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04/09/2014

CG4017
Bioprocess Engineering 2
Denise Croker, BM-028
Denise.Croker@ul.ie

Course Structure/Assessment
Course structure
Lectures/tutorials:

3 lectures/tutorials week

Labs:

Lab schedule on CES server, Friday week 1.


Labs are compulsory, no repeat facility available

Assessment
Final exam
Labs

70%
30%

Completion of both assessment components to a satisfactory standard


is compulsory in order to pass the module overall.

Attendance will be monitored on a random basis.

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Lab Sessions

4 Experimental Labs
EXP A: Determination of KLa
EXP B: Operation of a Lyophiliser
EXP C: Operation of a Bioreactor
EXP D: TBC

Complete 2,
Submit 2

3 Computational Labs
Complete as many as you can
EXP E: Fermentation simulation-Superpro Submit 2 ( 1 of each)
EXP F: Fermentation simulation-Polymath
EXP G: Activated Sludge Process simulation-Polymath
EXP H: Diffusion in a microbial film simulation-Polymath

Interview B.Eng. Only


Assessment
B.Eng.: 6% interview, 6 % per lab submission = Total 30%
B.Sc.: 7.5 % per lab submission = Total = 30%

Lab Safety
Lab Safety Guidelines available on the server.

Safety is the MOST IMPORTANT thing in the lab.


Print & Read the guidelines.
Keep a signed copy of the guidelines with you in
the lab.
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Syllabus
Revisit Bioprocess Engineering 1 (CG4003)

Biochemical kinetics: review of basics. Some advanced topics.

Material balances revisited. Mass transfer effects: bulk and internal.

Energy balances revisited. Heat transfer & heat exchanger design for
biochemical processing.

Bioreactor design, sizing, scale-up, operation & control.

Bioreaction product separation & purification processes 2.

Modelling & simulation of bioreaction processes.

Newer applications of bioprocess engineering.

Regulatory & licensing systems.

Learning Outcomes
On completing this module you should:
1.

Possess a knowledge of methodologies for the measurement and control of


oxygen mass transfer in aerobic fermentations.

2.

Understand and apply the principles of bioreactor scale-up.

3.

Demonstrate advanced skills in the design, sizing, operation and optimisation of


bioreactor systems.

4.

Demonstrate advanced skills in the selection, sizing and efficiency evaluation of


bioproduct separation and purification systems.

5.

Possess a knowledge of the regulatory and licensing systems used in the


biochemical industries.

6.

Be competent in the use of a computer package for the simulation of


bioprocessing systems.

7.

Show competence in the practical operation of bioprocessing units.

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Textbooks
CG4017 Outline course notes available from the CES server (DCroker) & SULIS
Recommended: P.M. Doran, 2012, Bioprocess Engineering Principles, Academic
Press, ISBN: 9780122208515. Available in print and electronic formats 65.
M. L. Shuler, and F. Kargi, 2001, Bioprocess Engineering: Basic Concepts, 2nd ed.,
Prentice Hall, ISBN: 0-13-081908-5. (ca. 97 hardback).

R. G. Harrison, P. W. Todd, S. R. Rudge, and D. P. Petrides, 2002, Bioseparations Science


and Engineering, Oxford University Press, ISBN: 978-0-19-512340-1.
G. Walsh, 2007, Pharmaceutical Biotechnology: Concepts and Applications, Wiley,
ISBN: 978-0-470-01245-1

1. REVIEW BIOPROCESS
ENGINEERING

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What is Bioprocess Engineering?


The application of process engineering principles to biochemical
systems on an industrial scale
An historical perspective

Bioprocess engineering essentially began with the requirement for industrial scale
production of antibiotics.
Penicillin discovered in 1928 by Alexander Fleming (UK). Discovery lay dormant for over
a decade.
Renewed interest during World War 2 to treat infection from battlefield wounds: clinical
trials very impressive.
Large scale production initially very difficult due to:
- low product concentration (only 0.001 g/dm3 !) in final fermentation broth
- requirement for large volumes of sterile air for the aerobic fermentation
- necessity for aseptic fermenter operation
- fragile nature of penicillin: recovery & purification challenges
Problems solved by US companies such as Merck, Pfizer, and Squibb: strain & fermenter
improvement gave over 50 g/dm3 product conc. Fermenter volumes of 40,000 dm3
used to meet capacity for treatment of 100,000 per year by 1945.

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Local Perspective MSD Brinny


Co. Cork
Product - Interferon alpha, 2 Beta
Process - Bacterial fermentation of a
strain of E. coli bearing a genetically
engineered plasmid containing an
interferon alfa- 2b gene from human
leukocytes

Treatment Hepatitis and


Rheumathoid Arthritis

Upstream Processing
Full Scale Bioreactor, 30,000L
< 25 Hours

Frozen Mother
Cell Strain
Shake Flask,
5 -6 hours
Seed Reactor
6-8 hours

Critical Process Parameters


- Oxygen Supply
- Medium quality
- Contamination

Process Checks
pH, dissolved Oxygen, temperature, agitation rate automatically
Cell density Off line Test aseptic sampling loop required

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Downstream Processing
Isolate product from the fermenter

Separate the product form the sludge


& Purify

Precipitation & Centrifugation

Cycles of Micro/Ultra Filtration &


Chromatography (15 process steps)

Product = 50 70 Kg of Sludge
All expressed proteins, cell debris

~ 15 L of liquid product solution,


11mg/ml interferon alpha 2 beta

Comparing Chemical/Biochemical Processes


Traditional Chemical Processes

Biochemical Processes

Non-biological reactant mixture, non sterile


facilities

More complex reactant mixture microbes etc,


sterile facilities

Reactant [] will decrease as the reaction


proceeds

Increase in reactant biomass concentration as


reaction progresses

Catalyst will be supplied if needed

Ability of microorganisms to synthesise their


own reaction catalysts (enzymes)

Extreme reaction conditions often needed

Mild conditions of temperature/pH

Typically organic solvents, or mixtures of same.

Usually restricted to aqueous phase

Should be robust crystal products

Mechanically fragile

Aiming for high [] of product in final reaction


mixture

Relatively low [] of product in reaction mixture


complex product solution

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Current status of biochemical engineering


This is a forefront area of modern technological activity, with many opportunities for
individuals who have competence in the field. It demands the following skills:

Numeracy
Understanding of biochemical systems
Knowledge and practice of process engineering
Conceptual/design/original thinking capabilities
Current status of this field can be classified by type of biochemical activity:
1. Microbial (fairly mature technology, with some new developments)
2. Animal cell culture (newer)
3. Plant cell culture (newer)
4. Genetically modified organisms (newer)
5. Medical applications: tissue engineering, gene therapy etc. (very new)
6. Mixed cultures: food products, waste treatment, etc. (old, but poorly understood,
yet important, technologies)

Single Use Technologies


Single use disposable technologies are
becoming popular in many process industries
including biochemicals.
Why?
Greater flexibility
Faster time to market
Cleaning time is the single biggest contributor to turnaround
time in the process industry
Fixed vessels represent large capital expense
Eliminates possibility of cross contamination

What does it look like?

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CSTR Type- Reaction in a Bag

Cell Culture Reactor Wave Reactor

https://www.youtube.com/watch?v=LiYT5b3CsLk&index=4&list=PLUSfjij8XMn7mLVIGnBoaeUf1zMBrK9HW

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2. BIOCHEMICAL KINETICS

2. Biochemical Kinetics
A quantitative knowledge of biochemical kinetics is essential in order to design,
size, and predict the efficiency of bioreactors:

Desired production rate

Bioreactor size
Speed of slowest kinetic step
2.1 Quantifying biochemical kinetics
Biochemical systems are complex in two major respects:
1. Structural complexity:

Some structural features


of a typical cell

http://teachernotes.paramus.k12.nj.us/nolan/cp%20bio.htm

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2. Segregational complexity:

Segregation of a cell culture into different functional units

Segregational complexity can also be modelled in terms of other parameters


such as cell age.
Degree of both structural and segregational complexity may also change with
time and culture environmental conditions.
Quantitative biochemical kinetic models may be:

Non-structured and non-segregated


Non-structured and segregated
Structured and non-segregated
Structured and segregated

Least realistic, least computationally complex

Most realistic, most computationally complex

2.2 Review of basic biochemical kinetics


Non-structured and non-segregated models: balanced growth (fixed cell
composition) is assumed. This assumption is normally valid for exponential
growth phase and for steady-state (single stage) continuous culture.
These models normally fail during transient conditions. In some cases
pseudobalanced growth can be assumed if cell response is fast compared to
speed of the environmental changes and if the magnitude of these changes is
not too large (<20% of initial condition values).

