Table of Content

Chapter 1. Microbial growth kinetics

Chapter 2. Sterilization
Chapter 3. Aeration and agitation

Reactor and Process Technology

Biochemistry I

Definitions (as used in this course)

• Fermentation (biochemistry):
o A metabolic process whereby electrons released from nutrients are
ultimately transferred to molecules obtained from the breakdown of
those same nutrients
o Degradation of organic matter under anaerobic conditions

Chapter 1. Microbial growth kinetics

Definitions (as used in this course) FERMENTATION

• Fermentation (industry): aerobic (O2)

or anaerobic
o Any production process using mass culture of microorganisms (aerobic or C-source (CO2 and H20)
anaerobic) to produce “useful end products” starting from raw materials
(other gases)
C source
Growing, living cells Fermentation
N source
product vitamins micro-organism heat
minerals

N-source FERMENTATION
PRODUCTS

biomass 4

An introduction to fermentation processes An introduction to fermentation processes

• Definition fermentation
• The range of commercially important fermentation processes
o Those that produce microbial biomass
o Those that produce microbial enzymes
o Those that produce microbial metabolites
o Those that produce recombinant products
o (Those that modify a compound which is added to the fermentation:
bioconversion
• The chronological development of the fermentation industry
• The components of a fermentation process
• Definition fermentation
• The range of commercially important fermentation processes
o Those that produce microbial biomass
o Those that produce microbial enzymes
o Those that produce microbial metabolites
o Those that produce recombinant products
o (Those that modify a compound which is added to the fermentation:
bioconversion
• The chronological development of the fermentation industry
• The components of a fermentation process
 Fermentation
• The term 'fermentation' is derived from the Latin verb “fervere”, to
boil or to bubble, thus describing the appearance of the action of
microorganisms in liquid fermentations, e.g. on extracts of fruit or
malted grains (wort).
Fermentation
• Fermentation has a different meaning to biochemists and industrial
microbiologists
o Biochemical meaning relates to the generation of energy (ATP) by
the catabolism of organic compounds under anaerobic conditions
(organic compounds are electron donors and acceptors)
o Industrial microbiologists have extended the term fermentation to
describe any process for the production of useful products by the
mass culture of a microorganism (aerobic or anaerobic) (often
under severely controlled conditions)

Fermentation (compared to respiration) Fermentation

Organic (compared to respiration)Organic
compound compound

Glucose is the Glucose is the Glucose is the Glucose is the

electron donor electron donor electron donor electron donor

NADH is the NADH is the NADH is the NADH is the

electron carrier electron carrier electron carrier electron carrier

O2 is the electron Pyruvic acid is the O2 is the electron Pyruvic acid is the
acceptor electron acceptor acceptor electron acceptor

Inorganic Organic
compoun compound
Biochemical Biochemical
d
meaning meaning
biochemical 21
meaning
 Fermentation

Biochemical
meaning

Biochemical
meaning
2
6

Microbial metabolites Microbial metabolites

1. Lag phase
• No increase in biomass/number of cells
• Adaptation of metabolism: time during which the microbes adapt to the
medium and synthesise the enzymes needed to break down the substances
in the growth medium. New, other conditions require other enzymes and
nutrients in the cell!
• Transfer of exponentially growing cells into the same medium does not show
a lag phase
• Influencing factors
Batch o Medium not optimal, e.g. enzyme synthesis, toxic compounds, spore germination
culture o Inoculum
o Culture in exponential phase => no lag phase if transferred to same medium
o Culture in stationary phase => relatively long lag phase

Inoculation of a nutrient medium – followed by four stages

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Microbial metabolites
2. Log phase or exponential phase
• Once the bacteria have adapted to the new medium they start to reproduce
quickly and their numbers multiply evenly for each increment of time. A plot
of the log number of cells against time gives a linear relationship.
• The cells are at their greatest activity in this phase.
• Transferring cultures to a fresh medium at regular intervals can maintain the
cells in an active state. An active culture can rapidly dominate any new
environment.
• Rate of exponential growth varies between micro-organisms
• Prokaryotes grow faster than eukaryotes
• Eukaryotes: smaller ones grow faster than large ones
• Influencing factors
o Temperature
o Composition culture medium
o Minimum living space
Microbial metabolites
3. Stationary phase
• As the bacteria dominate the growth medium, they deplete the available
nutrients or toxic waste products accumulate, slowing the rate of
reproduction. At the same time, cells are dying off: A state of equilibrium is
reached between the death of old cells and formation of new cells, resulting
in no net change in cell numbers.
4. Death phase
• In the next phase the formation of new cells ceases and the existing cells
gradually die off
The log phase can be prolonged by removing toxic waste, by adding more
nutrients or both = FED-BATCH culture or CONTINUOUS culture.

Microbial metabolites Microbial metabolites

• Primary products of microbial metabolism
oPrimary metabolites
• Secondary products of microbial metabolism
o Secondary metabolites
yeast molds

4
1
Microbial metabolites
• Primary products of microbial metabolism
o Products are essential for growth in all wild-type microorganisms
o Ex. amino acids, nucleotides, lipids, carbohydrates, alcohol,…
o Produced during the log phase (= exponential phase = trophophase)
o Are of economic importance
o Industrial productivity is improved by providing adequate cultural
conditions and/or modifying the wildtype microorganism

43

Microbial metabolites Microbial metabolites

• Secondary products of microbial metabolism
o Products do not have an obvious function in cell metabolism – very low
concentrations
o Ex. antibiotics, enzyme inhibitors, growth promotors,...
yeast molds o Produced during the stationary phase (= idiophase) – produced by slow-
growing or non-growing microorganisms
o Are of economic importance
o Especially produced by filamentous bacteria and fungi (molds)

4
1
Microbial metabolites Microbial secondary metabolites
• Interrelationship between primary and secondary metabolism Alkaloids
Antibiotics Pennicillins, cephalosporins, tetracyclines, …
o Primary biosynthetic routes are common to the vast majority of Antiprotozoals Spiramycine

microorganisms Pigments Carotenoids, monascin, …

Food preservatives Nisin, natamycine, …
o Secondary products are synthesized by only a few different microbial Feed promotors Bacitracin, viginamycine, tylosin, avoparcin, …
species Coccidiostats Monensin, lasalocid, salinomycin, ..
Antihelminics Avermectins, hygromycin, destomycin
Antifungals Griseofulvin, …
Plant disease controllers Blasticidin S, polyoxins,
Gibberellins
Enzyme inhibitors Clavulanic acis, acarbose, mevinolin, streptovaricins, …
Biopolymers Xanthan, poly-β-hydroxybutyrate, dextran, pullulan, hyaluronic acid, …
Anti-tumor-compounds Adriamycine, daunorubicine, sarkomycine, …
Bio-herbicides Bialaphos
Bio-tensides Emulsan
Immuno-modulators Cyclosporin
Hormonal Zearalonol

Recombinant products
• Recombinant DNA technology (genetic engineering):
o Microbial cells are capable of synthesizing ‘foreign’ (or heterologous)
proteins

Secondary products are elaborated from the intermediates and

products of primary metabolism
49
Recombinant products Operating modes
Batch culture

• Closed system where there are no additions of nutrients following

inoculation
o apart from acid or alkali for pH control and input of air for aerobic
fermentations
• Growth rate decreases by substrate limitation (one limiting nutrient is
enough)
• Definite beginning and end of the process
o Fermenter loaded, sterilized and inoculated
o Microbial growth through a typical batch profile
o Product or biomass harvested
o Fermentor cleaned and restarted

