of increasing C, T or V

 Role of diffusion in bioprocessing
• Scale of mixing
• Turbulence produces bulk mixing scale equal to smallest eddy size

• Within eddies streamlined flow mixing by mole diffusion (final step)

• Solid phase reaction

• Catalyst (solid) mediated reactions immobilization

• Unassisted bulk fluid convection  decides rate of reaction

• Mass transfer across phase boundaries

• O2 transfer, penicillin etc.
 Film theory
• Two film theory (usual model for MT b/w phases)
• Bulk to phase boundary  interface to II phase
• 2 immiscible liquids (water + chloroform / benzene)
- each phase is well mixed & turbulent flow
• Thin film – no turbulence, relatively stagnant fluid,
MT by mole diff only
• Assume  No resi. in bulk liq. & neg. resi. to
transport in the interface
• Diff. b/w CA1i and CA2i due to A’s solubility more in
any one of the phases
• Acetic acid in water and chloroform; CA1i higher by
5-10 than CA2i
 Convective mass transfer
• MT in the presence of bulk fluid motion

Conc. gradient  mole. diffusion

• Bulk fluid movement  over all MT rate higher due to convective currents

• Analysis of MT in multiphase system is important because resistance in

interfacial boundary layer is significant

Transfer rate α transfer area x driving force

Transfer rate = MT coeff. x transfer area x driving force

MT coefficient – proportionality constant

• Rate of mass transfer to a phase boundary

Rm - resistance to mass transfer

 Mass transfer situations

• Liquid – solid mass transfer

• Liquid – liquid mass transfer between immiscible solutions

• Gas – liquid mass transfer

 Liquid-solid Mass Transfer
• Transport of substrate to solid-phase cell or enzyme
catalyst; adsorption of mole onto surface 
chromatography like
• Near interfacial area, fluid vel ↓ and boundary layer
develops
• A is consumed at the surface
• C reduces  develops conc gradient - brings A from
liquid
NA= kLa (CAb – CAi)
kL – liquid phase MT coeff.
CAi estimation more difficult - correlated with conc. of A in
solid phase or reaction rate
Thank You
 Lecture 22

Liquid- liquid, gas-liquid and solid- liquid – Cellular

oxygen demand – Estimating demand – Mass transfer
correlations – Measurement of kLa – Methods

TB-2: 194-198; 210-213

 Liquid-liquid mass transfer
• Immiscible solvents  product
recovery stages of bioprocessing

• Organic solvents  isolate

antibiotics, steroids and alkaloids
– fermentation broth

• 2 phase aqueous system –

protein purification

• Eg. Pdn. of microbial biomass –

single cell protein
• I interface  NA1  kL1 a (CA1 - CA1i) • II interface  NA2  kL2 a (CA2i – CA2)

(NA1/ kL1 a)  CA1 - CA1i (NA2/ kL2 a)  CA2i – CA2

Assuming equilibrium at interface
Distribution law : CA1i/ CA2i  m
m – distribution coefficient / partition coefficient
CA1i  m CA2i ; CA2i  CA1i / m & NA1  NA2  NA

NA/ kL1 a  CA1 - m CA2i  (1) NA/ kL2 a  CA1i / m – CA2  (2)

x (1/m) xm
NA/ m kL1 a  (CA1 /m) - CA2i m NA/ kL2 a  CA1i – mCA2

NA/ m kL1 a  (CA1 /m) - CA1i / m  (3) mNA/ kL2 a  mCA2i – mCA2 (4)
• Add (1) & (4)
(NA/ kL1 a)  (mNA/ kL2 a)  CA1 - m CA2i  mCA2i – mCA2
 CA1 – mCA2  (5)
• Add (2) & (3)
(NA/ kL2 a)  (NA/ m kL1 a)  (CA1i / m) – CA2  (CA1 /m) - CA1i / m
 CA1 /m – CA2  (6)

Overall liquid phase MT coeff. KL

(5) gives NA [(1/ kL1 a)  (m/ kL2 a)  CA1 – mCA2
NA (1/ KL1a)  CA1 – mCA2  NA  KL1a [CA1 – mCA2 ]
(6) gives NA [(1/ kL2 a)  (1/ m kL1)]  CA1 /m – CA2
NA (1/ KL2a)  CA1 /m – CA2  NA  KL2a [CA1 /m – CA2]
Resistance – high  transfer – low
Based on the resistance any one of the formula can be used (a only diff to mea. k a)
 Gas – Liquid Mass transfer
• Oxygen from gas to liquid
• I interface  NAG  kG a (CAG - CAGi) • II interface  NAL  kL a (CALi – CAL)

(NAG/ kG a)  CAG – CAGi  (1) (NAL/ kL a)  CALi – CA2  (2)

CAGi/ CALi  m
m – distribution coefficient / partition coefficient
CAGi  m CALi & CALi  CAGi / m

• NAG/ kG a  CAG – m CALi • NAL/ kL a  CAGi / m – CA2

• x (1/m) • xm
• NAG/m kG a  (CAG /m) – CALi  (3) • mNAL/ kL a  CAGi – m CA2  (4)
Add (1) & (4)
(NAG/ kG a)  (mNAL/ kL a)  CAG – CAGi  CAGi – m CA2
(NAG/ kG a)  (mNAL/ kL a)  CAG – m CA2  (5)
Add (2) & (3)
(NAL/ kL a)  (NAG/m kG a)  CALi – CA2  (CAG /m) – CALi
(NAL/ kL a)  (NAG/m kG a)  (CAG /m) - CA2  (6) Overall gas-phase MT coeff. - K G

(5) gives NA [(1/ kG a)  (m/ kL a)]  CAG – m CA2

NA (1/ KG a)  CAG – m CA2
Overall liquid phase MT coeff. - KL
NA  KG a [CAG – m CA2]
(6) gives NAL [(1/kL a)  (1/m kG a)] (CAG /m) - CA2
NA (1/KL a)  (CAG /m) - CA2
 Oxygen uptake in cell culture

• NA – rate of O2 transfer per unit volume of fluid, gmol m-3 s-1

• kL – liquid phase mass transfer coefficient, m s-1
• A – gas liquid interfacial area per unit volume of fluid, m2 m -3
• CAL – O2 conc. in broth, gmol m-3
• C*AL – O2 conc. in broth in equilibrium with gas phase, gmol m-3
• (C*AL – CAL) – diff. b/w maximum possible and actual O2 conc. In liquid
 conc. Difference driving force for MT
 Problems in O2 transfer

• Very less solubility

• ↑ temperature  ↓ solubility

• Nutrients (energy/food source) in H2O – conc. more than pure H2O  solubility ↓

• Solubility of O2 in atm pr and T is  10 ppm  O2 supply 10-15 times

 Factors affecting cellular O2 demand

• Cell sp., culture growth phase, nature of C source

• Batch culture O2 uptake varies with time (O2 cons./
cell - uptake rate)
• QO = qO x (QO – g l-1 s-1; qO – g g -1 s-1)
• CCritical >CAL  qO - inherent demand of O2 for an
organism (qO) depends on biochemical nature of
cell and nutritional env.
• CCritical < CAL  linear dependant on O2 conc.
• Always maintain CAL>Ccrit
 Mass Transfer Correlation

• Calculation empirical correlation / experimental liquid-gas  kLa

• Depends on fluid properties and hydrodynamic conditions – correlation

Power dissipated, uG superficial gas vel – vol.
gas flow rate/c/s area, A, α, β const

• Viscous-non-Newtonian fluids
 Measurement of kLa

• Measurement method – match to fermenter conditions

Techniques

• Oxygen balance method

• Dynamic system (simple dynamic method)

• Sulphite oxidation method

 Oxygen balance method

V L - volume of liquid in the fermenter,

Fg - volumetric gas flow rate
CAG - gas-phase concentration of oxygen
i and o refer to inlet and outlet gas streams
PAG - oxygen partial pressure in the gas
T – absolute temperature

• Assumptions
• Liquid phase – well mixed
• Gas phase – well mixed
• Pressure – constant through out the vessel
• Not applicable for low cell growth and low O2 TR Ex. animal/plant cell
 Dynamic system (simple dynamic method)

• Easy to perform but inadequate data leads to inaccurate results

Final steady state

Cancelling kLaC*AL
 Variation of oxygen tension for dynamic measurement of kLa

Assuming kLa is constant with time and

integrating b/w t1 and t2
kLa - estimated using 2 points or more accurately, from several values of (CAL1, t1) and (CAL2, t2).
 Sulphite oxidation method

• Oxidation of sodium sulphite to sulphate in presence of Cu2+

• Depend on operating conditions in an unknown way and gives higher kLa

values than other values

• Application – discouraged as chemicals hinder bubble formation

Thank You
 Lecture 23

Heat transfer equipment – Heat transfer in bio processing

- Sterilization of Process Fluids - Kinetics of Death -
Sterilization of Liquids - Sterilization of Gases
TB-1: 314-320
TB-2: 164-168
 Heat transfer

• Heating and cooling requirements

• HT rates helps in calculating HT area

• HT - agitation and turbulent flow of the fluids

• HT Equipment

• Mostly fluids, no physical contact - hence solid metal wall separates the fluid streams
 Bioreactors
Two applications

• In situ batch sterilization of liquid medium

• Fermenter vessel + medium - sterilization with steam - a period of
time and to bring back the required temperature cooling will be
effected

• Temperature control
• T control during reactor operation
• Maintaining with in 1ºC range (normally 30-37ºC) considering heat
during metabolic activity
Jacketed vessel External coil Internal helical coil

Internal baffle-type coil External heat exchanger

 • Jacketed vessel • Lesser a  lab scale

• External coil • Large scale not sufficient, no homogeneity

• Internal helical coil Production  (fermenters)

• Internal baffle-type coil both heating & cooling

Temperature changes for control of

fermentation temperature using cooling water
 General equipment for HT
• Simplest form  Double pipe HE • Large scale  Shell and Tube
 Double pipe HT
• Cocurrent  Tco always < Tho Counter current tco can be > Tho
• Cocurrent  less heat is transferred and applied less frequently

Cocurrent flow Countercurrent flow

 Shell and tube heat exchangers
• HT area > 10-15 m2 area  shell and tube HE  large SA in smaller
volume
• Shell, tube, tube bundle, tube sheet, baffles
• Lesser HT capacities – single pass STHE
• Higher HT capacities, larger installations Double pass  multi pass
• Temperature curves  location of shell side entry nozzles
 Sterilization of process fluids
• Modern fermenters  larger capacities  10s of 1000s of l of media and
millions of l of air  needs sterility
• Disinfection  reduction of viable org. to possible lower not zero
• Sterilization  inactivating living cells, spores, viruses  death kinetics
• Probability of cell extinction P0 (t) = [1 - p(t)] N0

p(t) - probability of still viable @ t

N0 - initial number of viable spores
• Expected value of no. of cells present @ t  E [N(t)] = N0 p(t)

• Sp. death rate  kd = - d/dt (ln p(t)); p(t) = e-kd t

Decimal reduction time, D – time for no of viable cells to decrease ten fold

• E [N(t)] = N0 e-kdt ; 0.1 = e-kd D ; D = 2.303 / kd

 Survival Curve – ln N (t)/No vs time

Typical death-rate data for spores of Bacillus Typical death rate for E. coli in buffer
stearothermophilus in distilled water
 Sterilization of Liquids

• Heat, chemicals, radiation

• UV difficult for high suspended solid content fluids, X ray can do but safety
and cost are problems

• Chemicals  residue

• Ethylene oxide, sodium hypochlorite

• Temperature - Arrhenius Equation: kd =  e –Eod /RT

• E0d  E for death of org. (50-150)

• Most thermal sterilization 121°C
• high kd > 1010 min-1 vegetative cells;

• 0.5-5 min-1 Spores

• Factors – temp, time of exposure, initial no. of org.