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2.2.1 Generalised non-structured, non-segregated (balanced growth) model


Microbial floc
Cell inner metabolic
region: zone D

Cells

4
3

Cell wall: zone C

Intercellular gel: zone B

Substrate solution: zone A

Substrate molecule

Step 1: Transport of substrate from bulk liquid to floc surface


Step 2: 'Solid' phase diffusion of substrate through intercellular gel (or immobilising
medium)
Step 3: Transport of substrate through cell wall 'outer transport zone'
Step 4: Metabolic conversion of substrate to biochemical products and/or
cell reproduction, by biochemical reaction

2.2.1 Generalised non-structured, non-segregated (balanced growth) model


Microbial floc
Cell inner metabolic
region: zone D

Cells

4
3
2

Cell wall: zone C

Intercellular gel: zone B

Substrate solution: zone A

Substrate molecule

Step 1: Transport of substrate from bulk liquid to floc surface


Step 2: 'Solid' phase diffusion of substrate through intercellular gel (or immobilising
medium)
Step 3: Transport of substrate through cell wall 'outer transport zone'
Step 4: Metabolic conversion of substrate to biochemical products and/or
cell reproduction, by biochemical reaction

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2.2.1 Generalised non-structured, non-segregated (balanced growth) model


Microbial floc
Cell inner metabolic
region: zone D

Cells

4
3

Cell wall: zone C

Intercellular gel: zone B

Substrate solution: zone A

Substrate molecule

Step 1: Transport of substrate from bulk liquid to floc surface


Step 2: 'Solid' phase diffusion of substrate through intercellular gel (or immobilising
medium)
Step 3: Transport of substrate through cell wall 'outer transport zone'
Step 4: Metabolic conversion of substrate to biochemical products and/or
cell reproduction, by biochemical reaction

2.2.1 Generalised non-structured, non-segregated (balanced growth) model


Microbial floc
Cell inner metabolic
region: zone D

Cells

4
3
2

Cell wall: zone C

Intercellular gel: zone B

Substrate solution: zone A

Substrate molecule

Step 1: Transport of substrate from bulk liquid to floc surface


Step 2: 'Solid' phase diffusion of substrate through intercellular gel (or immobilising
medium)
Step 3: Transport of substrate through cell wall 'outer transport zone'
Step 4: Metabolic conversion of substrate to biochemical products and/or
cell reproduction, by biochemical reaction

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2.2.1 Generalised non-structured, non-segregated (balanced growth) model


Microbial floc
Cell inner metabolic
region: zone D

Cells

4
3
2

Cell wall: zone C

Intercellular gel: zone B

Substrate solution: zone A

Substrate molecule

Step 1: Transport of substrate from bulk liquid to floc surface


Step 2: 'Solid' phase diffusion of substrate through intercellular gel (or immobilising
medium)
Step 3: Transport of substrate through cell wall 'outer transport zone'
Step 4: Metabolic conversion of substrate to biochemical products and/or
cell reproduction, by biochemical reaction

Classification of biochemical reaction types for balanced growth kinetic models

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Reaction
type #

Zone(s) involved

General biochemical reaction name

Classification of biochemical reaction types for balanced growth kinetic models

Reaction
type #

Zone(s) involved

(1)

General biochemical reaction name

Cell-free enzyme reactions in non-viscous substrate


media

Classification of biochemical reaction types for balanced growth kinetic models

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Reaction
type #

Zone(s) involved

General biochemical reaction name

(1)

Cell-free enzyme reactions in non-viscous substrate


media

(2)

C+D

Single cell reactions in non-viscous substrate media

Classification of biochemical reaction types for balanced growth kinetic models

Reaction
type #

Zone(s) involved

General biochemical reaction name

(1)

Cell-free enzyme reactions in non-viscous substrate


media

(2)

C+D

Single cell reactions in non-viscous substrate media

(3)

B+C+D

Biological floc/film reactions in non-viscous substrate


media

Classification of biochemical reaction types for balanced growth kinetic models

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Reaction
type #

Zone(s) involved

General biochemical reaction name

(1)

Cell-free enzyme reactions in non-viscous substrate


media

(2)

C+D

Single cell reactions in non-viscous substrate media

(3)

B+C+D

Biological floc/film reactions in non-viscous substrate


media

(4)

B+D

Cell-free immobilised enzyme reactions in non-viscous


substrate media

Classification of biochemical reaction types for balanced growth kinetic models

Reaction
type #

Zone(s) involved

General biochemical reaction name

(1)

Cell-free enzyme reactions in non-viscous substrate


media

(2)

C+D

Single cell reactions in non-viscous substrate media

(3)

B+C+D

Biological floc/film reactions in non-viscous substrate


media

(4)

B+D

(5)

A+ D
A+ C+ D
A+ B+ C+D
A+ B+ D

Cell-free immobilised enzyme reactions in non-viscous


substrate media

Any of the above (1)-(4) in viscous substrate media

Classification of biochemical reaction types for balanced growth kinetic models

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2.2.2 Non-structured, non-segregated (balanced growth) kinetic models


Reaction type #1 - Cell-free enzyme reactions in non-viscous media
Time dependence of substrate concentration:
(Michaelis-Menten Equation or variant)

s
ds
max
dt
Km s

(1)

= rate of substrate utilisation = -ds/dt


Km = Michaelis constant t = time

s = concentration of substrate
max = maximum rate at high s

Reaction type #2 - Single cell reactions (exponential growth phase) in non-viscous


media
dx
x
dt

Time dependence of cell amount:


x = cell concentration

(2)

= specific growth rate of cells

Effect of substrate concentration on


(Monod Equation or variant):
max = maximum specific growth rate

Time dependence of
substrate concentration:

max s
Ks s

(3)

Ks = substrate utilisation constant

ds max qP

mS xo e max t
dt YXS
YPS

(4)

qp= specific rate of product formation


Ys = yield coefficients
ms= cell maintenance coefficient
xo = initial (inoculum) cell concentration
Time dependence of product concentration:

dp
qP xo e max t
dt

(5)

p = metabolic product concentration

Time dependence of cell amount on


substrate concentration (Logistic Equation):
Equations (2) and (3) can be combined to
give

dx max s

x
dt K s s

(6)

This can be then developed as follows,


to give a Logistic Equation that represents
the sigmoidal batch growth curve.

Sigmoidal shape of batch


cell growth curve

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The relationship between microbial growth yield and substrate consumption (yield
coefficient relationship) is:
x xo
(7)
YXS

so s

Combining this with (6) to eliminate s gives:

max YXS so xo x
dx

x
dt ( K sYXS YXS so xo x)

(8)

Integration of (8) yields a sigmoidal cell growth equation, graphically depicted in the
previous slide. Practical use of an equation such as (8) requires a predetermined
knowledge of the maximum cell amount produced, xmax, in a given reaction
environment. xmax is identical to the ecological concept of carrying capacity.

Logistic equations quantify cell growth in terms of


carrying capacity, usually by relating to the
amount of unused carrying capacity:

k 1
xmax

(10)

Thus from (2), we have a general form of the logistic equation:

dx
x

k x1
dt
x
max

(11)

where k = carrying capacity coefficient

Growth models for filamentous organisms: Here the organisms grow as microbial
pellets in submerged media, or as mold colonies on moist substrate surfaces. In
these cases the (normally linear) growth rate is expressed in terms of pellet or
colony, radius (R) or mass (M). Thus in the absence of mass transfer limitations:
2
dM
k p 4 R 2 M 3
dt

kp = growth rate coefficient

(12)

= pellet/colony density

= kp(36)1/3

Reaction type #3 - Biological floc/film reactions in non-viscous media

N s
aL s
max
s
s

(13)

R s
a s
max
s
s

(14)

Microbial film substrate utilisation flux, N:

Microbial floc substrate utilisation rate, R:

= effectiveness factor
a = area of active microorganism/unit film or floc volume
L = film thickness
= floc density
, = biological rate coefficients

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Reaction type #4 Cell-free immobilised enzyme reactions in non-viscous media


Sheet substrate utilisation flux, N:

k1 [e]L[ S ]
1 k3 [ S ]

Spherical particle substrate utilisation rate, R:

k1 [e][ S ]

= effectiveness factor

k1

max
Km

k 2 max
K
D
m e

k3

1
Km

(16)

= particle density

L = sheet thickness
0.5

(1 k3 [ S ])

(15)

(See equation 1. Note: k2 is part of .)

[e] = active enzyme concentration

Reaction types #5 Biochemical reactions in viscous media


Liquid phase substrate external mass transfer limitation, substrate transfer flux, N s:

Ns

ds
k s a sBulk liquid sSurface
dt

(17)

ks = liquid phase substrate mass transfer coefficient


a = (external surface area : volume) ratio of biochemically active particle
Gas phase substrate (O2) external mass transfer limitation, O2 transfer flux, NO2:

NO2

d [O2 ]L
.
k L a [O2 ]Sat
[O2 ]L
L
dt

(18)

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2.3 Advanced topics in biochemical kinetics


Basic non-structured/non-segregated models normally fail during transient
conditions. To address this issue, various workers have proposed more
sophisticated kinetic models, usually at the cost of increased computational
complexity, and difficulties in the experimental verification of key parameters
involved in these more complex mechanistic models.
For these reasons, it should be noted that, although such models may be of
use in representing transient (e.g. batch) behaviour, they are seldom used in
the design and control of steady state bioreactors.
Some examples are considered in the following sub-sections.

Cell reproduction video

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2.3.1 Structured non-segregated kinetic models


Generally applied to unicellular microbial systems. Essentially three types:
Compartmental models Biomass is divided into a set of compartments
relating to cell components, i.e. proteins, hydrocarbons, DNA, RNA, etc. Most
appropriate in genetic engineering and pharmacokinetic modelling.
Metabolic (biochemically) structured models Describe the carbon source
metabolism in the cell in order to predict product formation. Biomass is
considered as in non-structured models.
Chemically structured models Take account of both carbon and nitrogen
metabolism in the cell. Primarily for predicting growth, generally do not
consider product formation.