30

29

Operating modes Operating modes

Fed batch culture Batch and fed batch culture

• Variation of batch culture • Advantages of batch/ fed batch culture

• Extra nutrients are added as the fermentation progresses, increasing
the fermentation volume until the fermentor is full
o continuously
o intermittently
o as a single supplementation
→ often when a batch culture approaches the end of the exponential
phase
• Can extend the product formation phase and may overcome problems
associated with rapidly metabolized substrates
• May also be useful when a substrate is toxic at high concentrations
31
o Low investment
o In case of contamination, easy to restart
o Particularly efficient for production of secondary metabolites
• Disadvantages of batch/ fed batch culture
o Only a small fraction of the fermentation period is really productive,
as there may be a long lag phase
o Batch-to-batch variability
o Higher running costs for preparing and maintaining stock cultures
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Operating modes Operation modes fermentation processes
Continuous culture

• Open system where fresh medium is continuously added and culture

is simultaneously removed at the same rate, resulting in a constant
working volume
• Cells grow exponentially for extended periods
• More productive than batch cultures
• Particularly well suited for the production of biomass and primary
metabolites
• Disadvantages Batch Continuous Fed-batch
o Higher investment costs
o Sterility must be maintained and a continuous supply of media of Volume Constant Constant Increasing (until
constant composition fermentor is full)

33 34

Microbial growth kinetics Microbial growth kinetics

• Batch culture • Batch culture
• Fed-batch culture • Fed-batch culture
• Continuous culture • Continuous culture

35 36
 Growth rate = death rate
Batch culture Number of cells constant Batch culture
Some maths…. Growth equations…
Log (X)
• Lag phase
o Growth rate is essentially 0
o When an inoculum is placed into fresh medium, growth truly starts after
the lag phase
o Usually lasts from minutes to several hours
o Can be controlled to some extent
• Dependent on initial inoculum size
• Higher inoculum density, shorter lag phase
• Dependent on the stage the inoculum is in
Time • Taken from an exponential phase culture, and inoculated in the same fresh medium at a high
inoculum density, and incubated under the same growth conditions (T, shaking speed), no
Constant, exponential Constant, exponential noticeable lag phase
growth (max growth rate, dying • Taken from a stationary phase culture, long lag phase (cells need to shift physiologically from
max substrate removal) stationary phase cells to exponential phase cells)
• Dependent on the type of medium
Log scale Straight line up Straight line down
• Transfer into new medium requires a long lag phase, as the cells need to adapt to the new
37 38
conditions (produce the enzymes needed for growth under the new conditions)

Batch culture Batch culture

Some maths…. Growth equations… Some maths…. Growth equations…
• Log phase or exponential phase
o Characterized by exponential growth of the bacterial population, in which
the population is doubled at a constant time interval
o The rate of increase in cells (= growth rate of the bacterial population) is
proportional to the cell concentration (or biomass) present at any
particular time

o What happens? The number of cells increases in the following geometric

progression: 20 (1 cell), 21, 22, 23, … until 2n after n divisions
o Expressed in a quantitative manner:
The number of cells after n doublings is 2nX0, where X0 the initial cell
number

Exponential cell division: each cell division results in the doubling of the
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cell number 40
Batch culture Batch culture
Some maths…. Growth equations… Some maths…. Growth equations…
• Log phase or exponential phase • Growth rate directly proportional to cell concentration/biomass
o Characterized by exponential growth of the bacterial population, in which o dX/dt = µ X (Eq. 1)
the population is doubled at a constant time interval • Rearrange (divide by X, multiply with dt)
o The rate of increase in cells (= growth rate of the bacterial population) is o 1/X dX = µ dt (Eq. 2)
proportional to the cell concentration (or biomass) present at any • Integrate between time zero when X = X0 and time t when X = Xt
particular time xt t
o dX/dt α X o ∫1/X dX = µ ∫ dt (Eq. 3)
x0 0
o Equation of population growth during exponential phase:
𝒅𝑿
o Ln Xt – Ln X0 = µ t To calculate specific growth(Eq.
rate4)
𝒅𝒕
= µ𝑿 (Eq. 1) o Ln (Xt/X0) =vµ t To calculate generation time (Eq. 5)
where • Take exponential
• X: number or mass of cells (number of cells/volume; mass/volume) o Xt/X0 = eµ t (Eq. 6)
• t: time o Xt = X0 eµ t (Eq. 7)
• µ: specific growth rate (1/time) where
Can be used to calculate the specific growth rate (µ) or the generation time o X0: original biomass concentration
(doubling time) based on experimental data (see next slides).
41 o Xt: biomass concentration after42time t

Batch culture Batch culture

Some maths…. Growth equations… Some maths…. Growth equations…
• Calculate specific growth rate • Calculate generation time
o Ln Xt – Ln X0 = µ t (Eq. 4) o Ln (Xt/X0) = µ t (Eq. 7)
o Ln Xt = Ln X0 + µ t (Eq. 8) o For X to be doubled: Xt/X0 = 2 (Eq. 8)
o Thus, the natural logarithm of biomass concentration against time reveals o Ln (2) = µ t (Eq. 9)
a straight line, the slope of which is µ o So, t = ln(2)/µ where t = generation time (Eq. 10)
o So, µ = (Ln Xt – Ln X0) / t (Eq. 9)
µ can be calculated
ln (X) based on
µ experimental data at
any time in the
exponential phase

43
time 44
Batch culture Batch culture
Some maths…. Growth equations… Some maths…. Growth equations…
• During the exponential phase nutrients are in excess and the organism • Stationary phase
is growing at its maximum specific growth rate, called μmax, for the o State of no nett growth
prevailing conditions. o dX/dt = 0 (Eq. 11)
o Although no net growth, cells still grow and divide. Growth is simply
balanced by an equal number of cells dying

µmax: : characteristic for a microorganism in a certain medium

• >< In the lag phase, µ ~ 0

45 46

Batch culture Batch culture

Some maths…. Growth equations…
• Death phase • Xt = X0 eµ t (Eq. 7) predicts that growth would continue
o Nett loss of culturable cells indefinitely
o Even in the death phase there may be individual cells that are • Not in reality!
metabolizing and dividing, but more viable cells are lost than gained. o Growth in the exponential phase results ….
o The death phase is often exponential, although usually slower than the • in the consumption of nutrients
rate of growth during exponential phase • in the excretion of microbial products
o Equation of death phase:
o … in decrease of the growth rate
dX/dt = -kd X (Eq. 12) • due to the depletion of essential nutrients (substrate limitation), and/or
where • due to the accumulation of autotoxic products (toxin limitation)
• X: number or mass of cells (number of cells/volume; mass/volume)
• t: time o … resulting in the stationary phase
• kd: specific death rate (1/time)

47 48
 Batch culture
log phase
log x
time
max
time
What is happening in this phase
of deceleration of growth => see
next slides
49
Batch culture
How can we assess substrate limitation/ toxin limitation?
Over the zone A to B, due to an increase in
initial substrate concentration, a
X proportional increase in biomass
concentration at stationary phase is seen.
This relation between increase in initial
substrate concentration and proportional
increase in the biomass produced may be
described by equation:
X = Y(SR - s) (Eq. 13)
Where
SR X: concentration of biomass produced,
Y: yield factor (g biomass produced per g
substrate consumed),
Equation (13) may be used to predict SR: initial substrate concentration, and
the production of biomass from a s: residual substrate concentration
certain amount of substrate
51
Batch culture
How to differentiate between substrate limitation and toxin limitation?
• At which substrate concentrations does substrate limitation or toxin
limitation occur?
• The nature of the limitation of growth (substrate limitation or toxin
limitation) can be explored by growing the organism in the presence of a
range of substrate concentrations and plotting the biomass concentration at
stationary phase against the initial substrate concentration
Substrate
50
Batch culture
How can we assess substrate limitation/ toxin limitation?
• Over the zone A to B, s = 0 at the
X cessation of growth (all substrate
substrate consumed) (substrate limitation)
limitation • Over the zone B to C, an increase in
toxin initial substrate concentration does give
limitation a proportional increase in biomass
produced due to the exhaustion of
another substrate
• Over the zone C to D, an increase in
SR initial substrate concentration does not
result in substantially more yield, due
to the accumulation of toxic producs
(toxin limitation)
Ideal substrate concentration to
produce maximum amount of
52
biomass
Batch culture Batch culture
Monod equation Monod equation
• The decrease in growth rate and the cessation of growth due to the
depletion of substrate, can be described by the relationship
• How to determine?
between μ and the residual growth limiting substrate (s). o Start batch fermentation with SR and inoculum
• This relationship is represented by an equation given by J. Monod in o Measure s and µ in function of time
1942, known as Monod equation (actually describing the different o Plot µ in function of s
growth phases as a whole).
μ = μmax s /(Ks + s) (Eq. 14)
o s is residual substrate concentration
o Ks is substrate utilization constant (also known as affinity constant),
numerically equal to residual substrate concentration when μ is half of
μmax. μ = μmax s /(Ks + s) in function of time
• a measure of the affinity of the organism with the substrate Monod equation s
• tells about the relationship between the specific growth rate (µ) and the residual
growth limiting substrate concentration (s). 2
3
o
53 54