• Probability for unsuccessful fermentation  1 – Po(t)  1 – [1-p(t)]No

• Killing model in homogeneous population  1 – Po(t)  1 – [1- e-kdt]No

• n0 conc. of particle  Prob. of unsucc. Steriliz. in 1 l  1 – Po(t)  1 – [1-p(t)](1*no)

• P (unsuccessful sterilization) in 10000 l tank  1 – Po(t)  1 – [1-p(t)](10000*no)

• If kdt 15; n0 104 spores per l

• P(unsuccessful fermentation)  0.003 for 1 l ; 5x10-14 for 10000 l tank
• Sterilization protocols for bench and larger scale are different
Sterilization Chart
 Sterilization batch (insitu) or continuous
• Batch sterilization
• thermal lag and incomplete mixing, heating and cooling longer time
• kd is tenfold in 121°C than 110°C

• Elevated T damaging vit., protein, caramelization of sugars  alter medium

quality
Continuous sterilization

• Higher T, short exposure time  complete ster., lesser damage,

time & down time

• Disadv. medium dilution during steam injection and foaming

 Filter sterilization

• Medium containing heat sensitive materials

• eg. Medium used to grow animal cells

• Micro-porous filters <0.2 μm, absolute filters (narrow pore size)

• Pre-filter large particles

• All equipment sterilized

 Sterilization of gases
• 0.1 to 1 vol of air min-1 per vol of liquid, sterile air
• Microbes in air – 103-104/m3; 1-10 microbes / l
• 50000 l  7x106 l to 7x107 l per day
• Fermentation several days, 5 day -2x108 l
• Air compressors – adiabatic compression increases air T (150-220ºC)
• Dry heat is less effective than moist heat in effective killing
• To kill spores 220C - 30 s – compression helps but cools faster – filtration
reqd.
• Depth filters (glass wool) replaced with surface filters (membrane
cartridge)
 Glass wool – fibres
• Bacteria
• flow streamline around fibre
• high density part won’t follow stream line
• larger part. size – intercepted
• high mass part.  high intertia – st. line trajectory
– crash into the fibre
• Interception and interial effects are imp for
bacteria
• Virus
• diffusion eff. - deeper filters - smaller probability
for part. pass through
• Problems : fibres later shrink and harden – fibre
glass filters; wetting (condensation)
 Surface filters
• Sieving effect; Membrane cartridge and housing
• Membrane filters, sterilized and reused; condensate won’t
pass through
• Filtration – pr drop critical  air treatment 25% of total
prod. cost
• Testing – effectiveness, sterilization cycles, pr drop vs flow
• Exhaust gas pathogens – catalytic combustion – costlier;
filtration
Thank You
 Lecture 24

Unit operations – Filtration – theory – equipment

TB-2: 218-224
 Unit Operations
• Unit operation  physical steps in processes (chemical & biochemical
transformations)

• Downstream processing  treatment of culture broth after fermentation

• Downstream processing - steps

Cell removal
Primary isolation
Purification
Final isolation
 Filtration
• Fluid-solid mix  filter medium/cloth  filter cake (resi.)  + or – P  Δ
P
• Ease of filtration
 Properties of solid and fluid
 Filtration of crystalline
 Incompressible solids in low viscous fluid
• Fer. Broths – filtration difficult
 Viscous Non-Newtonian broth
 Cells - small size, gelatinous
• Microbial filter cakes – compressible
 Filter Aids
• Diatomaceous earth (kieselguhr)  
♦
♦
10 to 25 μm

♦
Very high porosity
15% total V of packed kieselguhr - solids

♦
• Precoat

♦
Wedge into pores of cloth
With fer. broth (1-5% wt.) ↑ cake porosity

♦
• Disadvantages

♦
Cost

♦
Liquid absorption

♦
Disposal of cell removal - difficult
Product is cell – X filter aid
 Filtration equipment
• Plate filters – small fermentation – freq. removal of cakes

• Rotary drum vacuum filters – widely used – fermentation industry

 Rotary Drum vacuum filters
• Horizontal drum : D - 0.5-3 m
• Cover – filter cloth - partially immersed - agitated
reservoir - 0.1-2 rpm - filtered
• Section of drum enters liquid - Vacuum - interior of
drum
• Cake – face of cloth - internal pipes to a collection
tank
• Drum rotates out – filter surface - sprayed - wash
liquid - cloth and collected - separate holding tank.
• After washing - dewatered - vacuum
• Vacuum - turned off - discharge zone – cake
removal - scraper, knife or strings
• Air pressure may also be - dislodge the filter cake
from cloth
• After cake removal - drum re-enters reservoir for
another filtration cycle
 Filtration Theory

• Rate of filtration = rate of filtrate collected

• Filtration rate depends on

Filter cloth area
Fluid viscosity

P across filter

Sp. cake resistance
Filter resistance offered by cloth and deposited filter cake
Vol. rate of filtration
• At any instant during filtration

Rate of filtration

A - Filter area,
Vf - Volume of filtrate
t - Filtration time
p - Pressure drop across the filter
f - Filtrate viscosity,
Mc - Total mass of solids in the cake
 - Average specific cake resistance
 particle shape & size, interstitial space, cake mech. stability
rm - Filter medium resistance
 negligible compared to cake resistance
• Filter cake – incompressible   - doesn't vary with P
• For compressible cake    ’ (P)s
s - cake compressibility;
• s  0  incompressible solids
• s  1 highly compressible material
’ – constant depends on cake particle size and morphology
•  - avg. properties of particles
  Kv a2 (1 - )
 3 p
• Kv - factor depending on particle shape
• a - sp. surface area of particle
surface area of a single particle / volume of a single particle
•  - porosity of cake
= (total vol. of the cake – vol. of solids in cake) / total vol. of cake
• p - particle density
 Improving the rate of filtration
• ↑A
• ↑Δp  α ↑ so Δp <0.5, filter aid ↓s
• ↓Mc - conti. & lesser scrap residue
• ↓μf
• ↓α
 ↑ε (filter aid)
↓Kv - shape fac.-cell morphology
↓ a - ↑av. par. size and min variation in par. size
• Sub. Mc = cVf in c-solid mass deposited per filtrate vol.

• Filtration rate

• Two ways of filtration : Cost. Δp – filtration rate ↓; Δp ↑ - const. filt. Rate

• Const. ΔP
(reciprocal form)

• At filtration beginning t=0, Vf = 0


  to calculate Vf or t (if all constants known)

For experimental determination of  & rm

Eqn. divide by A Vf 
Thank You
 Lecture 25

Centrifugation – theory and equipment – Cell disruption –

Two phase liquid extraction
TB-2: 225-233
 Centrifugation
• Separation of diff. density materials  Force > gravity
• Application
• to remove cells from fermentation broths
• to eliminate cell debris
• to collect precipitates and crystals
• to separate phases after liquid extraction
• Steam sterilizable – if recycling, Industries – heating ventilation
and cooling
• Effective
• Large particles, low viscous fluid, density diff b/w particles and fluid –
more
• Biological system : smaller particles, high viscous
• Disadvantages
• Lab centrifuge – high speed Industries – problems
• Fast spinning – aerosol (infectious)
• Centrifugation capacity
• cannot be ced – simply by  ing size of equipment
• Mechanical stress -  in proportion to r2  safe operating speed
• Overcome these difficulties  range of centrifuges developed
• Centrifuge classification  according to internal structure
• Tubular bowl centrifuge
• Disk stack bowl centrifuge
 Tubular bowl centrifuge

• Application: mainly for difficult separations

requiring high centrifugal forces
• Food / Pharma
• Spun out / collide
• Centrifugal force : 13000 - 16000 g
 Ultracentrifugation

• Narrow tubular bowl centrifuge

• Recover fine precipitates from high density media
• Breaking down emulsions
• Separation of colloidal particles (ribosome, mitochondria)
• Centrifugal force: 105 - 106 g
• Bowl : air driven & operated @ low P or N2 atm  reduce frictional heat
• Appn. Vaccine production (separate viral particles from cell debris)
• Batch /continuous
 Disc stack bowl centrifuge
• Discs - conical sheets of metal
• Disc clearance : 0.3 mm
• Disc - rotate with bowl  split the liquid
into thin layers
• Between discs
• heavy components - thrown outward
under g - at top or thro' nozzles
• lighter liquid - displaced towards bowl
centre
• Centrifugal force : 5000 to 15000 g
• Density difference : 0.01 to 0.03 kg m-3
• Minimum particle diameter : 0.5 m
Disc-stack bowl centrifuge with continuous discharge of solids
 Disc stack bowl centrifuge
• Principal difference in different types of disk stack bowl  method used
to discharge the accumulated solid

• Types based on solid removal

• Simple – removal periodically by hand
• Continuous or intermittent discharge – possible – without reducing
bowl speed
• Peripheral nozzles – continuous solid removal
• Concentrate solids in bowl periphery – discharge at top of centrifuge
with a paring device
 Centrifugation - theory
• Stoke’s law
Sedimentation (terminal) velocity under g

p – particle density; f – liquid density;  – liquid viscosity; D p – particle dia

• In centrifuge – corresponding terminal velicity

uc – particle velocity in centrifuge;  - angular velocity of bowl (rad s-1);

r – centrifuge drum radius
• Centrifuge effect or g-number (Z) : ratio of velocity in centrifuge to velocity
under gravity