An example of the second of these types can be found in the paper on ethanol
production by Kluyveromyces marxianus. Here metabolic production of ethanol
in an anaerobic batch reactor is described by a biochemically structured model:

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Cell metabolic reactions are specified as follows:

Solution of the following simultaneous differential equations for rates of


substrate consumption, and biomass and product formation, allows prediction
of the time-concentration profile of the batch fermentation:

Model prediction results (smooth curves) for product formation show good
agreement with experimentally determined data (points), but as expected, do
not show such good agreement for biomass production (see run 3):

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2.3.2 Non-structured segregated kinetic models


Normally involve the use of population balance equations (PBEs) to quantify the
distribution of different biochemical functional units (e.g. single cells, flocs,
vegetative cells, spores, etc.) and/or cells of different ages. PBEs are usually
complex integro-differential and/or partial differential equations involving three or
more interdependent variables, one of which is time. General form of PBE for a
bioreactor:
Rate of change
of cell
concentration

Cell
inflow
rate

Cell
outflow
rate

Cell
birth
rate

Cell
death
rate

(19)

Or, in quantitative terms:

dC ( y, t ) C in ( y) C ( y, t )

B ( y , t ) D( y , t )
dt

where:

(20)

C = cell concentration
t = time
y = segregated function, e.g. cell age function, or distribution of different
cell functional units (spores, vegetative cells, etc.)
= reactor space time = reactor volume/inflow rate

An example of a non-structured, segregated kinetic model can be found in the


paper on age segregated modelling of continuous production of Saccharomyces
cerevisiae (yeast) under aerobic fermentation conditions:

Such bioreactors
often show
oscillatory
behaviour:

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An example of a non-structured, segregated kinetic model can be found in the


paper on age segregated modelling of continuous production of Saccharomyces
cerevisiae (yeast) under aerobic fermentation conditions:
Vent
F

F = volumetric flow rate


V = reaction mixture volume
S = substrate concentration
DO = dissolved oxygen concentration
xL = living cell concentration
xD = dead cell concentration
y = cell age function (0 = birth, 1 = 1st reproduction)
t
= time

Sin(t)
xLin(y,t)
xDin(y,t)

V
S
DO
F
S(t)
xL(y,t)
xD(y,t)

Air

PBE to quantify living cell variations:

dxL ( y, t ) F in
d xL ( y , t )

xL ( y, t ) xL ( y, t ) K ( y, S , DO) xL ( y, t )dy D( y, S , DO) xL ( y, t )


0
dt
V
dy ( y)

(21)
Rate of
change
of living cell
conc.

Living cell inflow


rate outflow rate

Cell death rate

Cell birth rate


= reactor cell removal factor

= cycle length function

M.V.E. Duarte et al, Braz. J. Chem. Eng., vol.20, no.1, Jan./Mar. 2003. http://dx.doi.org/10.1590/S0104-66322003000100002

dxL ( y, t ) F in
d xL ( y , t )

xL ( y, t ) xL ( y, t ) K ( y, S , DO) xL ( y, t )dy D( y, S , DO) xL ( y, t )


0
dt
V
dy ( y)

(21)
where K = cell division probability density function:
2

y
y
K
K
K
K ( y, S , DO) K DO
1 DO
f DO
f SK f yK1 1 f yK2 exp P1 T 1
P2

P2

(22)

The fs in eq. 22 are sigmoidal functions, all other parameters are


constants/coefficients, e.g.

K
f DO

1 e

DO

(23)
K
f DO

DO

M.V.E. Duarte et al, Braz. J. Chem. Eng., vol.20, no.1, Jan./Mar. 2003. http://dx.doi.org/10.1590/S0104-66322003000100002

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K ( y, S , DO)

DO ( ppm)

Age, y (reproduction cycles )

Graphical representation of cell division probability density function, K.

M.V.E. Duarte et al, Braz. J. Chem. Eng., vol.20, no.1, Jan./Mar. 2003. http://dx.doi.org/10.1590/S0104-66322003000100002

dxL ( y, t ) F in
d xL ( y , t )

xL ( y, t ) xL ( y, t ) K ( y, S , DO) xL ( y, t )dy D( y, S , DO) xL ( y, t )


0
dt
V
dy ( y)

(21)
where D = cell death probability density function:

D
D
D
D( y, S , DO) 1D 2D 1 ( DO
(1 DO
) f DO
) f SD f yD1 1 f yD2

(24)

(Again the fs in eq. 24 are sigmoidal functions.)

D ( y, S , DO)

Age, y (reproduction cycles )

DO ( ppm)

Graphical representation of cell death probability density function, D.


M.V.E. Duarte et al, Braz. J. Chem. Eng., vol.20, no.1, Jan./Mar. 2003. http://dx.doi.org/10.1590/S0104-66322003000100002

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Full set of PBEs for this system:


Living cells (eq. 21):

dxL ( y, t ) F in
d xL ( y , t )

xL ( y, t ) xL ( y, t ) K ( y, S , DO) xL ( y, t )dy D( y, S , DO) xL ( y, t )


0
dt
V
dy ( y)

Dead cells:
dxD ( y, t ) F in

xD ( y, t ) xD ( y, t ) D( y, S , DO) xL ( y, t )
dt
V

(25)

Substrate:

S S
dS (t ) F in
S (t ) S (t ) M x
dt
V
KS S

DO DO
DO
xL ( y, t )dy
K DO DO 0

(26)

where Mx = total living cell mass.

Partial differential equations such as eqs. 21, 25, and 26 may be solved by a number of
methods such as:
Finite element/method of lines/Galerkin method
Method of characteristics
Finite difference method
M.V.E. Duarte et al, Braz. J. Chem. Eng., vol.20, no.1, Jan./Mar. 2003. http://dx.doi.org/10.1590/S0104-66322003000100002

Results give a successful replication of the experimentally observed periodic


behaviour:

xL ( y , t )

Age, y (reproducti on cycles )

Time (hr )

xD ( y , t )

Age, y (reproducti on cycles )

Time (hr )

Modelling results for time variation of live and dead cell concentrations
M.V.E. Duarte et al, Braz. J. Chem. Eng., vol.20, no.1, Jan./Mar. 2003. http://dx.doi.org/10.1590/S0104-66322003000100002

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Cell mass ( g / L)

Time (hr )

Substrate conc., S ( g / L)

Time (hr )

Modelling results for time variation of total cell and substrate concentrations

M.V.E. Duarte et al, Braz. J. Chem. Eng., vol.20, no.1, Jan./Mar. 2003. http://dx.doi.org/10.1590/S0104-66322003000100002

2.3.3 Structured segregated kinetic models


These comprise the most sophisticated representations of biochemical reactions,
involving both a structured model of the system biochemistry and a quantification
of the segregational nature of the biochemical functional units. Essentially they
involve a combination of the approaches used in sections 2.3.1 and 2.3.2.
The paper by Henson et al, on modelling of continuous culture of Saccharomyces
cerevisiae (budding yeast) in a chemostat under aerobic fermentation conditions,
provides an example of this type of approach. In this case the segregational
aspect involves the use of a PBE for the budding yeast cell reproduction cycle:

dW (m, t ) d K ( S ' )W (m, t )

dt
dm

m'

2 p(m, m' )(m' , S ' )W (m' , t )dm D (m)W (m, t )


(27)

W = cell mass distribution t = time S = intracellular substrate concentration


m = mass associated with mother cells
m = mass associated with daughter cells
p = newborn cell mass distribution function
= cell division intensity function
D = dilution rate (= volumetric flow rate/reaction mixture volume)
http://www.youtube.com/watch?v=FcV1ydls9hg

M. A. Henson et al , Biotechnol. Prog., vol. 18, 10101026, (2002).

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dW (m, t ) d K ( S ' )W (m, t )

dt
dm

m'

2 p(m, m' )(m' , S ' )W (m' , t )dm D (m)W (m, t )


(27)

The cell division intensity function, , is given by a sigmoidal-type function, e.g.,

(m, S ' ) e m md

* 2

m mt* mo , md*

when

(28)

m*t = cell transition mass


m*d = cell division mass
mo = mass above m*t that mother cell must have before division is possible
, = constant parameters
The newborn cell mass distribution function, p, is given by a Gaussian function:

p(m, m' ) A exp m mt*

A exp m m' m
2

* 2
t

(29)

A, = constant parameters.
This Gaussian function has two peaks in the cell number distribution, one at m*t ,
corresponding to mother cells, and one at m*t- m corresponding to daughter cells.