Batch culture Batch culture

Monod equation Monod equation

• 2 zones • 2 zones
• Zone A to B is equivalent to the • Zone A to B is equivalent to the
exponential phase where deceleration
exponential phase where deceleration
substrate concentration is in phase log phase substrate concentration is in phase log phase
excess, and growth is at μmax max excess and growth is at μmax max
• Zone C to A is equivalent to the • Zone C to A is equivalent to the
deceleration phase when deceleration phase when What happens after this
substrate concentration becomes substrate concentration becomes
limiting and will not support μmax limiting and will not support μmax point????
in function of time in function of time
s s
When there is no residual substrate 2 When there is no residual substrate 2
anymore left, growth stops and the 4 anymore left, growth stops and the 4
population enters the stationary phase population enters the stationary phase
55 56
 μ = μmax s /(Ks + s)
Batch culture Batch culture
Monod equation Growth rate of an organism with high substrate affinity
Ks = s when µ = µmax/2 • μ = μmax s /(Ks + s) (Monod eq.) (Eq. 14)
deceleration
log phase • dX/dt = µ X (Eq. 1)
phase
max →dX/dt = μmax s X /(Ks + s) (Eq. 15)

• If the organism has a very high affinity for the limiting substrate (a
low Ks value; Ks <<< s), the growth rate will occur at maximum speed,
max /2 and will not be affected until the substrate concentration has
declined to a very low level. Thus, the deceleration phase for such a
culture would be short.
in function of time
dX/dt = μmax X (Eq. 16)
KS s

57 58

Batch culture Batch culture

Growth rate of an organism with high substrate affinity Growth rate of an organism with low substrate affinity
µ Decel. Long log phase • μ = μmax s /(Ks + s) (Eq. 14)
µmax • dX/dt = µX (Eq. 1)
→ dX/dt = μmax s X /(Ks + s) (Eq. 15)
Ks very low;
Grows well • If the organism has a low affinity for the limiting substrate (a high Ks
with high an max /2
low value; Ks >>> s), the growth rate will be deleteriously affected by
substrate substrate concentration (Ks is in the numerator). Thus, the
amounts deceleration phase for such a culture would be relatively long.
in function of time dX/dt = μmax s X / Ks (Eq. 16)
KS s

59 60
 Batch culture Batch culture
Growth rate of an organism with low substrate affinity And what about the kinetics of product formation?
µ Long decel. Log phase • Growth-linked products are primary metabolites
µmax • Non-growth-linked products are secondary metabolites

Ks high;
Grows only
well at high max /2
substrate
amounts

in function of time

KS s

61 62

Batch culture Batch culture

Growth-linked products (primary metabolites) Growth-linked products (primary metabolites)
• Rate of product formation is proportional with the concentration of • dP/dX = Yp/x (Eq. 18)
biomass Multiply with dX/dt
dP/dt = qp X (Eq. 17) • dP/dt = Yp/x dX/dt (Eq. 19)
Where We know already that dX/dt = µ X (Eq. 1), therefore:
o P is the concentration product
• dP/dt = Yp/x µ X
o qp is the specific rate of product formation (g product per g
Combine with dP/dt = qp X (Eq. 17), gives: qp = Yp/x µ (Eq. 20)
biomass per h)
o X is the concentration of biomass
• Also, product fermentation is related to biomass production by the => When product formation is growth associated, the specific rate of
following equation: product formation increases with the specific growth rate
dP/dX = Yp/x (Eq. 18)
Where
o Yp/x is the yield of product in terms of substrate consumed (g product per
g biomass) 63 64
Batch culture Batch culture
Non-growth-linked products (secondary metabolites) Summarized
=> The specific rate of product formation (qp) may remain constant or it • May be used to produce biomass, primary metabolites and secondary
may vary in a complex manner. metabolites
o biomass production
= cultural conditions supporting the maximum cell population should be used
o primary cell metabolite production
= conditions to extend the exponential phase accompanied by product excretion
o secondary metabolite production
= conditions giving a short exponential phase and an extended stationary or
production phase should be used

65 66

Microbial growth kinetics Fed-batch culture

• Batch culture • Exponential growth in batch culture may prolonged by addition of
• Fed-batch culture fresh medium (= addition of growth limiting substrate)
• Continuous culture o Culture may not be toxin limited !
• Fed-batch culture
= Batch culture which is fed continuously, or sequentially with
medium, without removal of the medium, to expand the exponential
phase, wile volume increases with time
• A fed batch culture may reach a “quasi-steady state” in which the
specific growth rate (μ; 1/time) virtually is the dilution rate (1/time),
i.e. the ratio of medium flow rate to culture volume.
o In a quasi-steady state the specific growth rate gradually
decreases.
• Kinetics of a fed-batch culture are based on these of a batch culture
67 68
Start batch culture Add fresh medium

deceleratio deceleratio
log phase log phase
nphas nphas
max max
e e

Add fresh medium to

expand log phase/
slow down
deceleration!

in function of time in function of time

s s

69 70

Fed-batch culture Fed-batch culture

• Consider a batch culture in which growth is limited by the • If, at a time when X = Xmax a feed is started, such that the dilution rate is
concentration of one substrate, see Eq. 13 (substrate limitation), then less than µmax, virtually all substrate will be consumed immediately,
the produced biomass at any point in time can be described by thus:
X = Y * (SR - s) (Eq. 13) F * SR µ * X * V/Y (Eq. 22)
Where Where
X: produced biomass F: flow rate of the medium feed
Y: yield factor SR: initial substrate concentration
SR: initial substrate concentration X: biomass concentration at time t
s: residual substrate concentration V: volume of the vessel at time t
Y: yield factor
• The final biomass concentration, when s 0, can be described as
• Divide by V, multiply with Y => F/V * Y * SR µ * X (Eq. 23)
maximum biomass (Xmax)
• D = F/V (D = dilution rate) => D * Y * SR µ * X (Eq. 24)
Xmax = Y * SR (Eq. 21) • Xmax = Y * SR => D µ (Eq. 25)
• This situation is termed quasi-steady state: dX/dt 0 (no increase or
decrease in biomass concentration): cells continue to grow but are not
71
affected by negative effects of the72 medium
 Fed-batch fermentations
Dilution factor (D) Industrial examples
• Fed batch = a low residual substrate concentration
• D = F/ V • Advantageous in
Where F: flow rate of medium feed (l h-1) o maintaining conditions in the culture within the aeration capacity
V: volume (l) of the fermentor
D: dilution rate (h-1) o avoiding toxic effects of a medium component
o removing repressive effects of rapidly utilized C-sources
Examples: F = 20 l/h D=2/h
V = 10 l
F = 10 l/h D=1/h
V = 10 l

73 74

Microbial growth kinetics Continuous culture

• Batch culture • >< batch culture, continuous culture designed for long-term operation
• Fed-batch culture • Open system, with a continuous feed of influent with nutrients and
• Continuous culture substrate and a continuous drain of effluent with cells, metabolites,
waste products and unused nutrients and substrate
• Results in continous cell growth
• If medium is fed continuously at a suitable rate, a STEADY- STATE (no
increase or decrease in biomass) is achieved
• 2 important systems
o Chemostat
• Chemical environment is kept static, by which cells grow at a constant rate
o Turbidistat
• Biomass concentration in culture is kept constant by controlling turbidity
(photoelectric cell)