• Force developed in a centrifuge is Z times the force of gravity

• Z factor : Industry – 300 to 16000; Lab - 500000
• Sedimentation due to collide of part. with wall  ↑ vel of motion ↑ sedi.
• Collide depends on resi. time - ↓ flow rate
• uc can be increased by
•  centrifuge speed ()
•  particle dia (Dp)
•  density difference b/w particle and liquid (p - f )
•  viscosity of suspended fluid ()
• Performance of centrifuge  Sigma factor ()
•  - c/s area of gravity settler to centrifuge for same sedimentation
characteristics
• Continuous centrifuge
 - related to feed rate of material

Q – volumetric feed rate; ug – particle terminal velocity in g field

• If two centrifuge – equal effectiveness

 scale up centrifuge equipment

• Disc stack bowl centrifuge
 - angular velocity in rad s- 1
N - number of discs in the stack
r2 - outer radius of the disc
r1 - inner radius of the disc
 - half-cone angle of the disc

• Tubular bowl centrifuge

b - length of the bowl
r1 - radius of the liquid surface
r2 - radius of the inner wall of the bowl

• r1 and r2 in a tubular-bowl centrifuge are about equal

r - average radius roughly equal to either r1 or r2

 Cell disruption
• Down stream processing - filtration & centrifugation
• Next step - depends on location of desired product
• Ethanol, citric acid, antibiotics excreted from cells – recovered using unit
operation
• Enzymes, recombinant proteins remains in biomass and cells must be
broken open to release – cell disruption
• Cell disruption - variety of options
• Mechanical  grinding with abrasives; high-speed agitation; high-pressure
pumping and ultrasound
• non-mechanical  osmotic shock; freezing and thawing; enzymatic
digestion of the cell walls and treatment with solvents & detergents
 High pressure homogenization

• Widely used
• Shear force - disrupt the cells
• Manton - Gaulin homogenizer
• High pressure pump @ P 550 atm
• Adjustable valve - orifice
• Blocked highly filamentous org
• Cavitation, fluid shear, impact
and pressure shock
• Single / multi pass
• Equation - disruption of cells to operate conditions in Gaulin homogeniser

Rm - Max protein available

R - Protein released after N passes
k - temp. dependant rate const.
p - operating pressure
α - measure of resistance of the cells to disruption
• k & α - cell type dependant
• α = 0.9 (bacteria) to 2.9 (yeast)
• Sufficient Pressure  one pass - beneficial
• Reduction in no. of passes  higher passes  fine cell debris pdn.
 Aqueous two phase liquid extraction
Examples of aqueous two-phase systems

• Aqueous solvent – provides

two distinct phases –
protein, cell fragments,
organelles with protection
of biological activity
• 2 phase aqueous system -
Polymers or polymers and
salt dissolved in water
above certain
concentration
• Cell fragment in one phase and protein in another
• Extraction of enzymes and recombinant proteins from cell debris  then
• precipitation or crystallization
• Separation – concentrated bottom diluted upper
• Extent of differential partitioning b/w phases depends on equilibrium
relationship of system
• Partition coefficient

CAu - equ. conc. of A in upper phase; CAI - equ. conc. of A in lower phase;
k>1  A favours in upper phase; k<1  A favours in lower phase
k≥3 - single stage extraction of enzymes - required
• Even K - low; good product recovery or yield - achieved  using large
volume of phase preferred by solute
• Yield of A in upper phase (Yu )

Vu - volume of upper phase

Vi - volume of lower phase
CA0 - original pdt. conc. in that liquid
Yield of A in lower phase (Yl )
• Maximum possible yield for an ideal extraction stage
• Yu : divide both numerator and denominator by CAu & CAI / CAu = 1/K

• Yl : divide both numerator and denominator by CAI & CAI / CAu = 1/K
THANK YOU
 Lecture 26

Adsorption – Performance of fixed bed absorbers - Engineering

analysis – Chromatography – Migration and spreading

TB-2: 234-245
 Adsorption
• Components of gas or liquid concentrated on solid surface or at fluid
interfaces
• Result of electrostatic, van der Waals, reactive or other binding forces b/w
atoms/ions/molecules
• Types : Exchange, physical, chemical and nonspecific
• Application
• Medical and pharmaceutical products
• Ion-exchange ads. recovery of amino acids, proteins, antibiotics and
vitamins
• purification of citric acid  Adsorption on activated charcoal
• Waste water treatment  Adsorption of organic chemicals on
charcoal/porous polymer adsorbents
• Adsorption gaining increasing application suitability for protein isolation
• Absorbate – substance being concentrated on surface

• Absorbent – material to which adsorbate binds

• Ideal adsorbent  high surface area per unit volume – network of fine
internal pores  large internal surface area

• Adsorption of biological molecules – Carbons & synthetic resins based on

styrene, divinylbenzene, or acrylamide polymers

• Commercial absorbent – porous granular or gel resins (void vol. : 30-50%)

Pore dia. <0.01 mm

• Ex. Total surface area activated carbon : 450-1800 m2/g

 Adsorption Operations - Stages

1. Contacting - loading
2. Washing – residual unadsorbed material
3. Desorption/Elution - using solvent
4. Washing - residual eluant - pH
5. Regeneration
 Isotherm
• Adsorption equilibrium data – adsorption isotherm  useful to select
adsorbent & predicting performance of adsorption systems
• Langmuir isotherm – simplest form

• C*AS - equilibrium concentration or loading of A

on the adsorbent in units of kg solute kg-1 solid
or kg solute m-3 solid
• CASm - maximum loading of adsorbate
corresponding to complete monolayer coverage
of all available adsorption sites
• C*A - equilibrium concentration of solute in the
fluid phase in units of kg m-3
• K - constant
 Applications of Langmuir adsorption

• Adsorbed molecules form no more than a monolayer on the surface

• Each site for adsorption is equivalent in terms of adsorption energy

• There are no interactions between adjacent adsorbed molecules

• Freundlich isotherm – liquid solid systems

K F and n - constants characteristic of the particular

adsorption system

• n > 1  adsorption - favourable

• N < 1  adsorption - unfavourable
• Application: wide variety of antibiotics, hormones and steroids
 Performance characteristics of fixed bed
Adsorbers
• Equipment

Fixed bed Moving bed Fluidized bed Stirred tank contactors

• Fixed bed (most common) – higher adsorption area/m3

 Down-flow fixed bed adsorber

• Adsorption zone - moving down –

Top saturation
• Adsorption wave – movement of
adsorption zone - lesser speed than
fluid velocity thro’ bed
• Ads. zone reaches bottom – Breaking
point
• Breakthrough curve – f(t) or f(V)
 Relationship between the breakthrough
curve, loss of solute in the effluent, and unused column capacity

If adsorption continues until entire bed is saturated If adsorption halted at ‘t’, when [effluent] = C’A ,
and [effluent] = CAi , considerable solute - wasted only small amt. solute – wasted
 Engineering analysis of fixed bed adsorbers

• Design – quantity of resin and adsorption time for given quantity of solute
• Design procedure – predicting shape of breakthrough curve, time of
appearance of breaking pt.
• Factors – breakthrough curve
Feed rate
[solute]
nature of adsorption equilibrium
adsorption rate
• Aim – Effluent concentration as f(t)
 Column packed with adsorbent resin
• Mass balance equation
0 0

• When appropriate mathematical expressions for

rates of flow, axial dispersion and accumulation

- effective axial dispersion coefficient

 - void fraction
• Void fraction ()
VT - total volume of the column
Vs – volume of the resin particles
u - interstitial liquid velocity

• Interstitial liquid velocity (u)

FL -volumetric liquid flow rate

Ac – cross sectional area of the column
 Chromatography
• Separation process

• Differential migration – selective retardation of solute molecules during

passage thro’ a bed of resin particles
• At solvent flow – solutes diff speed – relative
affinities for resin particle Chromatographic separation of
components in a mixture
• Chromatogram - Pattern of solute peak emerging
from a chromatography column
• Mobile phase - Fluid carrying solute/elution–mobile
phase
• Stationary phase - Mat stay in column & effects
separation
• Gas chromatography - alcohol, ketones, aldehydes, organic /in-organic
compounds

• Liquid chromatography - bioprocess., large scale high purity

• Chromatography methods
• Adsorption chromatography
• Partition chromatography
• Ion exchange chromatography
• Gel (mole sieve) chromatography
• Affinity chromatography
 Adsorption chromatography Partition chromatography

Affinity chromatography

Gel (mole sieve) chromatography

Ion exchange chromatography
• Chromatography - Traditionally - atm P, vertical columns, manual sample
feed and gravity elution

• Normal phase chromatography – stationary phase is more polar than

mobile phase

• Reverse phase chromatography - When non-polar compounds are being

separated it is usual to use a stationary phase which is less polar than the
mobile phase

• Two chromatographic separations methods

• High performance liquid chromatography (HPLC)
• Fast protein liquid chromatography (FPLC)
• HPLC, FPLC - faster and better resolution using densely packed columns and high
flow rates
• HPLC – small scale, high resolution analytical – stationary phase part size 2-5 μm,
high P 5-10 MPa to achieve flow rates of 1-5 ml/min
• FPLC – large scale, preparative, 1-2 MPa, larger size packing, poor resolution

HPLC FPLC
 Differential migration
• Basis for chromatographic separation
• DM of Solutes A & B, Differential equilibrium affinities for stationary phase;
A adsorbed strongly than B
• Small quantity solution – limited depth of saturation of bed – both solutes
retained
• Eluant flow – adsorbed, desorbed; B easily desorbed; forward fast; washed
out
• Capacity factor k = (Ve-Vo)/Vo [ Ve-eluant; Vo-void]

• Two solutes-selectivity or relative retention δ=k2/k1

• Gel column
• Total volume, VT =Vo+Vi+Vs Vi – internal V of liquid in particle pores ; Vs – volume of gel

• Excluded from the stationary phase elute volume, Ve = Vo+KpVi

• Gel partition coefficient, Kp=(Ve-Vo)/Vi; V i=a Wr Wr - water regain value, a – const.

• Vi = Wrρg(VT-Vo)/(1+Wrρw) ρg – density if wet gel, ρw – density of water

 Zone spreading
• Elution bands spread out - each solute takes finite period-pass across
column
• Separatable molecules similar structure – zone spreading to be controlled
• Peak reaches 0 before and after if not spreading of peak – factors

• Axial diffusion, eddy

diffusion, local non-
equilibrium effect
• Equilibrium has to be
reached fastly – more sur
area, increased liquid flow
rate, heating – reduced
viscosity – reduced zone
spreading

Axial diffusion Eddy diffusion

THANK YOU
 Lecture 27

Overview of Reactor Types - Considerations on Aeration and

Agitation and Heat Transfer - Scale-up – of mixing systems -
Chromatography
TB-1: 285-301
 Scale up - Difficulties

• Performance – fungal culture – different - 10 l or 10,000 l ?