M. A. Henson et al , Biotechnol. Prog., vol. 18, 10101026, (2002).

The structured aspect of this kinetic model involves a quantitative description of


the extracellular environment. This comprises equations for reactor mass balance,
cell wall mass transfer, and biochemical reaction rate for each of the substrates
and metabolic products. For example, for glucose oxidation, we have:
Glucose mass balance

k gf (G' ) k go (G' )
dG
DG f G

W (m, t )dm
dt
Ygo
Ygf
0

(30)

G = extracellular glucose concentration


G = intracellular glucose concentration
Gf = reactor feed glucose concentration k gf = glucose fermentation rate
kgo = glucose oxidation rate
Ygf = glucose fermentation yield coefficient
Ygo = glucose oxidation yield coefficient
Glucose cell wall mass transfer

dG'
g G G'
dt

(31)

g = glucose first order cell wall mass transfer coefficient

M. A. Henson et al , Biotechnol. Prog., vol. 18, 10101026, (2002).

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Glucose oxidation rate:

k go (G' , O)

goG'

O
.
K go G' K gd O

(32)

O = dissolved oxygen concentration go = glucose maximum oxidation rate


Kgo = glucose substrate utilisation coefficient for glucose oxidation
Kgd = dissolved oxygen substrate utilisation coefficient for glucose oxidation

This structured segregated model, whilst complex to implement, was shown,


under certain conditions, to give a good quantitative representation of both
oscillatory and long term behaviour of the chemostat bioreactor.

M. A. Henson et al , Biotechnol. Prog., vol. 18, 10101026, (2002).

Modelling versus experimental results for chemostat yeast fermentation oscillatory behaviour
M. A. Henson et al , Biotechnol. Prog., vol. 18, 10101026, (2002).

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Dilution rate
increase here

Comparison of structured segregated model versus actual plant data showing


effect of dilution rate ramp increase at t = 96hours
M. A. Henson et al , Biotechnol. Prog., vol. 18, 10101026, (2002).

2.3.4 Biochemical kinetic models: the future?


The availability of cheap computational power, coupled with the intensive efforts
currently underway to understand more and more detail about the operation of cell
biochemistry, is paving the way for complete quantitative models of individual
microorganism species.
Scientists at Stanford University announced in July 2012, the first complete
computer model of the bacterium mycoplasma genitalium:

Mycoplasma genitalium

http://www.kurzweilai.net/first-complete-computer-model-of-an-organism

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3. BIOCHEMICAL MATERIAL
BALANCE

3. Biochemical Material Balances


Material balances: very important for quantifying and keeping track of the amounts of
reactants and products in a biochemical process. A fundamental tool of process
engineering.

3.1 Material balances revisited


From the law of conservation: for the quantity S in a system, where S = mass or
number of moles of a chemical or biochemical species:
Rate of accumulation
or dissipation of S

= Rate of input - Rate of output Rate of formation (33)


of S
of S
or consumption of S

The General Material Balance Equation

For systems at steady state (no accumulation/depletion) the total mass balance is:
Rate of input = Rate of output
of mass
of mass

(34)

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In the case of reactive species in a system at steady state, equation (33) can
also be simplified to:
0

Rate of input - Rate of output Rate of formation


of S
of S
or consumption of S

or:
Rate of input + Rate of formation = Rate of output + Rate of consumption (35)
(where S = mass of, or number of moles of, a chemical or biochemical species)

Essential good practice for carrying out material balance calculations


Draw a process diagram showing clearly all relevant information: a simple box diagram
showing all flows entering and leaving the system, together with the corresponding
known quantitative information (flow rates etc.).
Choose a consistent set of units and state it clearly. Units must be given for all
variables shown in the diagram.
Select a basis for the calculation and state it clearly. It is helpful to focus on a specific
quantity of material entering or leaving the system (flow rate for continuous processes,
total amount for batch or semi-batch).
State all assumptions made in order to carry out the calculation. For example: system
does not leak or cells do not burst during filtration
Identify which components of the system, if any, are involved in the reaction. This
is necessary for correct formulation of the material balance equation.

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Procedure for performing material balance calculations


A.

Assemble:

(1) Draw the flowsheet, showing all pertinent data with units.
(2) Define the system boundary and draw it on the flowsheet.
(3) Write down the reaction stoichiometric equation (if any).

B.

Analyse:

(4) State any assumptions


(5) Collect and state any extra data needed (e.g. constants, etc.).
(6) Select and state a basis for the calculation.
(7) List the compounds, if any, that are involved in reaction.
(8) Write down the appropriate material balance equation.

C.

Calculate:

(9) Set up a calculation table showing all components of all streams


passing across system boundaries.
(10) Calculate any unknown quantities by applying the material
balance equation.
(11) Check that your results are reasonable and make sense.

D.

Finalise:

(12) Answer the specific questions asked in the problem.


(13) State the answers clearly, using appropriate significant figures.
Important!!

From Bioprocess Engineering Principles by Pauline M. Doran

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From Bioprocess Engineering Principles by Pauline M. Doran

From Bioprocess Engineering Principles by Pauline M. Doran

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From Bioprocess Engineering Principles by Pauline M. Doran

From Bioprocess Engineering Principles by Pauline M. Doran

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From Bioprocess Engineering Principles by Pauline M. Doran

From Bioprocess Engineering Principles by Pauline M. Doran

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From Bioprocess Engineering Principles by Pauline M. Doran

From Bioprocess Engineering Principles by Pauline M. Doran

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From Bioprocess Engineering Principles by Pauline M. Doran

From Bioprocess Engineering Principles by Pauline M. Doran

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3.2 Metabolic stoichiometry for growth and product formation


Material balances require stoichiometric equations to quantify the changes involved in
reactions. Although these are more complex in the case of biochemical reactions,
nevertheless the law of conservation of matter is still obeyed, the advantage being that
the detailed intricacies of cell internal biochemisty can be overlooked and a
macroscopic quantification of the overall reaction can be achieved.
3.2.1 Growth stoichiometry and elemental balances

Basic stoichiometry for cell growth and primary metabolite formation

Taking one mole of substrate as the basis, we can write this as a balanced equation:
CwHxOyNz + aO2 + bHgOhNi
Substrate

Nitrogen
source

cCHONd + dCO2 + eH2O + fCjHkOlNm (36)


Biomass

Primary
metabolic
product

where a, b, c, d, e, and f are the stoichiometric coefficients.

In the chemical formula for substrate, e.g. for glucose: w=6, x=12, y=6, z=0
In the chemical formula for nitrogen source, e.g. for ammonia: g=3, h=0, i=1
The chemical formula for dry biomass is given as CHONd
Note: eq. 36 only applies to reactions involving growth and/or primary metabolic
product formation. Secondary metabolite formation requires separate stoichiometric
equations for growth and product formation.
Growth factors such as vitamins and minerals, additionally taken up in small amounts
during metabolism, are generally neglected in terms of their contribution to the
stoichiometry and energetics of the overall reaction.
Other substrates and primary products can easily be added to eq. 36, if appropriate.

Balancing eq. 36 requires a formula for the biomass involved.

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As can be seen from this table, over 90% of cell biomass can be accounted for by the
elements C, H, O and N, so cell biomass formulae are normally expressed in terms of
these elements only.
From Bioprocess Engineering Principles by Pauline M. Doran

CH1.8O0.5 N0.2
Average biomass molecular weight = 24.6 (+5-10% as residual ash/other elements)
From Bioprocess Engineering Principles by Pauline M. Doran

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CwHxOyNz + aO2 + bHgOhNi

cCHONd + dCO2 + eH2O + fCjHkOlNm (36)

In order to balance eq. 36 for a particular biochemical reaction, we must determine the
stoichiometric coefficients a-f. As in balancing chemical equations, elemental balances
can be done:
Carbon:
Hydrogen:
Oxygen:
Nitrogen:

w = c + d + fj
x + bg = c + 2e + fk
y + 2a + bh = c +2d + e + fl
z + bi = cd + fm

(37)
(38)
(39)
(40)

However we have six unknowns (a-f) and only four simultaneous equations, so these
cannot be solved for a-f. In addition, the fact that water is usually present in great
excess in biochemical reactions, often leads to difficulties in experimentally quantifying
changes in water concentration. This in turn means that H and O balances can be
unreliable.
Alternative stoichiometric information can be obtained by using an experimentally
determined respiratory quotient (RQ) for the reaction of interest:
RQ

moles CO2 produced d

moles O2 consumed
a

or aRQ = d

(41)

3.2.2 Electron balances and yield coefficients


Additional information for balancing stoichiometric equations can be obtained using
electron balances and yield coefficients. In the former, the principle of conservation of
reducing power or available electrons, is applied to obtain quantitative relationships
between substrates and products. An electron balance shows how the available
substrate electrons are distributed in the reaction.
Available electrons, = number of electrons available for transfer to oxygen on
combustion of a substance to CO2, H2O and N-containing
compounds.
Calculated from elemental valences: C=4, H=1, O=-2, P=5, S=6. For N, the number of
available electrons depends on the reference state: -3 if NH3, 0 if N2, 5 if NO3-.
Reference state for cell growth is normally that of the reaction N source. (Here well
take it as NH3).
Degree of reduction, = number of equivalents of available electrons in the amount of
material containing 1g atom of carbon
Thus for substrate CwHxOyNz: s = 4w + x 2y 3z, and s = s/w (or s = w s)
Note: degree of reduction relative to CO2, H2O, and NH3 is zero.

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Applying this idea (i.e. LHS = RHS) to eq. (36), for the case where there is no
product formation (cell growth only):
CwHxOyNz + aO2 + bHgOhNi
gives:

cCHONd + dCO2 + eH2O

wS 4a = cB

(36a)
(42)

where S and B refer to substrate and biomass respectively.