75
76
Continuous culture Continuous culture
Chemostat Turbidostat
air in

Feed reservoir feed

air out

photometer

fermentor = max
=D< max

Product reservoir product

1. Overflow 2. Peristaltic pump (circulation)

1. Medium filter 2. Feed reservoir 3. Flowmeter assembly 4. Peristaltic pump 3. Photometer/control 4. Peristaltic pump (feed)
5. Fermentor 6. Level control module 7. Double air filters 8. Peristaltic pump module
9. Air humidifier 10. Air Filter 11. Product reservoir
77 78

Start batch culture Add fresh medium…

deceleratio deceleratio
log phase log phase
nphas nphas
max max
e e

Add fresh medium to

expand log phase/
slow down
deceleration!

in function of time in function of time

s s

79 80
Then continuous culture Continuous culture
• … And take out spent medium
o Flow rate in = flow rate out • However, steady state can only be maintained as long as the dilution
rate does not exceed a critical rate, Dc
• Nett change in biomass with time is:
• Critical dilution rate Dc can be calculated based on the Monod
dX/dt = µ X – D X (= growth – drain) (Eq. 26) equation μ = μmax s /(Ks + s) (Eq. 14) and µ = D (Eq. 27)
Where:
X: cell concentration (mass/volume)
• Dc = μmax s /(Ks + s) (Eq. 28)
µ: specific growth rate (1/time) • Conditions can be optimized such that s >>> Ks (Dc µmax)
D: dilution rate (1/time) • However, note that when a fermentation is operating at Dc growth
• Steady state will be reached when rate will not be able to increase, and cells will become washed out…
dX/dt = 0 => when µ = D (Eq. 27) • Thus, Dc is an important parameter to keep an eye on:
• If µ > D: utilization of substrate exceeds supply of substrate, causing o If not high enough: operation efficiency not optimized
growth rate to slow down until µ = D o If too high: washout of cells
• If µ < D: amount of substrate added exceeds the amount utilized,
causing growth rate to increase until µ = D
• Thus either case will lead to steady
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state!
82

Continuous culture Continuous culture

• How does steady state X and s change in function of D for microbes
• How do X and s change in function of SR?
with a high (low Ks) or low affinity (high Ks) for the limiting substrate?
X
X Low Ks s X High Ks s SR SR

D
D D

Grow well irrespective of Dcritical Need high substrate

84
substrate concentration concentration
57
Continuous culture Continuous culture
Advantages Disadvantages
• Higher productivity • Susceptibility to contamination
• Uniformity of operation and ease of automation • Continuous culture
• Continuous culture • Duration is very long => risk of contamination from outside is greater
• Self-regulating system (D < µmax -> steady-state) - µmax contaminant < D => contaminant is washed out
• Constant labour demand - µmax contaminant = D => contaminant ‘co-exists’
• Steady-state = all parameters at a constant level => easy automation - µmax contaminant > D => contaminant replaces original organism
• Batch culture • Prevention of contamination by fermentor design, construction and operation
• Requires periods of intensive labour • Contamination by ‘strain degeneration’(= contamination from inside): more
probable in a long process
• Automation not so easy
• Batch culture
• Less risk of contamination

85 86

Table of contents
• Introduction
• Medium sterilization (by steam)
o The design of batch sterilization processes
o The design of continuous sterilization processes
• Sterilization of the fermentor
• Sterilization of the feeds
• Sterilization of the liquid wastes
Chapter 2. Sterilization
• Filter sterilization (air and media)

88
Table of contents
• Introduction
• Medium sterilization (by steam)
o The design of batch sterilization processes
o The design of continuous sterilization processes
• Sterilization of the fermentor
• Sterilization of the feeds
• Sterilization of the liquid wastes
• Filter sterilization (air and media)
Sterilization
• Sterilization: inactivation of ALL living microorganisms (both
vegetative cells and spores)
• Pasteurization: inactivation of microorganisms that can cause
disease (pathogens) or spoilage (food spoilage microorganisms),
often by applying a mild heat treatment.
o pasteurization (≠ sterilization) is not intended to kill all
microorganisms.
o pasteurization aims to achieve a significant reduction in cell
numbers so that they are unlikely to cause disease
• (Endo)spores are more resistant to extreme conditions such as high
temperature, high pressure, low pH, …
o Not killed by pasteurization

89 o Sterilization techniques needed

90 to kill these

Sterilization
Definitions
• Destructive sterilization
o Radiation - The use of high energy radiation to demolish unwanted
organisms
o Thermal sterilization - The controlled addition of heat to destroy heat
sensitive organisms
• Removal sterilization
o Filtration - The separation of microorganisms from a medium by
particle size
• Process sterilization
o Batch sterilization - Sterilizing the entire volume of medium at once
1862 using a heating-holding-cooling profile
o Continuous - Sterilizing only a fraction of the volume at a time by using
the medium as an internal heat exchanger and a heating source like
Louis PASTEUR 1822-1895 steam
92
Sterilization Sterilization
• Destructive sterilization: UV • Process sterilization – batch
(in the fermentor)

• Removal sterilization
• Process sterilization –
continuous (in the pipeline)

93 94

Introduction Introduction
Why should we sterilize? Why should we sterilize?
• Consequences of invasion by contaminating microorganisms: • Penicillin-resistant contaminating bacteria produce β-lactamase,
o Medium is used by both the production microorganism and the degrading penicillin
contaminant, resulting in loss of productivity
o Contaminant may ‘outcompete’ the production microorganism
o Contaminant may ‘contaminate’ the final product (e.g. SCP)
o Contaminant may interfere with ‘down stream processing’
o Contaminant may degrade the desired product (e.g. degradation of β-
lactam antibiotics by β-lactamase producing bacteria)
o Contamination of bacterial fermentations with bacteriophages can
result in lysis (killing) of the culture

95 96
Introduction Introduction
Why should we sterilize? Why should we sterilize?
• Bacterophages kill bacteria • Avoidance of contamination may be achieved by:
o Using a pure inoculum to start the fermentation
Cell lysis!!
o Sterilizing:
• the medium
• the fermentor
• all feeds, including air

o Maintaining aseptic conditions during the fermentation

Bacteriophages have both a lytic and lysogenic cycle. In the lytic cycle, the phage replicates and
lyses the host cell. In the lysogenic cycle, phage DNA is incorporated into the host genome,
where it is passed on to subsequent generations. Environmental stressors such as starvation
may cause the bacteriophage to enter the lytic cycle.
97 98

Table of contents Medium sterilization

• Introduction • Media may be sterilized by filtration, radiation, ultrasonic
• Medium sterilization (by steam) treatment, chemical treatment and heat (moist and dry)
o The design of batch sterilization processes
o The design of continuous sterilization processes • Out of these, steam (‘moist heat under pressure’) is the most useful
for the sterilization of fermentation media
• Sterilization of the fermentor
o Exception: media with heat labile components => then often filtration!
• Sterilization of the feeds
• Medium is heated to 121°C under pressure, for ca. 15-20 min
• Sterilization of the liquid wastes
• Filter sterilization (air and media)
• The increase in pressure (above atmospheric pressure) enables
steam to reach higher temperatures than boiling water (100°C). The
extra pressure brings the boiling temperature of water higher.