• Maintaining homogeneity
• Surface to volume ratio
• Change in culture  time length
 Overview of reactor type
• Large array of fermenter & basic design – basic types
Reactor with internal mechanical agitation
bubble columns  gas sparging for agitation
loop reactors  mixing & liquid circulation – induced by motion of
injected gas / mechanical pump/ combination of both
• Three phase reactions
• Difficult – design
• Mass Transfer control
• Complex fluid dynamics
 Reactor types

• Stirred tank reactor

• Bubble column reactor

• Airlift loop reactor with control draft tube

• Propeller loop reactor

• Jet loop reactor

Stirred tank reactor
Bubble column reactor
Airlift loop reactor with control draft tube
Propeller loop reactor
Jet loop reactor
 Traditional fermenter
• Stirred tank reactors – eg. with mechanical agitation

• Flexible, high kLa, 400 m3 antibiotic production + stirrer power 5 kW/m3,

viscosities – about 2000 centipoises (2 Pa sec)
• Sparger – gas under pressure  ring with holes or tube with single orifice
• Impeller  gas dispersion
• Impeller - types

Disc impeller Turbine impeller Marine propeller Paddle impeller

Mechanically stirred 100,000-liter fermenter
 Rushton Impeller Axial flow hydraulic impeller

• pump either down or up

• 6-8 blades • lower energy demand
• pump radial direction • reduced shear (sensible culture)
• predominant mid 80s • viscous mycelial fermentation
• 30 to 40 % of tank dia. • upto 50% of tank dia.
• Industrial and laboratory fermenters

Combined axial and radial impellers

• Baffles (w=8-10% cylinder dia)
• Vessel  Type 316 SS, covers & jacket (304), plant & animal (low C 316L)
• Bench scale fermenters  glass vessels with SS cover plates
• SS or glass (50 l to max 500 l) smooth surface – better sterility level
• Ht to dia 2-3; animal cell culture (closer to 1)
• Heat removal - Internal coils (rapid fouling)/jacketed vessel
• Foaming
• if escapes-contamination
• mechanical foam breakers
• surface active chemical agents (↓kLa, inhibition to cell growth)
• Working V – 75% of total V
• Sterility – O rings, flat gaskets
• Cleaning – ‘In Place’ tech. - Clean In Place tech. – highly alkaline detergents
• Bubble column high energy efficiency (O2 transfer/energy) than stirred tank
 Some considerations in aeration, agitation and
heat transfer
• O2 requirement – choice of organism

OUR = X qO2 = kLa (C* - CL)

Large scale 40-200 mmol/l/h (mostly 4-60)

• kLa – critical parameter

kLa = k (Pg/VR)0.4 (vS)0.5 (N)0.5

Pg - Power reqt. in aerated reactor, vg – superficial gas exit speed

Typical Respiration Rates of Microbes and Cells in Culture
Unsteady-state accumulation of oxygen in a large stirred tank
• Unsteady state method  change in DO

or

or

a plot of log (C* - CL) versus time will give an estimate of kLa
Sulfite method
• In the presence of Cu2+ , sulfur in sulfite oxidized to sulfate - 0 order rxn.

• Consequently CL approaches zero

• Sulfate formation rate  monitored & proportional to O2 consumption

rate(mol of O2 consumption  1 mol of SO42- production)

Rate of sulfate formation

• Steady state method – best way to kLa measurement

• Dynamic method
 Scale up
• Fermenter
• ht to dia ratio = 2:1 or 3:1
• scale up – Surface to Volume drastic ↓
• ↓ aeration to O2 supply or dissolved CO2 removal w.r.t sparging

• No duplicating physical conditions to geometric similarity

• Dia 5 folds  volume 125 times (if ht to dia - constant)
• Scale up – empirical and imprecise art
 Interdependence of Scale-up Parameters

• four scale up rules : P/V, N, NDi, Re – select appropriately

Some Time Constants
Time constants  20 m3 fermenter - gluconic acid pdn.
• Fermenter volume : 0.1 to 100 m3
• Mixing time

V – vessel volume; T - constant  fxn. of impeller type, placement & vessel design
• Scale up – empirical and imprecise art
THANK YOU
 Lecture 28

Scale–down of bioreactors – scale down models for validation

of bio processes – Validation tools – Dealing with the offsets
TB-1: 301-307
 Scale down
• Scale up models & use of characteristic time analysis – attractive
• Immediate approach to rational scaling of reactors  scale down
• Concept: provide @ providing smaller scale experiment system
duplicating real system environment
• Many cases – scale up  require using existing production facilities 
important to mimic production @ smaller scale
• Many parameters – Quick and inexpensive testing of expt. parameters
• Evaluate proposed changes – existing operating process
 Scale down
• Smaller scale apparatus  approximate types of variations in [S] & [DO] –
mixing time analysis & actual data on residence time distributions from
large reactor
• Estimate system’s response to changes in medium composition
• Growth rate, pdt formation & formation of contaminating by-products to a
new supplier of raw materials
• Introduction of modified production strains
• Use of diff. inoculum preparations
• New antifoam agents
• For testing for O2 & CO2 tolerance
• Correcting protocols  use in large system by stimulating response to pH
or O2 probe failure
• Complement to math models, scale-up rules & traditional pilot-plant
operation
Experimental setup – scale down apparatus
THANK YOU
 Lecture 29
BIOREACTOR INSTRUMENTATION AND
CONTROL
BIOREACTOR INSTRUMENTATION AND CONTROL
 The maintenance of optimal conditions for product formation →in a bioreactor
requires the control and measurement few parameters.
 Almost all fermenters have pH, temperature, and dissolved-oxygen control (by
control of agitator rotational speed).
 New probes and techniques →measurement of various ions, substrates, and
products → developed → implemented in particular situations.
 Actual process-control strategies for bioreactors are very primitive in comparison
to
the petrochemical industry→ because of a lack of sensors for on-line
measurements ,
reliable, quantitative, dynamically accurate models.

Instrumentation for Measurements of Active Fermentation

 The maintenance of sterility in a fermenter imposes a severe limitation →
measurements of fermentation parameters
 Some probes like pH and DO enter the reactor through penetrations in the
fermenter shell increases the probability of contamination.
 The probes themselves must also be sterilizable → steam and it should
withstand moderately high temperatures (121°C) in the presence of 100%
humidity.
 Chemical sterilization less desirable, may allow the use of a temperature-
sensitive device if it has sufficient chemical resistance.
 Lecture 30
Mixed cultures
Major Classes of Interactions in Mixed Cultures - Simple
Models Describing Mixed-culture Interactions - Mixed
Cultures in Nature - Industrial Utilization of Mixed Cultures
 The dynamics of mixed cultures →commercial fermentations and
understanding the response of many ecological systems to stress is important.
 Cheese manufacture and biological treatment of waste waters.
 Ratio of various species and composition of the population → failure
 Natural environments → cells exist in potentially mixed populations.
 Interaction of cells → natural cycles for the elements (C, N, and S), ecosystems
and the rate and extent of degradation of chemicals introduced into such
ecosystems.
 The major interactions between two organisms in a mixed culture are
competition, neutralism, mutualism, commensalism, amensalism and prey–
predator interactions.

 Competition → indirect interaction between two populations → negative effects

on both. Each population competes for the same substrate.
 Outcome of competition between two species for the same growth → limiting
substrate → by the specific growth-rate limiting substrate concentration
relationship.
1. µa is always greater than µb. The organisms with the fastest growth rate will
displace
the others from the culture. This is known as the exclusion principle.
2. Crossover in µ-S relationship. In this case, the faster-growing organism is
determined
by the dilution rate. Depending on the dilution rate, three different cases may
be identified:
a. At the crossover point D = µx; S = Sx, two species could be maintained in a
chemostat at
D = µx. (unstable operating point.)
b. If D > µx, then µa > µb, and B will be washed out; A will dominate.
c. If D < µx, then µb > µa, and A will be washed out; B will dominate.
 Neutralism is an interaction where neither population is affected by the presence
of the other → there is no change in the growth rate of either organism due to
the presence of the other. Relatively rare. (Yogurt starter strains of
Streptococcus and Lactobacillus in a chemostat).

 Mutualism and proto co-operation are more common than neutralism involving
different mechanisms.
 Both cases, the presence of each population has a positive effect on the other.
 Mutualism → the interaction is essential to the survival of both species.
 Proto co-operation → the interaction is non-essential.
 Mechanism → mutual exchange of required substances or the removal of toxic
end products by each organism.
 The metabolisms of partner populations must be complementary to yield a
mutualistic interaction.
 Phenylalanine-requiring strain of Lactobacillus and a folic-acid-requiring strain of
Streptococcus in a mixed culture.
 Mutualistic interaction exists between aerobic bacteria and photosynthetic algae.
 Bacteria → O2 and CHO for growth and produce CO2 and H2O. Algae convert CO2 to
CHO and liberate O2 in the presence of sunlight.
 Commensalism → interaction in which one population is positively affected by
the presence of the other. However, the second population is not affected by
the presence of the first population.
 Various mechanisms may yield a commensal interaction. Two common
mechanisms are the following:
1. The second population produces a required nutrient or growth factor for the
first
population.
2. The second population removes a substance from the medium that is toxic
to the
first population.
 An example of the first type of commensal interaction is the production of H2S
by
Desulfovibrio (through the reduction of SO42− ), which is used as an energy
source by
sulfur bacteria.
 Amensalism is the opposite of commensalism.
Population A is negatively affected by the presence of the other population (B).
Population B is not affected by the presence of population A.
Two common mechanisms are the following:
1. Population B produces a toxic substance that inhibits the growth of population A.
2. Population B removes essential nutrients from the media, thus negatively
affecting the growth of population A.
 One example of the first type of amensal interaction is the production of
antibiotics by certain molds to inhibit the growth of others.
 Some microbes excrete enzymes that decompose cell-wall polymers.
 Such organisms destroy their competitors and also utilize the nutrients released
by the lysed cells.
 The microbial synthesis of organic acids reduces pH and inhibits the growth of
other organisms.