Experimentally determined yield coefficients (Y) provide another source of quantitative
information to allow balancing of stoichiometic equations for biochemical reactions.
For biomass from substrate:

YXS

mass cells produced


mass substrate consumed

(43)

Many factors can affect the value of a yield coefficient including nature of C and N
sources, pH, temperature, and in aerobic cultures nature of the oxidising agent.
However, when the yield coefficient is constant, its experimentally measured value can
be used to determine c in equations (36) or (36a), since eq. (43) can be written in
stoichiometric terms (assuming 1 mole substrate as the basis) as:
YXS

c ( MW biomass)
( MW substrate)

(44)

where MW = molecular weight of substrate or biomass

Care must be exercised when using eq. (44), since it does not apply if a significant
amount of substrate is used for maintenance activities instead of growth. In such cases
the experimentally measured values of YXS must be adjusted to account for this.
We can also have a yield coefficient for primary metabolic product from substrate
YPS

mass product produced


f ( MW product )

mass substrate consumed


MW substrate

(45)

Again we must remember that eq. (45) only applies for primary metabolic product.
3.2.3 Metabolic stoichiometric calculations: summary
From our considerations in the previous two sections, we can see that it is now
possible to balance our stoichiometric metabolism equation (36) for all 6 unknowns:
CwHxOyNz + aO2 + bHgOhNi
cCHONd + dCO2 + eH2O + fCjHkOlNm (36)
Carbon balance:
Nitrogen balance:
Respiratory quotient:
Electron balance:
Yield coefficient biomass:
Yield coefficient product:

w = c + d + fj
z + bi = cd + fm
aRQ = d
wS 4a = cB
c = YXS (MW substrate)/(MW biomass)
f = YXP (MW substrate)/(MW product)

(37)
(40)
(41)
(42)
(44)
(45)

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3.3 Material balances for processes with recycle streams


In microbial reactions, bioreactor productivity can be significantly improved by retaining
the active biomass within the reaction system. One way to do this is to separate out the
cellular material from the reactor effluent stream and recycle it to the reactor.

Chemostat bioreactor with recycle of biomass

In performing material balances on such a system, we need to be aware that various


system boundaries may be chosen (e.g. around the entire system, around the reactor
only, or around the separator only).

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Assumptions: reaction occurs only in the chemostat; separation occurs only in separator; no
biomass in the feed or product streams (only in the cell waste stream). Let r X = biomass growth
rate and rS = substrate consumption rate.

Biomass balance around the entire system:

Accumulation/depletion = Input
dX
dX R
VR 1 VS
0
dt
dt

Output

Reaction

FW X R

rX VR

At steady state:
0

FW XR

rXVR

Thus wastage rate of biomass must equal growth rate of biomass to maintain s.s.

Assumptions: reaction occurs only in the chemostat; separation occurs only in separator; no
biomass in the feed or product streams (only in the cell waste stream). Let r X = biomass growth
rate and rS = substrate consumption rate.

Substrate balance around the entire system:


Accumulation/depletion = Input
dS
dS
VR 1 VS R
F S0
dt
dt
At steady state:
0
= FS0

Output

F1S P FW S R

F1SP - FW SR

Reaction

rsVR

+ rSVR

Here the consumption rate of S equals the difference between the inlet and outlet mass
flow rates of S.

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Material balances around individual units are often more useful for quantifying dynamic
(non-steady state) behaviour.
Biomass balance around the chemostat:
VR

dX 1
dt

FR X R

( F FR ) X 1

rX VR

Substrate balance around the chemostat:


VR

dS1
dt

F S0 FR S R

( F FR ) S1

rsVR

Biomass balance around the separator:


VS

dX R
dt

( F FR ) X 1 ( FW FR ) X R

Substrate balance around the separator:


VS

dS S
dt

( F FR ) S1

( FW FR ) S R F1S P

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Other important flow rate equations necessary to solve material balances with recycle:
Recycle flow rate:

FR

Cell concentrate flow: FW +FR =

R.F

where R = recycle ratio

(F+FR)/C

where C = settler concentration factor

Values of R and C must be chosen so that the cell waste flow rate (F W ) is positive.

Simulating Chemostat Operation with Polymath

Biomass balance around Chemostat

dX 1
VR
dt
dX 1
dt

( F FR ) X 1

FR X R
[ FR X R

( F FR ) X 1

rX VR
rX VR ] / VR

Substrate balance around Chemostat

dS
VR 1
dt
dS1
dt

F S0 FR S R
[ F S0 FR S R

( F FR ) S1
( F FR ) S1

rsVR
rsVR ] / VR

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POLYMATH model for chemostat with biomass recycle


#Chemostat with separation and biomass recycle

Chemostat with recycle.pol

d(x1) / d(t) = (fr*xr-(f+fr)*x1+rx*V)/Vr


# Reactor biomass balance
x1(0) = 2
d(s1) / d(t) = (f*so+fr*sr-(f+fr)*s1+rs*Vr)/Vr # Reactor substrate balance
s1(0) = 10
d(xr) / d(t) = ((f+fr)*x1-(fw+fr)*xr)/Vs
# Separator biomass balance
xr(0) = 10
d(ss) / d(t) = ((f+fr)*s1-(fw+fr)*sr-f1*sp)/Vs # Separator substrate balance
ss(0) = 5
fr=r*f
# Recycle flow equation
fw=((f+fr)/c)-fr
# Separator biomass concentrator efficiency
f1=f-fw
# Separator total mass balance
rs=-rx/Yxs
# Yield coefficient substrate to biomass
rx=mumax*x1*s1/(Ks+s1) # Biochemical kinetics
Vr=1000
# Operating parameters & constants
Vs=100
so=10
sp=ss
sr=ss
Yxs=0.6
mumax=0.5
Ks=0.5
c= 3
# Settler concentration factor
r=0.2
# Recycle ratio value
f=10
# Inlet feed flow rate
t(0) = 0
# Start time
t(f) = 100 # End time

POLYMATH model for chemostat with biomass recycle


#Chemostat with separation and biomass recycle

Chemostat with recycle.pol

d(x1) / d(t) = (fr*xr-(f+fr)*x1+rx*V)/Vr


# Biomass in Reactor
x1(0) = 2
d(s1) / d(t) = (f*so+fr*sr-(f+fr)*s1+rs*Vr)/Vr # Substrate in Reactor
s1(0) = 10
d(xr) / d(t) = ((f+fr)*x1-(fw+fr)*xr)/Vs
# Biomass in Separator
xr(0) = 10
d(ss) / d(t) = ((f+fr)*s1-(fw+fr)*sr-f1*sp)/Vs # Substrate in Separator
ss(0) = 5
fr=r*f
# Recycle flow equation
fw=((f+fr)/c)-fr
# Separator biomass concentrator efficiency
f1=f-fw
# Separator total mass balance
rs=-rx/Yxs
# Yield coefficient substrate to biomass
rx=mumax*x1*s1/(Ks+s1) # Biochemical kinetics
Vr=1000
# Operating parameters & constants
Vs=100
so=10
sp=ss
sr=ss
Yxs=0.6
mumax=0.5
Ks=0.5
c= 3
# Settler concentration factor
r=0.2
# Recycle ratio value
f=10
# Inlet feed flow rate
t(0) = 0
# Start time
t(f) = 100 # End time

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Chemostat with Biomass Recycle; Standard Operating Conditions, F = 10, R = 0.2, C = 3

At t = 60 min
X1 (biomass in Reactor) = ~8
S1 (Substrate in the Reactor = 0
XR (Biomass in Recycle Line) = ~25
SS (Substrate in Settler = 0
All substrate removed at ~ t = 20- mins

At t = 60 min
X1 (biomass in Reactor) = ~8
S1 (Substrate in the Reactor = 0
XR (Biomass in Recycle Line) = ~30
SS (Substrate in Settler = 0
All substrate removed at ~ t = 5 mins

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At t = 60 min
X1 (biomass in Reactor) = 0
S1 (Substrate in the Reactor = 0
XR (Biomass in Recycle Line) = 0
SS (Substrate in Settler = 10

At t = 60 min
X1 (biomass in Reactor) = ~8
S1 (Substrate in the Reactor = 0
XR (Biomass in Recycle Line) = ~30
SS (Substrate in Settler = 0
All substrate removed at ~ t = 18 mins

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At t = 60 min
X1 (biomass in Reactor) = ~8
S1 (Substrate in the Reactor = 0
XR (Biomass in Recycle Line) = ~38
SS (Substrate in Settler = 0
All substrate removed at ~ t = 22 mins

At t = 60 min
X1 (biomass in Reactor) = ~8
S1 (Substrate in the Reactor = 0
XR (Biomass in Recycle Line) = ~12
SS (Substrate in Settler) = 0
All substrate removed at ~ t = 25 mins

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4. MASS TRANSFER
Doran (2nd Edition), Chapter 13.

4. Mass Transfer
Due to the complex nature and rheological behaviour of many biochemical systems, a
detailed understanding of mass transfer is often important in order to be able to quantify
biochemical processing operations.
There are two major aspects to mass transfer in biochemical processes: internal and
external.
Internal mass transfer is concerned with the movement of substrates and products
within cellular agglomerates or immobilised enzyme systems, and is of major
importance in quantifying reaction rates.
External mass transfer deals with the transport of materials in the fluid phase(s)
surrounding the biochemically reactive entity, and features prominently in both
reactions (particularly in aerobic and/or highly viscous reaction media), and in
separation processes.