99 100
 Medium sterilization Kinetics of sterilization (steam, moist heat)
• Killing of microorganisms by steaming obeys the laws of a chemical degradation
reaction,
o i.e. a first order reaction in which the reaction rate, in each moment, is
proportional to the amount of product still to be degraded
Thus, the change in the number of viable cells versus a chosen time
of sterilization treatment t can be written as:
𝑑𝑁
= −𝑘𝑑𝑁 (Eq. 5.1)
𝑑𝑡

Where N: number of viable cells

t: time of sterilization treatment
kd: specific death constant

Boiling point water at 1 bar: 100°C

Boiling point water at 2 bar (i.e. 1 bar overpressure): 121°C

101

102

Kinetics of sterilization (steam, moist heat) Kinetics of sterilization (steam, moist heat)
• Killing of microorganisms by steaming obeys the laws of a chemical degradation
reaction, • Upon rearrangement of Eq. 5.1
𝑑𝑁 𝑑𝑁
= −𝑘𝑑𝑁 (Eq. 5.1) 𝑁
= −𝑘𝑑𝑑𝑡 (Eq. 5.3)
𝑑𝑡
• On integration of Eq. 5.3, from N0 when t=0, to Nt when t = t
Where N: number of viable cells
𝑁𝑡
t: time of sterilization treatment 𝑙𝑛 = −𝑘𝑑𝑡 (Eq. 5.4)
𝑁0
kd: specific death constant
• dependent on the species Where N0: ABSOLUTE number of viable cells present at
• dependent on the physiological form (vegetative or spore) the start of the sterilization period
• dependent on temperature (Arrhenius equation: kd = A e – Ed/RT) (Eq. 5.2)
• Where A = Arrhenius constant
Nt: ABSOLUTE number of viable cells present
• Ed = activation energy for thermal cell death after a period t
• R = ideal gas constant
• T = absolute temperature
• On taking the exponential of Eq. 5.4:
𝑁𝑡
= 𝑒 −𝑘𝑑𝑡 (Eq. 5.5)
𝑁0

104

103
 Kinetics of sterilization (steam, moist heat) Kinetics of sterilization (steam, moist heat)
Exponential decline • The relationship presented in the previous Figs will only be
observed with the sterilization of (i) a pure culture in (ii) one
physiological form, under (iii) ideal sterilization conditions, because
the specific death constant kd is:
• dependent on the species (i)
…
• dependent on the physiological form (vegetative cell, spore) (ii)
𝑁𝑡 𝑁𝑡 • dependent on the temperature (iii)
𝑙𝑛 = −𝑘𝑑𝑡 = 𝑒 −𝑘𝑑𝑡 (Eq. 5.5)
𝑁0 𝑁0 • Importance of physiological form
• This kinetic description makes 2 predictions: o Endospores are far more heat resistant than vegetative cells, and may
o Sterile conditions (i.e. Nt = 0) are achieved in an infinite time germinate upon heat treatment
o After a definite time (close to infinity), number of cells present will be less o Therefore, a deviation from theory may be experienced in practice
𝑁
than 1 (when 𝑡 approaches 0)
𝑁0
• Meaning of Nt when <1: “risk of contamination”
• E.g. Nt = 0,001 => the probability of an organism surviving the treatment is 1 to
105 106
1000

Endospores Endospores

Spore coat

Vegetative cell

Peptidoglycan layer
Endospore

Bacterial chromosome

107 108
Effect of heat treatment on the survival of bacterial Effect of heat treatment on the survival of bacterial
endospores endospores
• Moist heat may have a double effect on endospores:
o Destruction Heat activation of spores > heat desactivation of spores
o Induction of spore germination by the heat and moisture in the initial period of the
sterilization process

• Fig. Left: Activation of spores is MORE than spore death during the early stages.
Therefore, viable numbers increase before exponential decline
• Fig. Middle: Activation of spores is BALANCED by spore death during the early stages Heat activation of spores = heat desactivation of spores
• Fig. Right: Activation of spores is LESS than spore death

Heat activation of spores < heat desactivation of spores

109 110

!!!Deviation from theory!!!

Effect of heat treatment on the survival of mixed Sterilization of a pure microbial culture
cultures
• Example: Mixed cultures of 2 bacteria having different heat Which combination of time (t) and temperature (T)?
sensitivities 𝑁𝑡
𝑙𝑛 = −𝑘𝑑𝑡 (Eq. 5.4)
𝑁0
• Fig. Left: The culture consists mainly of a heat sensitive organism 𝑁0
𝑙𝑛 = 𝑘𝑑𝑡 (Eq. 5.6)
• Fig. Right: The culture consists mainly of a heat resistant organism 𝑁𝑡
𝑘𝑑 = 𝐴𝑒 −𝐸𝑑Τ𝑅𝑇 (Eq. 5.2)
𝑁0
𝑙𝑛 = 𝐴𝑡𝑒 −𝐸𝑑Τ𝑅𝑇 (Eq. 5.7)
𝑁𝑡
𝑵𝟎
𝒍𝒏 is a sterilization criterion, known as the Del factor or the
𝑵𝒕
Nabla factor, represented by , and defined as a measure of the
fractional reduction in viable counts produced by a certain heat
and time regime

 = 𝐴𝑡𝑒 −𝐸𝑑Τ𝑅𝑇 (Eq. 5.8)

111

112
 Sterilization of a pure microbial culture Sterilization of a pure microbial culture
Which combination of time (t) and temperature (T)? Which combination of time (t) and temperature (T)?
= 𝐴𝑡𝑒 −𝐸𝑑Τ𝑅𝑇 (Eq. 5.8)
• On taking natural logarithm and rearranging: The same degree of sterilization
( ) can be obtained over a wide
𝐸𝑑 ln t
ln = ln(𝐴𝑡𝑒 −𝐸𝑑Τ𝑅𝑇 ) => ln = ln(𝐴) + ln(𝑡) − range of time and temperature
𝑅𝑇
𝐸𝑑
regimes.
ln 𝑡 = + ln( ) (Eq. 5.9)
𝑅𝑇 𝐴

A plot of the natural logarithm of the time required to achieve a

certain degree of sterilization against 1/T results in a straight
line, the slope of which is dependent on the activation energy Short time, high temperature
(R is constant) =
long time, low temperature

1/T

113 Del 2.3 or 4.6= ?? 114

Sterilization and nutrient quality Sterilization and nutrient quality

And what about influence on nutrient quality?
• Fermentation medium is not an inert mixture of components and deleterious
reactions may occur in the medium during the sterilization process, leading to a
loss of nutritive quality, and thus less yield of a subsequent fermentation
• So, the choice of the sterilization regime (T and t) is dictated by the
requirement to achieve the desired reduction in microbial content with the
least impact on the medium
And what about influence on nutrient quality?
• Explanation: loss of nutrients by:
o Interactions between nutrient components of the medium
Example: Maillard reaction (reaction of reducing sugars with the amino
groups of amino acids and proteins)

The initial rise in yield is due to

some components in the
medium that have become more Solution: sterilizing N- and C-source separately
available through the sterilization
o Degradation of heat labile components
process
Example: degradation of vitamins, amino acids and proteins
Solution: sterile membrane filtration or judicious choice of steam
sterilization regime

115 116
 Sterilization and nutrient quality Sterilization and nutrient quality
Kinetics of thermal destruction of essential medium components Kinetics of thermal destruction of essential medium
• First order reaction kinetics components
𝑑𝑥
= −𝑘𝑥 (Eq. 5.10) • The effect of T on the reaction rate constant may be expressed by the
𝑑𝑡
𝑥
Arrhenius equation
rearrange, integrate and take exponential 𝑥𝑡 = 𝑒 −𝑘𝑡 (Eq. 5.11)
0 𝑘 = 𝐴𝑒 −𝐸Τ𝑅𝑇 (Eq. 5.12)
Where x0: original nutrient concentration (at onset of sterilisation) 𝐸
xt: nutrient concentration after a heat treatment period, t take natural logarithm: ln 𝑘 = ln 𝐴 − 𝑅𝑇
(Eq. 5.13)
k: reaction rate constant Where A: Arrhenius constant
• The effect of T on the reaction rate constant may be expressed by the Arrhenius E: Activation energy
equation
𝑘 = 𝐴𝑒 −𝐸Τ𝑅𝑇 (Eq. 5.12)
𝐸 Thus, a plot of the natural log of the reaction rate against 1/T
take natural logarithm: ln 𝑘 = ln 𝐴 − (Eq. 5.13)
𝑅𝑇 gives a straight line, with slope –E/R. As R is constant, the
Where A: Arrhenius constant slope is determined by the activation energy E
E: Activation energy

117 118

Sterilization and nutrient quality Steam sterilization in the lab: autoclaves

ln kspore = ln A – Ed/RT

High activation energy Ed (actvation energy thermal destruction Bacillus

spores) = 67,7 kcal mole-1 => steaper slope!
spore destruction
ln(kspore )
vs
Low activation energy ln knutrient = ln A – E/RT
ln(knutrient)
nutrient destruction
E (actvation energy thermal destruction
ln(knutrient) nutrients) = 10 - 30 kcal mole-1

vs
ln(kspore)
An increase in temperature would
accelerate spore destruction more
than medium destruction
Higher temp.