 Predation and parasitism are interactions → one population benefits at the

expense of the other.
 These two interactions are distinguished by the relative size of organisms and the
mechanisms involved.
 Predation involves the ingestion of prey by the predator organism.
 A good example of prey–predator interaction is the ingestion of bacteria by
protozoa.
(aerobic waste-treatment reactors such as activated sludge units).
 A common example of parasitism → destruction of microorganisms by
SIMPLE MODELS DESCRIBING MIXED-CULTURE INTERACTIONS

 Multiple interacting species can give rise to very complex behavior

 In some cases, coexistence of species is prohibited or complex sustained
oscillatory behavior may be observed leading to multiple steady states.
 A balance must be written for each species (organism, rate-limiting substrate, or
product), and these balances will be the same as we have discussed or used
previously.
 In mixed cultures → unstructured models are used for each species.
 The population model is still structured in the sense that the whole biomass is
divided into distinct subpopulations.
 These equations are often applied to chemostats that mimic many ecosystems
and yield multiple steady states.
 For each steady state we need to analyze its stability. It is often easier to test a
steady state for its local stability than for its global stability.
 With this background, we will consider some representative examples.
Competition of two species for the same growth-rate-limiting substrate is common.
Determine when the two organisms may stably coexist if both A and B follow
Monod kinetics.
Solution For this situation, the following equations describe the dynamic situation:
 Equation 16.5 is meaningful only if S ≥ 0. Consider the two cases in Fig. 16.1.
 In case (a), µmA KSB > µmB KSA and µmA > µ mB ; consequently, S is always less than
zero, and coexistence is impossible.
 In case (b), however, a value of S can be found from eq. 16.5 that will allow the
populations to coexist. The corresponding D for the crossover point is Dc.
 Although this coexistence is mathematically obtainable, not physically
attainable
in real systems. In real systems, the dilution rate will vary with time, and
variation
will show a bias.
 It can be demonstrated that one competitor will be excluded from the
chemostat if the intensity of the “noise” in D and the bias of the mean of D
away from Dc are not both zero. Also, it is possible for either competitor to be
excluded, depending on how D varies.
 Two competitors can coexist if we modify the conditions of the experiments.
Examples of such modifications include allowing spatial heterogeneity or
another level of interaction is added.
 Also, operation of the chemostat in a dynamic mode (D is a function of time)
can sometimes lead to coexistence.
 It is also interesting to note that the use of other rate expressions (e.g.,
substrate inhibition) can lead to multiple crossover points and potentially
multiple steady states.
MIXED CULTURES IN NATURE

 Mixed cultures of organisms are common in natural ecological systems.

 Microorganisms are involved in the natural cycles of most elements (e.g.,
carbon, nitrogen, oxygen, and sulfur).
 Organisms living in soil and aquatic environments actively participate in carbon
and nitrogen cycles.
 For example, certain organisms fix atmospheric CO2 to form carbohydrates,
while others degrade carbohydrates and release CO2 into the atmosphere.
 Similarly, some organisms fix atmospheric nitrogen (N2) to form ammonium
and proteins, while others convert ammonium into nitrite and nitrate
(nitrification), and others reduce nitrate into atmospheric nitrogen
(denitrification).
 Sulfur-oxidizing organisms convert reduced sulfur compounds (sulfur and
sulfide) into sulfate, and sulfate-reducing organisms reduce sulfate into
hydrogen sulfide.
 The aforementioned interactions among different species take place in natural
systems in a more complicated manner. Understanding such complex cycles
is
important to understanding the capacity of natural ecosystems to degrade
organic
pollutants.
INDUSTRIAL UTILIZATION OF MIXED CULTURES

 Defined mixed microbial populations →cheese making

 Cheeses of various types → inoculating pasteurized fresh milk with appropriate
lactic acid organisms.
 The bacteria used for lactic acid production are various species of
Streptococcus and Lactobacillus in a mixed culture.
 Organisms are used to develop flavor and aroma →Brevibacterium linens,
Propionibacterium shermanii, Leuconostoc sp., and Streptococcus diacetilactis.
 After inoculation of pasteurized milk, a protein-rich curd is precipitated by the
acidity of the medium, and the liquid is drained off.
 The precipitated curd is allowed to age by action of bacteria or mold. Some
molds used in cheese making are Penicillum camemberti and Penicillum
roqueforti.
 Lactic acid bacteria → whiskey manufacture.
 Lactobacillus added to the yeast reduces pH and contributes to the flavor and
aroma of whiskey.
 A favorable interaction between yeast and lactic acid bacteria exists in ginger-
beer fermentation.
 The utilization of undefined mixed microbial cultures in waste-treatment
processes
 Cellulase producers, acid formers, and methane producers are typical
organisms involved in the anaerobic digestion of cellulosic wastes.
 Corn and pea wastes →mixed culture of Trichoderma viride and Geotrichium sp.
 T. viride → cellulase to break down cellulose into reduced sugar molecules
 Geotrichium → produces amylases .
 Both organisms utilize reduced sugar molecules for growth.
 A mixed culture of Candida lipolytica and Candida tropicalis → hydrocarbons, n-
paraffins, or gas oil → single-cell protein (SCP) production.
 The utilization of a mixed culture of yeasts was proved to yield better product
quality as compared to pure yeast strains.
 Gaseous hydrocarbon substrates like methane → bacteria to produce SCP.
 Mixed cultures of methane utilizing organisms grow faster than pure cultures.
 Pseudomonas oxidize methane to methanol. However, Pseudomonas is inhibited
by the end product, methanol.
 Inclusion of a methanol utilizing bacteria such as Hyphomicrobium into the growth
medium eliminates the problem of methanol inhibition.
 This relationship is mutualistic in the sense that Pseudomonas supplies carbon
source (CH3OH) for Hyphomicrobium, and Hyphomicrobium removes the growth
inhibitor (methanol) of Pseudomonas.
 Lecture 31
Biological Waste Treatment - Example of
the Industrial Utilization of Mixed Cultures
– Monod kinetics
 Biological waste treatment
Industrial wastes - HCs, carbohydrates, alcohols, lipids and aromatic organics
Rich C – Deficient N leads to High C/N
Domestic waste – sewage, municipal, human activity
Agricultural waste – C rich, cellulosic
Treatment depends on characteristics of waste.
Physical - screening, flocculation, sedimentation, filtration and flotation
Chemical - oxidations (chlorination, ozonation) and precipitation using CaCl2, FeCl3,
Ca(OH)2 or Al2(SO4)3
Biological – aerobic and anaerobic treatment by microorganisms

Characterization of waste water

Physical - colour, odor, pH, temperature and solids contents (suspended and dissolved
solids)
Chemical – organic (major - carbohydrates, lipids, HCs, proteins; minor - phenols,
surfactants,herbicides, pesticides and aromatic), inorganic (nitrogenous, sulfur,
phosphorus, heavy metals)
C content – BOD (strength of waste water 20ºC, 5 days), COD (organic compounds
chemically oxidizable), TOC (TOC analyzer)

C6H12O6+ 6O2 → 6CO2 + 6H2O

(1g glucose needs 1.07g O2 for complete oxidation - BOD)
CaHbOc + Cr2O72- + H+ → Cr 3+ + CO2 + H2O (Dichromate oxidizing agent)
Typical waste water treatment operations
Primary - coarse solids and suspended matter (screening, sedimentation, filtration)
and conditioning by pH adjustment and nutrient additions
Secondary - major step; bio oxidation or anaerobic treatment of soluble and
insoluble org compounds; org comp – oxidized to CO2 + H2O in aerobic; Unoxidized
org and solid in aerobic to CH4, CO2 and H2S in anaerobic / sludge
Tertiary - remaining inorganic comp (phosphate, sulfate, ammonium) and other
refractory org comp by one or more physical separation methods such as carbon
adsorption, deep-bed filtration, membrane techniques (RO, electrodialysis)

Biological waste treatment process

Mixed culture of organisms
Aerobic – Activated sludge, trickling filter, rotating biological contractors, oxidation
ponds

Activated sludge
Well aerated, agitated, PFR/CFSTR, mixed culture organisms produces polymers →
help organism to agglomerate – good floc – good performance; large dense floc in
sediment recycle
Mechanical surface aerators, liquid spray – change in air water interface – better O2
transfer
Shock loading – sludge bulking
Modifications → step feeding along the length;
Solids reaeration (contact stabilization) – Two tanks : 1 h – adsorption of organisms
in floc, 3-6 h – reaeration – Conversion of org to CO2, H2O; concentration of
organisms before aeration – volume red as 50%
Specific growth rate of miroorganisms is given by Monod

Trickling biological filters (TBF)

Packed bed of inert mat (sand/plastic) covered with mixed organims culture
Loosely filled column (0.4-0.5 void), shallow bed (D/H=3)
Low flow rate to avoid shear of film; Air from bottom and natural circulation
Heterogeneous medium environment; Liquid flows downwards – org (S) diffuse
though microbial film – utilized by organisms
Liquid film thick 0.01 mm, biofilm thick 0.25 mm, HRT – 0.5-4 h,
Rotating biological contactors (RBC)
Rotating disc (polystyrene/poly ethylene) (2-4 m dia) – periodical contact with
waste water – biofilm (disc surface) nutrients (org and DO) diffusion
More compact and effective than TBF

Oxidation ponds
Shallow (like natural aquatic ecosystem) – Bacteria and algae (symbiotic)
Bacteria utilizes org consuming algae produced O2, algae consumes bacteria
produced CO2 release O2 by photosynthesis
Large area, less efficient, toxic comp no degradation-collection with sediment
Anaerobic digestion (biological treatment)
Solid/excess sludge in aerobic
Particulate (screening/sedimentation) in primary/biomass in sludge → anaerobic →
methane – slow process (30-60 days)
Steps:
1. Solubilization of insoluble organics: cellulose / starch by hydrolysis readily
utilizable form.
2. Formation of volatile acids: 1 metabolized by acid formers to acetic, butyric,
formic, propionic and short chain fatty acids, pH 4-6, T 35, ethanol, butanol and
proponal
3. Methane formation: 2. To CH4 + CO2 by methanogenic bacteria; T 35-40, pH
7-7.8
Low cell yield – 0.05 g/g of COD; Biogas comp, HHV
H2S generation problem, solid red to 50-60%
Digested effluent – dewatered dried fertilizer
 Lecture 32
Advanced Waste-water Treatment
Systems - Conversion of Waste
Water to Useful Products
Advanced waste water treatment system
 Removal of residual Nitrogen, Phosphorous and refractory carbonaceous
compounds

Nitrification and denitrification

 Amino acids, proteins → biologically oxidized to ammonium oxidized to nitrite
(Nitrosomonas) and nitrate (Nitrobacter) [Ammonia to nitrate – nitrification]

 Growth yield of Nitrifying bacteria is low (0.2 g/g N)