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Int. m.t.
Ext. m.t.

Substrate concentration profile for


a spherical biocatalyst particle
Immobilised biocatalysts:
(a) cells; (b) enzymes

From Bioprocess Engineering Principles by Pauline M. Doran

4.1 Review of mass transfer concepts previously encountered


4.1.1 Internal m.t.: the effectiveness factor,
Recall eq. 14 for the substrate utilisation rate, R, in a microbial floc :

R s
a s
max
s
s

(14)

a = area of active microorganism/unit floc volume


= floc density , = biological rate coefficients
is a complex function of many of the chemical and physical properties of the reaction
system, including:
Substrate concentration
Diffusion coefficient(s) for transport through the intercellular gel/immobilising
medium
Physical structure of the intercellular gel/immobilising medium
Floc/film size/thickness
Microorganism effective surface area
Reaction rate coefficients

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The effectiveness factor can be defined in practical terms:

actual rate of substrate utilisatio n


(46)
rate of substrate utilisatio n in absence of gel / immobilising medium

is related to the tortuosity factor (Thiele modulus), , of the gel/medium, for example
for 1st order reaction kinetics in a flat plate:

tanh

4.1.2 External mass transfer


The two major situations in biochemical processing where external mass transfer is of
importance are with viscous substrate media (liquid-solid m.t.) and in aerobic
fermentations (gas-liquid m.t.).
Liquid-solid mass transfer in viscous substrate media

Bulk substrate (A) solution


Concentration of A in bulk = CAb

Substrate concentrations near


biochemical solid surfaces

Floc,
film,
or particle

CAs

Liquid boundary layer at


particle surface

Concentration of A at
particle surface = CAs

Poorly mixed and/or high viscosity substrate soln: CAs Cab :

NA

dC A
k s a C Ab C As
dt

(17)

NA = rate of substrate mass transfer


ks = liquid phase substrate mass transfer coefficient
a = (external surface area:volume) ratio of biochemically active particle

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Gas-liquid mass transfer of oxygen in aerobic fermentations


[O2]G
[O2]G(i)

Liquid
film

Bulk liquid
(substrate
solution)

Oxygen concentrations at
the gas-liquid interface

Gas
film

Bulk gas
(bubble)

[O2]L(i)

[O2]
[O2]L
Gas-liquid
interface

NO2

d [O2 ]L
.
k L a [O2 ]Sat
[O2 ]L
L
dt

(18)

NO2 = oxygen transfer rate


kL = oxygen mass transfer coefficient
a = gas bubble/liquid interfacial area per unit liquid volume
[O2]Lsat = dissolved oxygen concentration when liquid is saturated with oxygen
Major factor affecting NO2: agitation efficiency in the aerated medium

4.2 Internal m.t. concepts as applied to heterogeneous reactions


4.2.1 Ficks law of diffusion
Consider a binary mixture of molecular component A and B. In the case where
concentration of A is non-uniform, and where there is no large-scale fluid motion e.g.
due to stirring, then mixing occurs by random molecular motion.

Ficks law of diffusion states that mass


flux is proportional to the concentration
gradient:

JA

NA
dC A
DAB
a
dy

(47)

JA = mass flux of A
NA = rate of mass transfer of A
a = mass transfer area
DAB = diffusivity of A in a mixture of A and B
Concentration gradient of component A
inducing mass transfer across area a
From Bioprocess Engineering Principles by Pauline M. Doran

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4.2.2 Steady-state shell mass balance on a biocatalyst particle


The exact equations describing internal mass transfer
depend on the particle geometry and the reaction
kinetics. Consider first a spherical particle.
A mass balance may be performed by considering
the processes of mass transfer and reaction occurring
in the shell of radius r.
Shell mass balance on
a spherical particle

Recall the general material balance equation:


Rate of accumulation
or dissipation of A

= Rate of input - Rate of output Rate of formation (33)


of A
of A
or consumption of A

We can apply this with the following assumptions:


(i) The particle is isothermal
(vi) The particle is homogeneous
(ii) Mass transfer occurs only by diffusion
(v) The partition coefficient for A is unity
(iii) Ficks law applies with constant DAe
(vii) The system is at steady state
(iv) [A] varies with a single spatial variable
From Bioprocess Engineering Principles by Pauline M. Doran

Accum.
/dissip.
of A
0

Input
of A

= DAe

Output
of A

dC A
dC A
- DAe
4 r 2
4 r 2
dr
dr
r
r r

Formation/ (33)
consumption
of A

rA 4 r 2 r
(CA = concn. of A)

Dividing by 4r gives:

DAe dC A r 2
DAe dC A r 2

dr
dr
r r
r

rA r 2
r
or

dC A 2

DAe
r
dr

r r2
A
r

where the numerator term refers to the difference in the two numerator terms from
the previous equation.

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As r tends towards 0, we can write:

dC A 2

d DAe
r
dr

r r2
A
dr

Since DAe is independent of r, it can be moved outside the differential:

dC

d A r2
dr

r r2
DAe
A
dr

The bracketed term is the derivative of a product, d(u.v)/dx, where u = dCA/dr and
v = r2. Thus we can expand this derivative to give:

d 2C A 2
dC
DAe
r 2r A
2
dr
dr

rA r 2

(48)

Integration of this 2nd order differential equation yields an expression for how C A varies
with distance (r) inside the particle. This must be done on a case-by-case basis
according to the reaction kinetics however, since rA is a function of CA in most cases.

4.2.3 Substrate concentration profile: 1st order kinetics & spherical geometry

d 2C

dC

A 2
With 1st order kinetics, eq. (48) becomes: DAe
r 2r A k1C A r 2 0
2
st
dr
dr
(k1 = 1 order rate constant)

(49)

According to assumptions (i), (iii) and (vi) above, k 1 and DAe can be considered
constant. Since this is a 2nd order differential equation, two boundary conditions are
required:
dC A
CA = CAs at r = R, and
0 at r = 0

dr

where CAs is substrate concentration at the particle surface. The latter boundary
condition is called the symmetry condition: the substrate concentration profile is
symmetrical at the centre of the particle, thus the slope (dC A/dr) is zero at r=0.

From Bioprocess Engineering Principles


by Pauline M. Doran

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04/09/2014

Integration of (49) with these boundary conditions yields:

C A C As

R sinh r k1 / DAe
r sinh R k1 / DAe

(50) ,

where

sinh( x)

e x e x
2

Eq. (50) can be used to calculate the particle substrate concentration profile.
4.2.4 Substrate concentration profile: zero order kinetics & spherical geometry
In this case eq. (48) becomes:
(k0 = zero order rate constant)

d 2C A 2
dC
DAe
r 2r A
2
dr
dr

k0 r 2 0

(51)

Assuming that CA can be zero only at r = 0 (centre of the sphere), integration of eq. (51)
with the same boundary conditions as before, gives:

C A C As

k0
r 2 R2
6 DAe

(52)

It is important, from a practical perspective, that the particle core does not become
starved of substrate. This is more likely with larger particles. Here we can calculate
the maximum particle size, Rmax where CA > 0 (depletion only occurs at r = 0) from
eq. (51), since then CA = r = 0, and:
6 DAe C As
(53)
Rmax

k0

4.2.5 Substrate concentration profile: Michaelis-Menten kinetics & spherical


geometry

d 2C

dC

A 2
In this case eq. (48) becomes: DAe
r 2r A max A r 2 0
2
dr K m C A
dr

(54)

Analytical integration of eq. (54) is difficult,


so it is usually resolved using numerical
computational means. Remembering that the
limiting cases of the M-M equation are zero
and 1st order kinetics, we can expect that the
solutions found in the previous two sections
can be used to determine the extremities of
the M-M solution.
Experimental verification of these types of
calculated concentration profiles has been
done in a number of studies, using microanalytical techniques (e.g. opposite).

From Bioprocess Engineering Principles by Pauline M. Doran

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4.2.6 Concentration profiles with other geometries


Similar treatments may be applied as in
sections 4.2.3 4.2.5, for other biocatalyst
geometries. The most important of these
is flat-plate geometry.
Here it is assumed that plate length and width
are infinite (or at least that these are much
greater than its thickness), in order to keep the
single spatial variable assumption.
The boundary conditions in this case are:

and

CA = CAs

at

z = b,

dC A
dz

at

z=0

Substrate concentration
profile in an infinite flat plate
From Bioprocess Engineering Principles by Pauline M. Doran

4.2.7 Steady-state substrate concentration profiles: summary


These can be summarised in the following:

(50)

(55)

(52)

(56)

From Bioprocess Engineering Principles by Pauline M. Doran

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4.2.8 Prediction of observed reaction rate


Equations such as (50), (52), (55) and (56) allow prediction of the overall reaction
rates, rA,obs, over the entire particle. Consider the case of spherical particles.

rA,Obs

1st order reaction:

rR

r 0

k1C A dV p

(57)

Substituting eq. (50) for CA in (57), and integrating gives:

rA,Obs 4 R DAe C As R k1 / DAe coth R k1 / DAe 1

(58) , where: cothx

e x e x
e x e x

Zero order reaction: Assuming CA > 0 everywhere in the particle, then the rate will be
constant and independent of CA. Thus the overall rate is simply the rate constant
multiplied by the particle volume:

rA,Obs

4
R 3 k0
3

(59)

Michaelis-Menten kinetics: Since CA cannot be expressed explicitly as a function of r,


then numerical methods must again be used.