Portable
autoclave
Short time, high temperature ≠ long time, low temperature
k: reaction rate 119
constant
120
Steam sterilization in the lab: autoclaves Steam sterilization in the industry

121 122

Monitoring sterilization Monitoring sterilization

• Biological indicators • Chemical indicators
o Biological indicator vials contain spores from Bacillus
stearothermophilus, a microorganism that is inactivated when
exposed to 121°C saturated steam for a minimum of 20 minutes.
o Change of color (pH) from purple to yellow indicates growth

Color change

123 124
Table of contents Batch and continuous sterilization processes
• The same degree of sterilization (Del factor) can be achieved over a range
• Introduction of temperature-time regimes
• Medium sterilization (by steam) • Therefore, it would be advantageous to employ a high T for a short time
o The design of batch sterilization processes to achieve sterility yet causing minimum nutrient degradation.
o The design of continuous sterilization processes • Thus, the ideal technique would be:
• Sterilization of the fermentor o To heat the fermentation medium to a high temperature,
To hold it for a short time, and
• Sterilization of the feeds o

o To rapidly cool to the fermentation temperature

• Sterilization of the liquid wastes
• Batch sterilization: sterilization of the whole fermentation medium at
• Filter sterilization (air and media) once (as a batch)
• Problem: not possible to sterilize a huge batch of medium without the
heating and cooling periods contributing considerably to the total
sterilization time (i.e.)
o Long heating and cooling time may also cause nutrient degradation. Should be taken
into account!!!
125

126

Batch and continuous sterilization processes

• The only method to achieve the objective of a short-time high T
treatment is to sterilize the medium in a continuous stream, i.e. to
Batch sterilize the medium in the pipeline when it is transported to the
sterilization fermentor
o Using heat exchangers
The larger the holding o Nowadays the method of choice (especially spiral heat exchangers, see
volume, the
further)!
longer the
heating and
cooling time

127

128
 Batch and continuous sterilization processes Batch and continuous sterilization processes
temp Advantages
Batch
“in the • Batch sterilization • Continuous sterilization
fermentor” o Lower equipment costs o Superior maintenance of
o Lower contamination risk medium quality
o Easier manual control o Easy scale-up
o For media with solid matter o Easier automatic control
Continuous o Reduction of sterilization
“in the pipeline” cycle time
o Reduction of fermentor
corrosion

Heating a batch (steam Continuous sterilizers

injection in pre-heated
medium)
129 130
31

Design of a batch sterilization process Design of a batch sterilization process

• Objective: to design a sterilization process to obtain the desired • Knowing the original number of organisms present and the risk of
degree of sterility with minimum loss of nutritive quality contamination considered acceptable, the desired Del factor can be
• Commonly used sterilization temperature: 121°C (overpressure of 1 calculated
bar; saturated steam (no air)), hold for 15-20 min • Example:
• Crucial information to design a batch sterilization process: o N0 = 1011
o Profile of the increase and decrease in the temperature of the o Nt = 10-3; commonly accepted risk of contamination is 1 in 1000
fermentation medium during the heating and cooling periods o Question: how long should the medium be hold at 121°C to obtain
o Number of cells (spores) originally present in the medium (N0) this risk of contamination: calculate holding time
1011
o The risk of contamination considered acceptable (Nt) o overall = ln( − ) = 32,2
10 3
o Thermal death characteristics of a model organism relevant for the BUT! Destruction of cells not only occurs during the period of
fermentation, e.g. spores of Bacillus stearothermophilus (kd = 2,538
121°C, but also occurs during the heating and cooling of the medium,
min-1 for 121°C)
thus
o = heating +
overall holding+ cooling
o How to calculate heating and cooling ???
131 132
 Design of a batch sterilization process Design of a batch sterilization process
Calculation of heating and cooling Calculation of heating and cooling
• For a constant temperature • Divide heating time in equal time intervals
𝑁0 • For each increment, the temperature
)=At𝑒 −𝐸𝑑 Τ𝑅𝑇 =kdt
=ln( corresponding to the mid-point time is
𝑁𝑡
recorded
• But: in a BATCH STERILISATION PROCESS temperature during heating and • Total Del factor of the heating-up period is
cooling is not constant. So the total should be calculated as an integration equal to the sum of the Del factors of the
of the equation: mid-point temperatures for each time
increment
𝑡 𝑡 • 1 = k1 t; 2 = k2 t; 3 = k3 t; etc, often until
= ‫׬‬0 1 𝐴𝑡𝑒 −𝐸𝑑Τ𝑅𝑇 + 𝐴𝑡𝑒 −𝐸𝑑Τ𝑅𝑇 + ‫ 𝑡׬‬3 𝐴𝑡𝑒 −𝐸𝑑Τ𝑅𝑇 30
2

Heating Holding Cooling • k values can be deduced from the Arrhenius

equation using the thermal death
characteristics of
• Using graphical method of integration = practical and easy method B. stearothermophilus spores at the
given temperature (eq 5.13)
• Similar for Del factor cooling down period

133 134

Design of a batch sterilization process Design of a batch sterilization process

Rapid method
• Back to our example… • Graphical integrations: time consuming…
• Example • Rapid method available, based on the assumptions:
o N0 = 1011 o All spore destruction occurs above 100°C
o Nt = 10-3 o Parts of heating and cooling above 100°C are linear
1011
o overall = ln( −3) = ln 1014 = 32,2 • Table with Del factors for B. stearothermophilus spores available which
10
would be obtained when heating and cooling a broth to holding
o overall= heating + holding + cooling , Suppose heating = 9,8 and cooling = 10,1 temperatures of 101-130°C based on a temperature change of 1°C per
o holding = overall - heating - cooling = 32,2 - 9,8 - 10,1 = 12,3
minute.
o = kd t; and kd for B. stearothermophilus spores at 121°C is 2,54 min-1
12,3
o Thus t = holding = = 4,84 min
kd 2,54
o If the contribution of heating and cooling was ignored:
holding 32,2
t= = = 12,68 min
kd 2,54
➔ Ooops…. Will cause medium degradation

135 136
 Del Values for B. stearothermophiles spores for the heating-up period over a temperature range of 100
to 130°C, assuming a rate of temperature change of 1°C min-1 and negligible spore destruction at Design of a batch sterilization process
temperature below 100°C.
Rapid method
• Example: calculate Del factor for
o Heating from 100 to 121°C in 30 min
o Cooling from 121°C to 100°C in 17 min
o heating = 12,549 * 30/21 = 17,93

21°C, 21 min
o cooling = 12,549 * 17/21 = 10,16

Temperature Correction for the fact that

change of 1°C / the Del factor in the Table
min was based on an increase of
1°C / min

137
13
7 138

Design of a batch sterilization process Design of a batch sterilization process

Scale up of batch sterilization process Methods of batch sterilization
o Del factor does not include a volume term! Instead ABSOLUTE
numbers of cells are included. [ = ln (N0/Nt)]
o Bath sterilization of the medium
o When volumes are taken into account the formula should be • In the fermentation vessel
adapted. • In a separate batch cooker
o Example: Calculate Del factor for
• 1 000 l medium containing 106 cells/ml and Nt = 1/1000

• 10 000 l medium containing 106 cells/ml and Nt = 1/1000

Del factor is affected by the size of the fermentor volume.