 Nitrification units → (BOD5/TKN - >5 single stage or <3 double stage)
 Anaerobic conditions – some bacteria utilize nitrate as final e- acceptor instead of
O2 → Denitrification
 Two types of nitrate reductase → Assimilatory – nitrate to ammonia to cell mass;
dissimilatory – nitrite to elemental Nitrogen
 Denitrifiers (bacteria) - Pseudomonas, Acaligenes, Arthobacter and Corynebacter
Phosphate removal
 Assimilation – Aerobic/anaerobic – 3% cell mass is made of phosphorus
 Luxury phosphate uptake – Acinetobacter – anaerobic condition – utilize acetate
and fatty acids - synthesis polyhydroxybutyrate (PHB) – energy obtained by break
down of polyphosphates
 Aerobic – organisms utilize PHB to synthesis polyphosphate and store as granules
inside the cell
 Organisms remove phosphate from liquid when synthesis polyphosphate in
aerobic
 In anaerobic phosphate in recycled sludge released to liquid media and
polyphosphate is organic fertilizer

 The A/O process is used for combined BOD and phosphate removal.
 It is a twostage process incorporating anaerobic and oxic (aerobic) steps in
sequence.
 In the anaerobic stage, the phosphate in the recycled sludge is released into liquid
media.
 The released phosphate is taken up by the cells under aerobic conditions along
with phosphate in the feed waste-water stream and is stored as polyphosphate
granules.
 Polyphosphate is removed from the system by the waste sludge, which has a high
value as fertilizer.
 When the BOD/P ratio in the feed wastewater is ˃10, the effluent phosphate levels
can be lower than 1 mg/l.
 The PhoStrip process consists of an aerobic stage and an anaerobic one on the
sludge recycle stream.
 The waste-water stream is fed to the aerobic stage for polyphosphate synthesis,
the effluent of which is fed to a sedimentation tank.
 The underflow of the sedimentation tank is fed to the anaerobic stage for
phosphate release.
 The released phosphate is precipitated in a sedimentation tank with the addition
of lime in form of calcium phosphate.
 Part of the sedimented sludge and anaerobic reactor sludge is recyled back to
the aerobic stage.
 Phosphate is removed from the process in the form of calcium phosphate. The
effluent water stream would contain less than 1.5 mg/l phosphate in a well-
operating PhoStrip process.
Combined nitrogen and phosphate removal
 Combinations of anaerobic, anoxic and aerobic zones accomplish simultaneous
BOD removal A2/O and five-stage Bardenpho processes, UCT (The University of
Cape Town) and VIP (Virginia Initiative Plant) processes
 The A2/O process includes an anoxic zone for denitrification in addition to A/O
process.
 Hydraulic residence times in anaerobic and anoxic zones are less than 1.5 h, and
in the aerobic zone 4–6 h.
 The effluent of the aerobic stage is recycled back to the anoxic zone for
denitrification purposes.
 Phosphate release takes place in the anaerobic zone. Denitrification and BOD
removal are the major functions of the anoxic zone.
 Phosphate removal in the form of polyphosphates, nitrification, and some BOD
removal take place in aerobic zone.
 Phosphate is removed from the system in form of waste sludge; nitrogen is
released in form of N2 (gas); and BOD is converted to CO2 and H2O.
 The five-stage Bardenpho process includes two additional anoxic and aerobic
zones as compared to the A2/O process (A2/O/A/O process).
 The effluent of the aerobic zone is partly recycled back to the anoxic zone for
denitrification purposes.
 The last two zones are for additional denitrification and polyphosphate removal
to further reduce nitrate and phosphate levels in the effluent.
 Hydraulic residence times in anaerobic and anoxic zones are less than 4 h and
in the first aerobic zone 4–12 h.
 The UCT and VIP processes employ similar zones and recycle schemes in
different orders, resulting in low nitrogen, phosphate, and BOD levels in the
effluent.

 Elemental sulfur in waste water can be oxidized to sulfate by Thiobacillus

species.
 Sulfate can be further reduced to sulfides by anaerobic sulfate-reducing bacteria,
such as Desulfovibrio, and sulfides can be precipitated out of waste water in the
presence of certain metal ions, such as Fe2+, Zn2+, and Pb2+.
 Sulfates can also be precipitated with the addition of limestone (CaCO3) and
Ca(OH)2 and can be filtered out of waste water.
Conversion of waste water to useful product

 Cellulose or starch in waste – high protein food stuff, SCP, ethanol, org acids,
methane and ethanol
 Yeast Saccharomyces for protein production
 Waste to SCP – SYMBA, PEKILO process
 The SYMBA →potato wastes to SCP and consists of two stages.
 The first stage is for enzymatic hydrolysis of starch by Endomycopsis sp. and
the second is for SCP production on glucose by Saccharomyces sp.
 PEKILO → based on growth of Paecilomyces sp. on waste sulfite liquor.
 Starch to fermented to glucose using enzymes α, β, gluco amylase
 Cellulose to ferment glucose by cellulase (Tricoderma, Clostrida)
 Lecture 32

Microbial Growth Kinetics in biological waste water treatment

RB-4: 580 - 588
 Microbial Growth Kinetics
• Performance of biological process in waste water treatment
 dynamics of substrate utilization &microbial growth
Terminology
• Organic compounds concentration
 biodegradable COD (bCOD) or Total or ultimate carbonaceous (UBOD) - both soluble
biodegradable soluble COD (bsCOD)
• Total suspended solids (TSS)
• Volatile suspended solids (VSS)
• Mixed liquor suspended solids (MLSS)
 Mixture of solids resulting from combining recycled sludge with influent waste water
• Mixed liquour volatile suspended solids (nbVSS)
• Inert inorganic total suspended solids (iTSS)
 Rate of utilization of soluble substrates
• Saturation type equation
rsu = - kXS
Ks + S

rsu = rate of [S] change due to utilization, g/m3.d

- ve sign substrate mass decreases with time due to S utilization
K = max specific substrate utilization rate g substrate/g microorganisms.d
X = [biomass(microorganism], g/m3
Ks = half velocity constant, [S] at one-half the max. specific substrate utilization
rate, g/m3
S = growth limiting [S] in solution, g/m3
 Rate of change of substrate
utilization Vs bsCOD

If k = m /Y  rsu = - m XS
Y(Ks + S)
m max sp. Growth rate, g new cells/g cells.d
Y = true yield coefficient, g/g
 Rate of soluble substrate production from
biodegradable particular organic matter

Rsc P = - kP (P/X) X
Kx + (P/X)

Rsc P = rate of change of particular [S] due to conversion to soluble substrate, g/

m3.d
KP = max specific particular conversion rate, g P/g X.d
P = particular [S], g/m3
X = [biomass(microorganism], g/m3
Kx = half velocity degrade coefficient, g/g
 Rate of biomass growth of soluble substrates
• Biomass growth rate  substrate utilization rate by the synthesis yield coefficient
• Biomass decay  biomass present
• Relationship b/w rate of growth and rate of substrate utilization  applicable in both
batch and continuous culture systems

• Above eqn  by biomass conc.,X,  Specific growth rate

 = specific biomass growth rate, g VSS/g VSS.d

• Specific growth rate corresponds to the change in biomass per day relative to the amt. of
biomass present & f( [S] and kd )
Kinetic coefficients for substrate utilization and
biomass growth
 Rate of oxygen uptake

• Rate of oxygen uptake is related to stoichiometrically to the organic

utilization rate and growth rate
Oxygen uptake rate can be defined as
r0 = rsu – 1.42 rg
• r0– oxygen uptake rate, g O2/m3d
• rsu– rate of substrate utilization, g bs COD/m3d
• 1.42 – cod of cell tissue, g bs COD/ g VSS
• rg– rate of biomass growth, g VSS/
 Effect of temperature
• Temperature dependence of the biological reaction rate assessing the
overall efficiency of a biological treatment process
• Temperature
 influences the metabolic activities of the microbial population
effect on gas-transfer rates
 effect on settling characteristics of the biological solids

kT = k20 (T-20)

kT = rxn. rate coefficient @ T, C

K20 = rxn. rate coefficient @ 20 C
 = Temp. – activity coefficient
T – temp, C
 Total volatile suspended solids
VSS production rate in the aeration tank  sum of the biomass production
(net nbVSS from soluble bCOD), nbVSS production from cells and nbVSS in
the influent
rxr.VSS = Y rsu – kd X + fd (Kd) X + Q Xo,t / V

rxr.VSS - total VSS production rate, g/m3.d

Q – influent flow rate, m3/d
Xo,t – influent nbVSS concentration, m3/d
V- volume of reactor, m3
kd – endogenous decay coefficient, g VSS/g VSS.d
fd – fraction of biomass that remains as cell debris
rru – rate of substrate utilization
Y – synthesis yield coefficient, g VSS/g bsCOD
 Active biomass

• Active biomass in the MLVSS  ratio of the sum of the growth and decay
term divided by the total MLVSS production

F X,act (g/g)  ( - Yrsu – kdX) / rxr.VSS

 Net biomass yield and observed yield

• Net biomass yield ratio of the net biomass growth rate to the substrate
utilization rate

Ybio (g biomass/g substrate used) = - rg/rsu

• Observed yield accounts for the actual solids production that would be
measured for the system

Yobs (g VSS produced/g substrate removed) =- rXt.VSS/rsu

 Determine biomass and solids yields
For an industrial wastewater activated sludge process, the amount of
bsCOD in the influent wastewater is 300 g/cm3 and the influent nbVSS
concentration is 50 g/cm3 . the influent flowrate is 1000 m 3 /d, the
biomass concentration is 2000 g/cm3 , the reactor bsCOD concentration is
15 g/cm3 , and the reactor volume is 105 m3 . if the cell debris fraction, fd
is 0.10, determine the net biomass yield, the observed solid yield and the
biomass fraction in the MLVSS. Use the table for the coefficient value.
• Net biomass yield
• VSS production rate

• Observed solids yield

• Active biomass fraction in MLVSS

THANK YOU
 Lecture 34
Plant cells in Bioreactors
Plant cells in culture compared to microbes

 The plant kingdom → produces thousands of chemicals.

 Only few plants have been scientifically named and screened for the production
of novel and useful compounds.
 The great genetic potential of plants to produce compounds of use to humans →
little exploited.
 The rapid destruction of forests worldwide → the extinction of many plants
without preservation of their genomes.
 Plant products as a medicinal value → practicing worldwide.
 In addition to uses as medicinals, plant products → dyes, food colors, food flavors,
fragrances, insecticides and herbicides.

The production of chemicals from plant cell tissue culture offers a number of
important advantages:
1. Control of supply of product independent of availability of the plant itself.
2. Cultivation under controlled and optimized conditions.
3. Strain improvement with programs analogous to those used for microbial systems.
4. With the feeding of compounds analogous to natural substrates, novel
compounds not present in nature can be synthesized.
PLANT CELLS IN CULTURE COMPARED TO MICROBES
 The primary difference between plant cells and microbes is the ability of the cells
to undergo differentiation and organization even after extended culture in the
undifferentiated state.
 The capacity to regenerate whole plants from undifferentiated cells under
appropriate environmental conditions → Totipotency
 This capacity is essential to plant micro propagation and is often associated with
secondary metabolite formation.
 A callus can be formed from any portion of the whole plant containing dividing
cells.
 The excised plant material is placed on solidified medium containing nutrients and
hormones that promote rapid cell differentiation.
 The callus that forms can be quite large (> 1 cm across and high) and has no
organized structure.