4.2.9 Thiele modulus () and effectiveness factor ()


Recall eq. (46):

actual rate of substrate utilisatio n


rate of substrate utilisatio n in absence of gel / immobilising medium
r

which in our present considerations can be written as: i A,Obs


(60)
rAs
At this stage it is useful to distinguish between
internal effectiveness factor, i, and external
effectiveness factor, e, to be used in later considerations of external mass transfer.
From section 4.2.8, for a given kinetics and particle geometry, we have an expression
for rA,Obs, so combining this with a term for rAs allows the formulation of an expression
for the effectiveness factor from eq.(60). In the case of 1 st order kinetics and spherical
geometry:

4
rAs R 3k1C As
3

(61),

since the rate is k1CAs multiplied by the particle volume.

Thus, substituting (58) and (61) into (60) gives:

i ,1

3 DAe
R k1 / DAe coth R k1 / DAe 1
R 2 k1

(62)

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In general terms, i, depends only on four types of parameter:


(i) reaction kinetic constants/coefficients
(ii) surface concentration of substrate
(iii) effective diffusivity
(iv) particle size
The Thiele modulus or tortuosity factor, , for a given kinetics and particle geometry, is
a dimensionless combination of the important parameters that quantify mass transfer
and reaction in a heterogeneous catalyst system. The generalised Thiele modulus is
given as:

V p rAs C As

C DAe rA dC A
S x 2 A,eq

(63)

where: Vp = particle volume, Sx = external surface area, CA,eq = equilibrium [A] (=0 for
most biochemical reactions), and rA = reaction rate. From geometry, Vp/Sx = R/3 for
spheres, and = b for flat plates.
For 1st order kinetics with spherical geometry, we have:
and:
1
(65)
31 coth 31 1
i ,1

R
3

k1
DAe

(64)

312

and i for other kinetics and geometries can be quantified by similar equations or
numerical methods.

Effectiveness factor versus generalised


Thiele modulus for 1st order kinetics,
e.g. from eq. (65). (Note: )

From Bioprocess Engineering Principles by Pauline M. Doran

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04/09/2014

Generalised Thiele moduli for various


kinetics and particle geometries.

From Bioprocess Engineering Principles by Pauline M. Doran

Internal effectiveness factor versus generalised Thiele modulus for


Michaelis-Menten kinetics, obtained by numerical methods
(Note: and =Km/CAs)
From Bioprocess Engineering Principles by Pauline M. Doran

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04/09/2014

4.2.10 The observable Thiele modulus ()


This is used when k1 or other kinetic parameters are not known, as is often the case.

Vp

Sx

rA,Obs

DAe C As

(66)

All the parameters for are usually easily obtained experimentally.


Once is known, i can be determined from the corresponding equation/plot.
To assess whether or not internal diffusion is important in determining the overall rate
determining step, apply the Weisz Criteria:

If < 0.3, i 1: internal mass transfer is fast.


If > 3, i << 1: internal mass transfer is slow.

For typical particle geometries we can write:


2

R rA,Obs
Sphere
3 DAe C As

Flat plate b 2

rA,Obs
DAe C As

(R = sphere radius, b = plate thickness)

Internal effectiveness factor versus observable Thiele modulus, .


(Note: and =Km/CAs)

From Bioprocess Engineering Principles by Pauline M. Doran

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4.3 External m.t. concepts as applied to heterogeneous reactions


4.3.1 Liquid-solid mass transfer correlations
L-S mass transfer equation: N A

dC A
k s a C Ab C As
dt

(17)

Since ks depends on reactor hydrodynamics and liquid properties, it is often difficult to


measure accurately, especially for neutrally buoyant entities such as microbial flocs.
Values of ks, accurate to within 10-20%, can however be estimated using various
correlations available in the literature. These correlations are expressed in terms of
dimensionless groups or numbers.
Sherwood number:

Sh

Schmidt number:

Sc

ks Dp

L
L DAL

Particle Reynolds no: Re p


Grashof number:

(67)

DAL

(68)

D p u pL L

L
gD L ( p L )
Gr
L2
3
p

(69)
(70)

Dp = particle diameter, DAL = diffusivity of component A in the liquid,


upL = particle linear velocity relative to the liquid (slip velocity), L = liquid density,
L = liquid viscosity, p = particle density, g = gravitational acceleration.

Sh

ks Dp
DAL

Sc

L DAL

Re p

D p u pL L

Gr

gD 3p L ( p L )

L2

Sh:

ratio of overall to diffusive mass transfer across the boundary layer

Sc:

ratio of momentum (viscous) diffusivity to mass diffusivity

ReP:

ratio of inertial to viscous forces acting on the particle

Gr:

ratio of gravitational to viscous forces acting on the particle (important with


neutrally buoyant particles)

The form of the correlation(s) used to estimate k s depends on the configuration of the
mass transfer system, the flow conditions and other factors. In all cases, ultimately the
Sherwood number, Sh, must be evaluated.

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Sh

ks Dp
DAL

Sc

L DAL

Re p

D p u pL L

Gr

gD 3p L ( p L )

L2

Free-moving spherical particles. For this situation, the rate of mass transfer depends
on the slip velocity, upL, which is difficult to measure and must be estimated before
calculating ks from Sh.

1. Calculate Gr:

For Gr<36

Re p

Gr
18

(71)

For 36<Gr<8x104

Re p 0.153Gr 0.71

(72)

For 8x104<Gr<3x109

Re p 1.74Gr 0.5

(73)

2. Calculate Sh: For RepSc<104


For Rep<103

Sh 4 1.21Re p Sc

(74)

Sh 2 0.6 Re0p.5 Sc 0.33

(75)

0.67

3. Calculate ks from eq. (67)

Sh

ks Dp
DAL

Sc

L DAL

Re p

D p u pL L

Gr

gD 3p L ( p L )

L2

Spherical particles in a packed bed. Here, ks depends on the liquid linear velocity, u,
around the particles. This is measureable and replaces the slip velocity u pL, in the
calculation of Rep.
1. Calculate Rep: For 10<Rep<104

Sh 0.95 Re0p.5 Sc 0.33

(76)

2. Calculate ks from eq. (67)

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4.3.2 Observable external mass transfer modulus


From eq. (17), if external mass transfer is rate limiting then rA,obs = NA, and:
rA,Obs k s

Sx
C Ab C As
Vp

V p rA,Obs
C As
1
C Ab
S x k s C Ab

, or

(77)

Define as , the observable external mass transfer modulus:

V p rA,Obs

(78)

S x k s C Ab

All of the rhs terms are usually measurable. To assess whether or not external diffusion
is important in determining the overall rate determining step, apply the following criteria:
If << 1, then CAs CAb, and external mass transfer is not rate limiting.
If is significant (typically greater then 0.05), then CAs < CAb, and external
mass transfer is limiting.
For typical particle geometries we can write:
R rA,Obs
Sphere
3 k s C Ab

Flat plate b

rA,Obs
k s C Ab

(R = sphere radius, b = plate thickness)

4.3.3 Total effectiveness factor


For reactions affected by both internal and external mass transfer limitations, we can
define the total effectiveness factor, T:

rA,Obs
rAb

observed rate
rate if C A C Ab throughout the particle

(79)

T can be related to the internal effectiveness factor, i, by:


r

T A,Obs As i e
rAs rAb

(80)

where e is the external effectiveness factor. Thus e is defined:

rAs rate if C A C As throughout the particle

rAb rate if C A C Ab throughout the particle

(81)

Considering e for different reaction kinetics:

Zero order (rA k ) e,0 1

First order (rA kCA ) e,1

C As
C Ab

C
C K C Ab
Michaelis Menten rA max A e, M As m
K

C
C Ab K m C As
m
A

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4.4 Minimising mass transfer resistances


2

V p rA,Obs
(66)

S x DAe C As
The observable Thiele modulus, , indicates that internal mass transfer rate can be
improved by:
Internal mass transfer

Decreasing particle size. This has the biggest effect on , since the latter is
proportional to the square of particle size (R2 for spheres and b2 for plates).
There may be practical limitations however on this, due to poorer mechanical
strength and increased difficulty in retention of the particles within the reactor
system.

Reducing the observed reaction rate, rA,Obs. This is a paradoxical situation,


where, although the mass transfer rate is improved, the overall rate must be
decreased to achieve this. In practical terms, this can be achieved by reducing
the cell/enzyme loading in the solid particle. However this results in a larger
number of particles necessary, and hence a larger reactor volume.

Increased diffusivity may be achieved by changing the particle pore structure.


Increasing CAs may be best achieved by minimising external mass transfer
resistance across the liquid boundary layer at the particle surface.

External mass transfer

V p rA,Obs
S x k s C Ab

(78)

The observable external mass transfer modulus, , indicates that external mass
transfer rate can be improved by:

Decreasing the particle size. This increases the external particle surface area
and reduces the thickness of the boundary layer.