139 140
 Design of a batch sterilization process
Sterilization in a separate batch cooker
• Advantages
o One cooker for several fermentors
(saving time: sterilization while
cleaning!)
o Cooking in a more concentrated
form; then dilution with sterile
water prior to inoculation
o In some cases the medium is most
viscous during sterilization =>
Sterilization requires a more
powerful motor for agitation
o Production fermentor is spared
corrosion which may occur with
medium at high temperature
• Disadvantages
o Construction costs of a cooker are
comparable to the production
cost of a fermentor
o Pipework from cooker to
fermentor: risk of contamination
o Mechanical failure in a cooker
would cause production stop
(especially when serving several
fermenters)
Table of contents
• Introduction
• Medium sterilization (by steam)
o The design of batch sterilization processes
o The design of continuous sterilization processes
• Sterilization of the fermentor
• Sterilization of the feeds
• Sterilization of the liquid wastes
• Filter sterilization (air and media)
142

141

Design of a continuous sterilization process Design of a continuous sterilization process

• As in a batch sterilization process, the continuous system includes:
o Heating: a time period during which the medium is heated to the
sterilization temperature
o Holding: a holding time at the desired temperature
o Cooling: a cooling temperature to bring the medium to the
fermentation temperature
But: in this case the sterilization is not performed in the
vessel, but in the pipeline in which a small fraction is
sterilized at a time
• Practical:
o Heating: 2 sequential heat exchangers, the first one using the
outcoming sterilized medium as heating source (= pre-heat heat
exchanger), the second one using steam (indirect or direct heat
exchanger)
o Holding: insulated holding coil; length of the holding period
determined by length of the coil and flow rate of the medium
o Cooling: 2 sequential heat exchangers, the first one using the incoming
medium as cooling source, the second using cooling water
• Advantages:
o A much higher temperature may be utilized, reducing holding time
and nutrient degradation
o ‘Heating up’ and ‘cooling down’ periods are very small – nearly
negligible

143 144
Design of a continuous sterilization process Design of a continuous sterilization process
Types of continuous sterilizers
• A much higher temperature can be used, reducing the holding time
! • Indirect heat exchanger
o Example: For the same Del factor (i.e. 45,7), the following o Plate heat exchanger
combinations can be used, bringing the holding time to only a few o Spiral heat exchanger
seconds when T = 150°C !

o Cold and warm flow in opposite

direction next to each other, during
which heat is exchanged

• Direct heat exchanger

o Steam injector

145 146
14 14
5 6

Design of a continuous sterilization process Design of a continuous sterilization process

Plate heat exchanger Plate heat exchanger
Mechanism:see next slide Heated medium
out

Steam in

Unsterile cool
medium in
(cooled)
steam out

• https://www.youtube.com/watch?v=Jv5p7o-7Pms
Flow diagram of a typical continuous plate heat exchanger sterilizator • Disadvantages:
• Cross contamination possible (through the plates)
147 • May be blocked be solid particles
148
14 14
7 8
 Design of a continuous sterilization process
Spiral heat exchanger
Heated medium
out

Unsterile
medium in

Steam in

Advantages
(cooled)
steam out
• Suitable for suspended solids
• No cross contamination

149 150
15
DOI:10.3390/en10091398
0

Design of a continuous sterilization process Design of a continuous sterilization process

Spiral heat exchanger Steam injector

• Advantages
o Very short heating up times
o Suitable for media with suspended solids
o Low capital costs
o Easy cleaning and maintenance
o High steam utilization efficiency

• Disadvantages
o Foaming during heating
o Direct contact between medium and steam requires ‘clean
steam’ (without anticorrosion additives)

151 152
15
2
Continuous sterilization Continuous sterilization
Which time-temperature regime? Which time-temperature regime?
• First define a Nutrient Quality Criterion (Q), based on similar logic than Del • First define a Nutrient Quality Criterion (Q), based on similar logic than
factor but not scale dependent (sterilization criterion) Del factor (sterilization criterion)
𝑥 𝑥
𝑄= ln( 0) (Eq. 5.14) 𝑄 = ln( 0) (Eq. 5.14)
𝑥𝑡 𝑥𝑡
Where x0: the concentration of essential nutrients at the start • Destruction of a nutrient is a first-order reaction (see before)
of the sterilization 𝑥𝑡
= 𝑒 −𝑘𝑡 (Eq. 5.11)
𝑥0
xt: the concentration of essential nutrients after a
Where k: reaction rate constant
sterilization time t 𝑥0
• Destruction of a nutrient is a first-order reaction (see before) • Taking the natural logarithm: ln 𝑥𝑡
= 𝑘𝑡 (Eq. 5.15)
𝑥𝑡
= 𝑒 −𝑘𝑡 (Eq. 5.11) Thus : 𝑄 = 𝑘𝑡 and 𝑘 = 𝐴𝑒 −𝐸Τ𝑅𝑇 (Eq. 5.16; Eq. 5.17)
𝑥0
Where k: reaction rate constant → 𝑄 = 𝐴𝑡𝑒 −𝐸Τ𝑅𝑇 (Eq. 5.17)
𝑥0 • Taking natural logarithm and rearrangement:
• Taking the natural logarithm: ln 𝑥𝑡
= 𝑘𝑡 (Eq. 5.15)
Thus : 𝑄 = 𝑘𝑡 and 𝑘= 𝐴𝑒 −𝐸Τ𝑅𝑇 (Eq. 5.16; Eq. 5.17) 𝐸 𝑄
−𝐸 Τ𝑅𝑇
ln 𝑡 = + ln( ) (Eq. 5.18)
→ 𝑄 = 𝐴𝑡𝑒 (Eq. 5.17) 𝑅𝑇 𝐴

153 154
15 15
3 4

Continuous sterilization Continuous sterilization

Which time-temperature regime? Which time-temperature regime?
• Time required to achieve a certain Q value against 1/T
→ straight line microbe destruction
𝐸 𝑄
(high E)
ln 𝑡 = + ln( ) (Eq. 5.18) nutrient destruction
𝑅𝑇 𝐴
(low E)

• Remember time required to achieve a certain Del value against 1/T

→ straight line
𝐸𝑑
ln 𝑡 = + ln( ) (Eq. 5.9)
𝑅𝑇 𝐴

• E for nutrient destruction is low compared to E for microbe

destruction
→ different slopes
156
74
155
 Continuous sterilization Table of contents
Which time-temperature regime?
• Introduction
microbe destruction • Medium sterilization (by steam)
(high E)
nutrient destruction o The design of batch sterilization processes
(low E) o The design of continuous sterilization processes
• Sterilization of the fermentor
t1 • Sterilization of the feeds
V
t2 (low E) • Sterilization of the liquid wastes
• Filter sterilization (air and media)

1/T2 < 1/T1

The only way in which Del factor may be increased without any change in
157 157
nutrient quality criterion is to increase the temperature and to decrease the 158
7
74
holding time 5

Sterilization of the fermentor Table of contents

• Any joints, crevices or flange joints are potential hazardous points • Introduction
where nutrients and microorganisms can stay and contamination • Medium sterilization (by steam)
can take place
o The design of batch sterilization processes
• Fermentor should be free of crevices and stagnant areas and have a
o The design of continuous sterilization processes
minimum number of joints, and joint points should be as smooth as
possible • Sterilization of the fermentor
• Industrial fermenters are designed for in situ steam sterilization • Sterilization of the feeds
under pressure • Sterilization of the liquid wastes
• After sterilization, the fermentor should be flushed with sterile air • Filter sterilization (air and media)
to keep positive pressure in the fermentor
• After sterilization, medium and air entering the fermentor should
be sterile. The transport line should be maintained under aseptic
conditions
160