 For both callus and suspension cultures→ chemically defined medium is used.
 Suspension cultures→ maintained in the dark and while exposure to light may be
used to regulate expression of specific pathways, light is rarely used solely to
support growth as most cells are incapable of sustained photoautotrophic growth.
 Typical media use a carbon/energy source such as sucrose, inorganic
nutrients, vitamins, and hormones (auxins, cytokinins, and giberellins).
 The establishment of suspension cultures from callus is generally
straightforward if the callus is friable.
 A piece of callus is placed in a liquid medium in a shake flask with gentle to moderate
agitation, cells or small aggregates of cells will slough off and maintained at 27°C
and a pH of 5.5 in the dark.
 After two or three weeks→ transferred to fresh medium and large aggregates or
residual callus are discarded.
 Suspensions can grow to high cell densities → Aggregates
 Plant cells can be very large (diameters 10 to 100 mm), grow slowly with doubling
times (20 to 100 h).
 Growth is usually non-photosynthetic, sucrose or glucose supplied exogenously
→carbon and energy source with respiration rates are roughly 0.5 mmol O2/h-g dry
weight or about 5% to 15% of that in E. coli.
 Plant cells can often be cultivated at very high densities (70% of the total reactor
volume).
 Plant cells contain a higher percentage of water than bacterial cells (90% to 95%
versus
80 %) → presence of the central vacuole.
 Plant cells tend to secrete relatively few compounds of commercial interest →
cytotoxic if not removed from the cytoplasm.
 In whole plants, there are a number of secondary metabolites which functions as
defensive mechanisms against the attacks from pathogenic fungi, bacteria, and
 The exposure of suspension cultures products to elicitors → rapid increases in
the accumulation of secondary products.
 The use of elicitors has been an important breakthrough in improving volumetric
productivities in bioreactors.
 The most useful elicitor is methyl jasmonate → relatively inexpensive, readily
introduced into large bioreactors and regulates expression of a wide variety of
plant genes.

Bioreactors for Suspension Cultures

 Many of the differences between plant cell cultures and microbes → design and
scale-up of suspension cultures Plant cells are large, and when they are exposed
to turbulent shear fields → twisting motion → damage them.
 Lower levels of shear appear to affect cell surface receptors and nutrient
transport.
 Reactors with high shear must be avoided. However, plants cells → with stand
far more shear than animal cells and shear-tolerant lines can sometimes be
developed.
 Stirred tanks designed for the culture of bacteria are not good choices, but
modified stirred tanks can be suitable. Reactors up to 75000 l have been used
successfully.
 Plant cell cultures can achieve high cell densities and viscosities.
 Airlift reactors for low or moderate cell densities or helical-ribbon impellers for
high-cell-density systems → need for good mixing and the shear sensitivity of
plant cells.
 The formation of aggregates appears to be necessary to achieving the mix of cell
types essential for good secondary-product formation.
 The degree of aggregate formation is influenced by the degree of mixing →
achieve equivalent oxygen transfer or equivalent shear changes upon scale-up,
the degree of aggregation and productivity may change.
 Mixing depends on a combination of sparging and mechanical agitation.
 Plants make at least one volatile hormone, ethylene, and its rapid removal can
affect productivity.
 The low growth rates of plant cells → problems for large-scale systems.
 The primary problem is maintenance of aseptic conditions for the two to four
weeks, low growth rates reduce volumetric productivities and genetic instability
of many cell lines.
 Cryopreservation of cell lines is not possible in all cell lines must be maintained
through routine subculture.
 One strategy → increasing productivity has been the use of a two-phase culture.
 The first phase → uses a medium optimized for growth and second phase →
uses a different medium optimized for product formation.
 The first commercial process with plant cell culture → shikonin production
(utilizes two batch reactors in series)
Comparison of yield (g/l, mmol/l) and productivity (g/l/day, mmol/l/day) for biomass
(Morinda citrifolia) and anthraquinone in different culture systems: (1) Shake flask
(2) flat-blade turbine (3) perforated disc impeller (4) draft tube reactor with Kaplan
turbine
(5) airlift reactor.
Reactors Using Cell Immobilization
 Further improvements in productivity may be possible due to cell immobilization.
 Immobilized cell reactors inherently follow the two-phase approach.
 Cells are grown and then immobilized; once immobilized, conditions for product
formation are optimized. Immobilized cell systems are advantageous when
continuous operation is possible and if the product is, or can be made, extracellular.
 Plant cells → self-immobilize by preferentially attaching to or within a porous
matrix.
 Plant cells have also been entrapped in gels or between membranes.
 Immobilization generates concentration gradients that alter the biosynthetic
capacity of the culture.
 The cell-to-cell contact due to immobilization or the contact of the cell surface with
the surrounding gel phase may also alter cell physiology.
 The production of ajmalicine from periwinkle (Catharanthus roseus).
 Ajmalicine is stored primarily in the vacuole in cells in suspension culture, small
amount (10%) is excreted.
 Ajmalicine has a pKa of 6.3, so that at the growth pH (5.6) much of the alkaloid is
in the neutral form.
 A neutral resin can be used to remove ajmalicine in situ.
 The in situ removal of the product can enhance formation of a product by relief of
feedback inhibition or protection from degradation or further conversion.
 Results of experiments involving combinations of in situ adsorption, use of a
fungal elicitor, immobilization in calcium alginate gels, and the use of a production
medium.
 Using all four approaches increases extracellular concentrations almost 100-fold
and the purity is high.
 The closely related alkaloid, serpentine, has a much higher pKa (10.8) and will not
adsorb into the resin.
Bioreactors for Organized Tissues
 In many cases, neither suspension nor immobilized-cell cultures will produce
satisfactory amounts of a desired metabolite.
 Organ cultures from the same plant may give good yields. In addition to high yields,
organ cultures have a number of distinct advantages
 Some plants will respond to infection by Agrobacterium rhizogenes with rapid root
proliferation → hairy roots.
 The best doubling times for hairy root cultures approach or exceed typical values
for many suspension cultures. Even in species not susceptible to A. rhizogenes
infection, recent results indicate that proper control of hormone content can
accelerate growth rates to acceptable levels.
 The biggest difficulty with the large scale culture of roots is the formation of root
mats which restrict internal mass
 transfer, entrap gas and float, and present significant problems in maintaining a
scalable uniform environment.
 Large-scale units for root culture have been built (20000 l for ginseng roots in
Japan).
 Laboratory-scale reactors using a mist or forced convection of liquid nutrients
appear promising and provide better mass transfer in the center of root mats.
 Advantage of using organ cultures over whole plants is the possibility of using
precursor feeding and elicitors.
 The use of precursors can greatly enhance the formation of flavor compounds in
onion and garlic.
 Shoot cultures present additional problems. Light may be required for some shoot
cultures, while roots can be grown easily in the dark.
 Shoot cultures may be mixotrophic, involving the exogenous supply of sugars as
well as some photosynthesis.
 The most crucial need for light comes from the role that light often plays in cellular
regulation.
 Exposure to light of certain wavelengths is essential to induce synthesis of some
enzymes and these enzymes play a crucial role in secondary metabolism.
 The maintenance of uniform light intensity in a large reactor is a challenging and
partially unsolved problem.
 Shoot cultures has been the belief that they could not be grown under totally
submerged conditions and recent experiments → tobacco shoots will grow as well
in submerged culture as a standard shake flask (3day doubling time).
 In Israel, commercial production of plantlets using totally submerged culture has
been reported and organ cultures are promising vehicles for the production of
some important secondary metabolites.
ECONOMICS OF PLANT CELL TISSUE CULTURES
 Far fewer commercial processes for plant cell culture have been established
than for bacterial, fungal, or animal cell cultures.
 Currently, four large-scale systems have been constructed in Japan and South
Korea (1200 to 30,000 l) and one in Germany (75,000 l).
 In North America, the possible commercial development of three products is
under active consideration.
 As a rule of thumb, most commercial fermentations yield revenue of about 20 ¢/
l-day.
 If the volumetric productivity of a process is known, the wholesale price
necessary to achieve this level of revenue can be estimated.
 The table below lists the published values of the volumetric productivities
reported for some of the more intensely studied cell lines.
 Bulk prices in excess of $220/kg would be necessary to yield 20 ¢/l-day for these
products.
 Recap

Microbial Growth - Batch Growth - Quantifying Cell Concentration - Growth Patterns and Kinetics
Effect of environmental Conditions on Growth Kinetics - Heat Generation by Microbial Growth - Cell
kinetics and cell mortality - Yield
 Cell growth

Substrate + cells  extra cellular products + more cells

ΣS + X  ΣP + nX
 Microbial growth  Ex. For auto catalytic reactions
 Rate of growth α Cell concentration
 Cellular reproduction  normal outcome
 dX/dt α X X – cell mass conc. (g/l); t – time (h)
 dX/dt = μX
 1/X dX/dt = μnet  net specific growth rate
 Net specific growth rate
μnet = μg – kd
• μg – gross specific growth rate

• kd – rate of loss of cell mass due to cell death or endogenous metabolism (h-1)