Increasing the mass transfer coefficient, ks. This may be best done by
increasing the liquid (linear) velocity adjacent to the particle surface. Changing
the liquid viscosity and density, or increasing the substrate diffusivity in the liquid
will also increase ks.

Increasing the substrate bulk concentration. This increases the driving force for
external mass transfer according to eq. (17)

Reducing the observed reaction rate, rA,Obs. See similar comments on this,
under improving the internal mass transfer rate.

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Chapter 5 + Chapter 6, Bioprocess Engineering Principles, 2nd Ed., P.M. Doran.

5. BIOCHEMICAL ENERGY
BALANCES

5. Biochemical Energy Balances


Although bioprocesses in general are not as energy intensive as chemical processes,
energy effects are nevertheless important since biologically active species are very
sensitive to heat. Heat released during reaction or generated during separation
operations, can cause cell death and denaturation of enzymes, if it is not quickly
removed. For good design of heat exchange equipment, energy flows in the system
must be evaluated using energy balances.

Energy in/out by
shaft work, Ws

Energy of
in-flowing
materials

The bioprocessing
system

Energy of
out-flowing
materials

Energy in/out by
heat transfer, Q

Energy changes in a bioprocessing system

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5.1 Law of conservation of energy


In the case of reaction systems, we are usually interested only in the total energy of
the system, rather than the energy of individual biochemical species, so a situation
analogous to that for total mass applies:
Energy accumulated or
depleted within system

Energy in through - Energy out through


system boundaries
system boundaries

(79)

Referring to the various energy transfer modes possible, we can quantify eq. (79) to
obtain a series of general energy balance equations:
E = Mi (Ek + Ep + U + pV)i - Mo (Ek + Ep + U + pV)o - Q + W s

(80)

where E = energy accumulated/depleted, M = mass, Ek = kinetic, Ep = potential, and


U = internal energies, pV = flow work, Q = heat exchanged, W s = shaft work, and
subscripts i and o refer to the inlet and outlet streams.
Enthalpy, h, can be defined as: h = U +pV
so we have for equation (80):

(81)

E = Mi (Ek + Ep + h)i - Mo (Ek + Ep + h)o - Q + W s

(82)

In the case (true for many biochemical reaction systems) where there is little change in
kinetic or potential energy, and where the system is operating at steady state (no
energy accumulation/depletion), we can further simplify (82) to give:
Mi hi - Mo ho - Q + W s

= 0

or, in cases where there may be more than one input and one output streams:

(M h)inlet streams - (M h)outlet streams - Q + W s

= 0

(83)

Steady-state Energy Balance Equation


Another case can be considered when no heat is transferred into or out of the system
(i.e. Q=0):
(M h)inlet streams - (M h)outlet streams + W s

(84)

Adiabatic Energy Balance Equation


These equations or variations of them can be used in performing energy balance
calculations for chemical and biochemical systems.

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5.2 Calculation of enthalpy changes


Equation (81) showed that enthalpy is comprised of both the internal energy (U) of a
substance and the flow work term (pV). It is not possible to have an absolute measure
of U, therefore it is also not possible to know absolute enthalpy values. In energy
balance calculations, what is more important is to determine enthalpy changes as the
substance is processed in a given plant unit. This can be done if the calculations are
performed with respect to a chosen reference state, at which the enthalpy is assigned
a value of 0.
Enthalpy changes can occur as a result of:
i.
ii.
iii.
iv.

Temperature changes
Change of phase e.g. liquid to gas
Mixing or dissolution
Reaction

5.2.1 Change in temperature


Sensible heat: this is the term given to heat exchanges to raise or lower the
temperature of a substance. The corresponding change in enthalpy of a system
due to temperature change is called sensible heat change.

Changes in enthalpy, H, due to temperature change, T, are given by:


H = M.Cp.T
where:

(85)

M = mass
Cp = heat capacity at constant pressure.

The heat capacity value for a given substance can vary with temperature. These are
often given in the literature as a polynomial function of temperature, such as :

Cp,i i i T i T 2

(86)

where the coefficients , and are tabulated for difference substances.


In other cases, mean heat capacity values ,C pm, are quoted as a function of
temperature relative to a reference temperature (usually 0 oC) in the literature. To
calculate H for a temperature change from T1 to T2, the corresponding values are
inserted into:
H = M[(Cpm)T2 (T2 - Tref) - [(Cpm)T1 (T1 - Tref)]

(87)

Since large temperature changes do not often occur in bioprocessing, in many cases
Cps are assumed constant.

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5.2.2 Phase changes


Relatively large changes in internal energy and enthalpy occur as intermolecular
bonds are broken during phase changes such as evaporation or melting. Latent heat
is the term given to the heat exchange that occur during phase changes at constant
temperature and pressure.
Enthalpy changes that result from phase changes can be calculated directly from the
corresponding latent heat literature values for that substance. For example, for
evaporation of a mass of liquid, M:

H = M. hv

(88)

where hv is the latent heat of vaporisation.


Literature values of latent heats are usually given for substances at their normal
boiling, melting or sublimation points at 1 atm. pressure. Latent heat, like heat capacity
can also be temperature dependent, so when phase change occurs at some nonstandard temperature, e.g. evaporation of water at 70 oC, the corresponding enthalpy
change must be calculated by including the relevant sensible heat changes into an
appropriate energy cycle.

5.2.3 Mixing and dissolution


In a similar vein to phase change, enthalpy changes can also occur on mixing of two
liquid components or on dissolution of substances in a solvent. Thus for example
dissolution of sodium hydroxide pellets in water can release enough heat to boil the
water in some cases.
Heat of mixing is property of the mixture components, their concentrations and the
temperature, and can be quantified from integral heats of mixing data, available in
the literature. Since most biochemical processes operate with dilute aqueous mixtures,
enthalpy of mixing effects can often be assumed to be minimal in energy balance
calculations.
5.2.4 Reaction enthalpy changes
For chemical reactions, Hesss Law allows us to calculate reaction enthalpy change,
Hr :
H r0 ( Ni H 0f ,i ) products ( Ni H 0f ,i ) reactants
(89)

where:

Ni = number of moles of component i involved in the reaction


H0f,i = standard enthalpy of formation of component i.

Whereas H0f,i values are often available for chemical components, this is often not
the case for biochemicals.

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Instead heats of combustion, h0c,i are used. These are relatively easily measured
since all biochemical materials are combustible, giving simple gases such as CO 2, H2O
vapour and N2. Thus:

H r0 ( Ni hc0, i ) reactants ( Ni hc0, i ) products

(90)

Equation (90) allows calculation of the standard enthalpy of reaction (i.e. normally at
25oC). For reactions carried out at other temperatures, then sensible heat changes
have also to be taken into account, as outlined in section 5.2.1. For most biochemical
reactions, this is not an issue. However one major exception is in the case of single
enzyme conversion reactions. These have very small reaction enthalpy changes, and
hence any additional sensible heat effects can be quite significant in determining the
overall enthalpy change.
The enthalpy of reaction at non-standard conditions can be quantified by considering
the hypothetical path that involves the same initial and final states of the reaction
mixture, but takes place via the standard reaction conditions.

Actual path

Hypothetical path for calculating Hrxn


at non-standard conditions

0
H rxn (at T ) H1 H rxn
H 3

From Bioprocess Engineering Principles by Pauline M. Doran

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In aerobic fermentations, where oxygen is the main oxidising agent in cell


metabolism, it is possible to calculate the enthalpy of reaction, based on the amount of
oxygen consumed:
(91)
H
460 kJ per mole O consumed
r , Aerobic

Thus either equation (91) (for aerobic fermentations) or equation (90) (for all
fermentation types) can be used to calculate Hr if either the fermentation oxygen
consumption or the reaction component heats of combustion are known.
Heat of combustion of biomass have been found experimentally to fall into two
broad groups:
Bacteria

h0c 23.2 kJ.g-1

Yeasts

h0c 21.2 kJ.g-1

5.2.5 Energy balance equation for cell bioreactors


In fermentation reactors, reaction enthalpy changes usually dominate the energy
balance, compared to the small enthalpy contributions due to sensible heat and heat of
mixing changes, so that the latter terms can often be ignored. (Note: this only applies
to cell bioreactors, not to other process units such as heat exchangers, etc.).
Thus the only significant factors to be accounted for in the energy balance for such
reactor units are:
reaction enthalpy changes, Hr
latent heat enthalpy changes (usually due to evaporation), Hv (=Mv.hv, eq.88)
shaft work, W s (usually due to stirring/agitation)
Recalling the steady state energy balance equation:
(M h)inlet streams - (M h)outlet streams - Q + W s

= 0

(83)

it is possible to formulate a useful version for this particular case (cell bioreactors):
-Hr

Mv.hv - Q + W s = 0

(92)

since reaction and latent heat are the only significant contributors to enthalpy change
between the inlet steams and the outlet streams.

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5.2.6 Essential good practice and procedure for performing energy balance
calculations
These are essentially the same as for material balances (section 3.1).

From Bioprocess Engineering Principles by Pauline M. Doran

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From Bioprocess Engineering Principles by Pauline M. Doran

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