Net specific replication rate

μR = 1/N dN/dt

• N – cell number concentration

• if kd = 0  μR = μ’R
 Quantifying cell concentration
Petroff – Hausser slide or hemocytometer - Direct cell counting
Calibrated grid microscope no. of cells/grid
Statistical Atleast 20 grids (clear non-aggregated cultures)
Plate count method
• Plates suitable (careful selection) growth medium gelled with agar
• Viable organisms (capable of reproduction) culture samples diluted, spread
on agar surface
• Colonies are formed (Colony Forming Units)
• Plate counts suitable for bacteria, yeast / not suitable for molds
Slide count method
• Plates with growth medium - Microscopic slide
• Agar gel medium placed in a small ring mounted on a microscope slide cells
are spread after incubation microscope to count cells
Commercial particle counter
• Electrical resistance of cells
• 2 electrodes and one electrolyte 1 electrode in tube with orifice and vacuum
created to suck electrolyte with cells
• Based on size and no of cells R increases during application of electrical
potential application between electrodes ht of pulse determined
• Phototube (nephelmetery)
• Scattering of light intensity with respect to cell concentration number of
particles
 Cell mass concentration
Direct method
• Dry weight method
• suitable for solid free medium. No noncellular solids.
• Culture broth centrifuged/filtered washed with buffer 80°C dry for 24 h
• Packed cell volume
• Rough cell concentration in fermentation broth
• Using tapered graduated tube under std rpm and time cell volume
• Rapid method – OD measurement
• Spectrometer absorption of light be suspended solids
• Absence of other solids / light absorbing cells
 Cell mass concentration
Indirect Method
 substrate consumption
 product formation
 intracellular compounds like RNA, DNA, protein, ATP
 nutrients used for cell mass production (nitrate, phosphate, sulphate)
 utilization of carbon source
 oxygen uptake rate
 product of cell metabolism like ethanol, lactic acid
 carbon dioxide production
 changes in pH
 viscosity of fermentation broth (ex. Starch / cellulose biohydrolysis
reduction of viscosity)
Growth pattern and Kinetics in Batch Culture
 Lag phase
• Immediate after inoculation; adaptation of cells to new environment
• Optimum inoculum  minimum lag phase
• To minimize lag phase duration, young and active cells  larger inoculum size (5%
to 10% by volume)
Diauxic growth
• Medium contains more than one carbon
source
• One C source is exhausted, the cells adapt
their metabolic activities to utilize 2nd C
source shift in metabolic pathways in
middle of a growth cycle
 Exponential (log) growth phase
• Cell mass and cell number density increase exponentially with time
• Balanced growth cell grow @ same rate avg. composition of a single cell  Constant
• μnet of cell no. = μnet of cell mass
• Independent of nutrient concentration
• Exponential growth rate – 1st order rxn

Integrate, X = Xo at t = 0 

• Exponential growth - St. line on a semi-log plot of ln X Vs t

• Doubling time
if X = 2 Xo ; t = td  ln (2 Xo/Xo)  μnet td

t'd Based on net specific

td replication rate
 Deceleration growth phase
• Growth decelerates due to either depletion of one or more essential nutrients or the accumulation of
toxic by-products of growth

• Very short period of t

• Unbalanced growth cell composition and size will change and td ≠ t’d

• Due to nutrient depletion or waste accumulation – cell restructuring  increase prospects of cellular
survival
 Stationary phase
• Deceleration phase end, when net growth rate = zero (no cell division) or when growth rate = death rate
• Cells - metabolically active and produce 2o metabolites
• 1o metabolites - growth-related products
• 2o metabolites - nongrowth-related
• Certain metabolites pdn. - during the stationary phase (e.g., antibiotics) due to metabolite deregulation
• Total cell mass concentration - constant, but no. of viable cells may decrease
• Cell lysis may occur and viable cell mass may drop
• 2nd growth phase may occur and cells may grow on lysis products of lysed cells (cryptic growth)

• Endogenous metabolism : cell catabolizes cellular reserves for new

building blocks and for energy-producing monomers
• Maintenance energy
• Cell expend energy to maintain an energized membrane (proton-
motive force)
• Transport of nutrients
• For essential metabolic functions (motility &repair of damage to
cellular structures)
• Appropriate eqn. – describe conversion of cell mass into maintenance energy or
loss of cell mass  cell lysis in stationery phase

dX/X = - kd dt

ln (X/XSo) = - kd t

X = XSo e - kd t

• kd – 1st order rate constant for endogeneous metabolism

• Xso - cell mass concentration at the beginning of the stationary phase
• S = 0  μg = 0 in stationary phase
 Death (decline) phase

• Reason for termination of growth may be either exhaustion of an essential nutrient or accumulation
of toxic products
• Rate of death

• Ns - concentration of cells at the end of the stationary phase

• k’d - 1st order death-rate constant
 Description of growth kinetics
• Stoichiometrically related parameters

• Different yield coefficients

• At end of batch growth period  apparent or observed growth yield

• Ex. Substrate as Glucose (Carbon & energy source)

• On continuous growth culture  true growth yield & apparent yield  differentiated

• Yield coefficient – other substrates or pdt. formation

• Maintenance coefficient,

specific rate of substrate uptake for cellular maintenance

• Cellular maintenance - energy expenditures to repair damaged cellular

components, to transfer some nutrients and products in and out of cells,
for motility, and to adjust the osmolarity of the cells’ interior volume.

• Microbial growth, product formation, and substrate utilization rates -

expressed in the form of specific rates (e.g., normalized with respect to X)
 bioreactions - autocatalytic
 Microbial products can be classified in three major
categories
• Growth associated pdts - produced simultaneously with microbial
growth
μp α μg
Specific rate of product formation,

• Non growth associated pdt. formation takes place during stationary

phase when growth rate = 0

Ex. Many secondary metabolites i.e.. antibiotics (penicillin)

• Mixed-growth-associated pdt. formation takes place during slow growth

and stationary phases
 Effect of environmental Conditions on Growth Kinetics
Temperature
• Organisms classification
(1) psychrophiles (Topt < 20°C)
(2) mesophiles (Topt = from 20° to 50°C)
(3) thermophiles (Topt > 50°C)
• Upto optimal T, 10ºC increase growth doubles, then thermal death

• Higher T kd exceeds μR N reduces

• Activation for growth and thermal death are 10-20; 60-80 kCal/mol
• T also affects yield coefficient
pH
• pH affects activity of enzymes and there by cell growth
• Different organisms have different pH optima
• Bacteria 3 to 8
• Yeast 3 to 6
• Molds 3 to 7
• Plant cells 5 to 6
• Animal cells 6.5 to 7.5
• Fermentation pH vs nitrate
• If ammonia - sole source for nitrogen, H ions will be released to the medium 
pH reduces
• Nitrates - sole sources  pH increases
• Also based on the production of acids, utilization of acids or production of bases
• Evolution or supply of CO2 also changes pH
• pH control by buffer or an active pH (control – important)
DO
• Important in aerobic fermentations
• Limiting substrate as sparingly soluble in water
• Higher cell concentrations, O2 consumption will be more than O2 supply
• Below critical concentration, growth or respiration will be first order dependence on DO
• Above critical concentration, growth rate is independent of O2 conc.
• If the DO < critical O2 concentration, another component  growth-extent limiting like
glucose, ammonium
• Ex. Azotobacter vinelandii at a DO = 0.05 mg/l, μ @ 50% large amount of glucose
• If glucose totally consumed, μ = 0, even if DO = 0.05 mg/l.
• Critical O2 conc.
@ 5% to 10% of the saturated DO concentration for bacteria and yeast
@ 10% to 50% for mold cultures
• Saturated DO concentration in water
• at 25ºC and 1 atm P - 7 ppm
• Presence of dissolved salts and organics, increase in T decreases saturation value
• Fermentation
• O2 sparging air
• O2 transfer from gas bubble to cell  O2 transfer thro’ liquid film surrounding gas bubbles
• Rate of oxygen transfer from the gas to liquid phase

kL - oxygen transfer coefficient (cm/h)

a - gas–liquid interfacial area (cm2/cm3)
kLa - volumetric oxygen transfer coefficient (h-1)
C* - saturated DO concentration (mg/l)
CL - actual DO concentration in the broth (mg/l)
NO2 - rate of oxygen transfer (mg O2/l.h)
• Rate of oxygen uptake or OUR (oxygen uptake rate)

qO2 - specific rate of oxygen consumption (mg O2/g dw cells. h)

YX/O2 - yield coefficient on oxygen (g dw cells/g O2)
X - cell concentration (g d cells/l)
• O2 transfer - rate-limiting step
• Rate of O2 consumption = rate of O2 transfer
• If the maintenance requirement of O2 is negligible compared to growth

or

• DO limitations
To increase O2 transfer rate  O2 rich gas or pure O2 at high P (2-3 atm)
 Redox potential
• Acquire e- and thereby reduce

• Complex function of DO, pH, reducing and oxidizing agents

• Electrochemical potential of fermentation medium

 F – Faraday constant
 Electrochemical potential – mV
 H+ - pH meter
 PO2 in atm pr.

• Redox can be reduced by passing N2 or adding cysteine, HCl, Na2S

• To increase, O2 or oxidizing agents may be passed

 Dissolved CO2 content

• [CO2] α performance of organisms

• Certain level - proper metabolism

• Very high may be toxic to some cells

• Controlled by CO2 in air supply and agitation speed

 Ionic strength
• Affects the transport of certain nutrients in and out of cell
• Metabolic function of cell
• Solubility of certain nutrients like O2
• Ionic strength

• C - concentration of an ion
• Zi - its charge
• I - ionic strength of the medium
 Inhibitory to cellular functions
 High substrate concentrations
 Depending on the type of cells and substrate
• Glucose - above 200 g/l (e.g., ethanol fermentation) probably due to a reduction in
water activity
• NaCl - above 40 g/l due to high osmotic pressure
• Refractory compounds (phenol, toluene, and methanol) - lower conc. (e.g., 1 g/l)
• Maximum non-inhibitory concentrations
• Glucose -100 g/l
• Ethanol - 50 g/l for yeast, much less for most organisms
• Ammonium -5 g/l
• Phosphate - 10 g/l
• Nitrate - 5 g/l
• Substrate inhibition can be overcome by intermittent addition of the substrate to
the medium
 Heat Generation by Microbial Growth
• aerobic metabolism  @ 40% to 50% of energy stored in a C and energy source 
biological energy (ATP) and rest  heat
• For actively growing cells, maintenance requirement - low and heat evolution 
cell growth
• Heat generated during microbial growth  Heat of combustion of the substrate
and of cellular material
• Heat of combustion of the substrate = metabolic heat + heat of combustion of the
cellular material

• ΔHs - heat of combustion of the substrate (kJ/g substrate)

• YX/S – substrate yield coefficient (g cell/g substrate)
• Δ Hc - heat of combustion of cells (kJ/g cells)
• 1/YH -metabolic heat evolved per gram of cell mass produced (kJ/g cells)
Rearranging

• Δ Hs and Δ Hc can be determined from the combustion of substrate and cells

• Δ Hc values for bacterial cells - 20 to 25 kJ/g cells
• YH
• Glucose - 0.42 g/kcal; malate - 0.30 g/kcal; acetate - 0.21 g/kcal; ethanol - 0.18 g/kcal;
methanol - 0.12 g/kcal; methane - 0.061 g/kcal.
• Degree of oxidation of the substrate - strong effect on the amount of heat released
• Total rate of heat evolution in a batch fermentation

• VL - liquid volume (l)

• X - cell concentration (g/l)
• In aerobic fermentations, rate of metabolic heat evolution can roughly be
correlated to the rate of O2 uptake, since O2 - final electron acceptor

• QGR - kcal/h, while QO2 - millimoles of O2/h

• Metabolic heat released - removed by circulating cooling water or cooling jacket
• Temp. control (adequate heat removal) - important limitation on reactor design
• Proper reactor design - ability to estimate heat-removal requirements
Thank You