159
Sterilization of the feeds Table of contents
• E.g. water, additional raw materials, antifoam mixture etc. • Introduction
• Large quantities: continuous sterilization • Medium sterilization (by steam)
• Heat labile nutrients: sterile membrane filtration o The design of batch sterilization processes
• Batch sterilization of feeds in storage vessel o The design of continuous sterilization processes
• Feed pipework must be sterilizable • Sterilization of the fermentor
• Sterilization of the feeds
• Sterilization of the liquid wastes
• Filter sterilization (air and media)

162

161

Sterilization of the liquid wastes

• Genetically modified microorganisms: regulations !
• Then: sterilization of waste biomass (batch or continuous)
• See sterilization of medium
• Calculation sterilization process
o Kinetic thermal death characteristics of the process organism
o High Del factor => try to kill them all

164

163
Table of contents Filter sterilization
• Introduction • Separation of suspended solids from a fluid (gas or liquid)
• Medium sterilization (by steam)
o The design of batch sterilization processes
o The design of continuous sterilization processes
• Sterilization of the fermentor
• Sterilization of the feeds
• Sterilization of the liquid wastes
• Filter sterilization (air and media)

165

166

Filter sterilization Filter sterilization

Medium sterilization Air sterilization

• Filter sterilization of incoming and exhaust air

• Filter sterilization of medium containing heat sensitive compounds
o Membrane and depth filters
o Membrane filters (surface filtration)
o Incoming air
o Membrane: made of cellulose esters or other polymers, pore diameter
• Air contains 103 – 104/ m3 microorganisms
between 0.2 and 0.45 µm
• Method of air sterilization:
o Membrane itself must be sterilized by steam or radiation before use
• Sterilization by heating (economically impractical)
o Bacteria and other particles with dimensions greater than the pore • Radiation (UV)
size are screened out and collected on the surface of the membrane • Use of germicidal sprays (e.g. phenol, formalin,…)
o Filtration is not as effective or reliable as heat sterilization • Sterilization by filtration: often used!
o Off-gasses leaving the fermentor
• The concentration of cells in unfiltered fermentor off-gasses is several times
greater than in air
• Some organisms may be harmful to plant personnel or environment

167 168
Filter sterilization Filter sterilization
Types of filters Types of filters

• Two types of filters

‘Fixed pore’ filter Surface ‘Non-fixed pore’ filter
o Surface (membrane) filters (absolute filter): filters with pores that are
smaller than the size of the microorganisms to be removed filter Depth filter
o Depth filters (fibrous filter): filters with pores that are bigger than the
size of the microorganisms to be removed; microbes are collected in
the filter
Filter Type Surface filter Depth filter
Application filtration of liquids filtration of gasses
and gasses (and liquids)
Filter material membranes (polymer, …) package of fibers

Pores < dimension of > dimension of

retained particles retained particles
Particle removal absolute rating probability

Disadvantage large pressure drop small pressure drop

across the filter across the filter
(minimized by large
surface area) 170

169

Filter sterilization And what is a HEPA filter?

Types of filters

To qualify as HEPA (high-efficiency particulate arresting) by US government standards,

membrane depth filters the filter must remove (from the air that passes through) 99.97% of all particles that
filters (fibrous material) have a size of 0.3 µm or more.
171
(pleated sheet)
172
 Content lectures

• PART 1: THE HISTORICAL DEVELOPMENT OF BIOTECHNOLOGY

• PART 2: FERMENTATION TECHNOLOGY
Chapter 3. Aeration and agitation
1. Introduction to fermentation processes
2. Microbial growth
3. Isolation, preservation and improvement of industrial micro-
organisms
4. Media for industrial fermentations
5. Sterilization processes
6. The development of inocula for industrial fermentations
7. The fermentor design
8. Instrumentation and control
9. Aeration and agitation
10. Downstream processing

• PART 3: BIOCONVERSION TECHNOLOGY

174

Table of contents Table of contents

• Introduction
• Oxygen requirements of industrial fermentation
• Oxygen supply
• Determination of KL a values
• Factors affecting KL a values in fermentation vessels
• Introduction
• Oxygen requirements of industrial fermentations
• Oxygen supply
• Determination of KLa values
• Factors affecting KLa values in fermentation vessels

175 176
 Introduction Table of contents
• Most fermentations are aerobic, and require oxygen • Introduction
• Productivity is often limited by oxygen availability • Oxygen requirements of industrial fermentations
• Oxygen must be supplied during growth at a rate sufficient to satisfy • Oxygen supply
the organisms’ demand • Determination of KLa values
• Normally satisfied by aerating and agitating the broth • Factors affecting KLa values in fermentation vessels
• This chapter:
o Oxygen requirements of industrial fermentations and oxygen
uptake
o Quantification of oxygen transfer (KLa values)
o Factors affecting KLa values in fermentation vessels

177 178

Oxygen requirements Oxygen demand dependent on carbon source

• Darlington expressed the composition of 100 g (dry weight) of
• Example: using stocheometry of the respiration of glucose yeast cells as C3,92H6,5O1,94
C6H12O6 + 6 O2 → 6 H2O + 6 CO2 o Equations have been proposed for yeast production from

180 g 108 g 264 g

carbohydrate and hydrocarbon:
• Carbohydrate (CH2O):
192 g
6,67 CH2O + 2,1 O2 → C3,92H6,5O1,94 + 2,75 CO2 + 3,42 H2O
o Both components must be in solution before they are available • Hydrocarbon (CH2):
for a microorganism (O2 is 6000x less soluble in H20 than C6H12O6…) 7,14 CH2 + 6,135 O2 → C3,92H6,5O1,94 + 3,22 CO2 + 3,89 H2O
o Formula gives no indication of an organism’s true oxygen
demand as it does not take into account the carbon that is The more reduced the carbon source, the greater the oxygen
converted in biomass and products demand
o Production of 100 g biomass from hydrocarbon requires 3x
more oxygen to produce the same amount of biomass from
➔ What if we take biomass production into account?
carbohydrate
➔ Oxygen demand is dependent on the carbon source!
179 180

Atomic mass C: 12; H: 1; O: 16

Effect of DOC on oxygen uptake Effect of DOC on oxygen uptake
• BUT: not correct to derive the supply of oxygen for a
fermentation based on an estimation of the overall oxygen QO2 max
demand, as microbial metabolism is affected by the
concentration of dissolved oxygen (DOC) in the broth
• Effect of DOC concentration on the Specific Oxygen Uptake is the DOC for QO2 = 0,99 QO2 max
Rate (SOUR; QO2) (aka specific respiration rate)?
o Specific oxygen uptake rate: mmol of oxygen consumed per Ccrit
gram of dry weight of cells per hour.
QO2 max
Specific oxygen uptake rate increases with increase in DOC
concentration up to a certain point, i.e. Ccrit (critical dissolved
Michaelis-Menten type
oxygen concentration) above which no further increase in
oxygen uptake rate occurs
Ccrit

181 182

10

Critical dissolved oxygen concentrations Effect of DOC on oxygen uptake and biomass and
product formation
• QO2max gives maximum biomass production

o DOC < Ccrit:

• Biomass production disturbed (less biomass)
• Product formation may or
o DOC > Ccrit:
• Biomass production not influenced (= maximum)
• Product formation may

➔ Aeration conditions necessary for optimum production of a product

may be different from those favouring biomass production

183 184

10
Effect of DOC on oxygen uptake and product
formation
• Example: production of amino acids by Brevibacterium flavum is
strongly dependent on DOC
• Especially members of the glutamate and aspartate families are
detrimentally affected by DOC levels below Ccrit
Effect of DOC on oxygen uptake and product
formation
• Example: production of amino acids by Brevibacterium flavum is
strongly dependent on DOC
• Especially members of the glutamate and aspartate families are
detrimentally affected by DOC levels below Ccrit

Oxygen
requiring
Krebs cycle
185 Ccrit 186

Effect of DOC on oxygen uptake and product Table of contents

formation
• Introduction
• Example: production of amino acids by Brevibacterium flavum is
strongly dependent on DOC • Oxygen requirements of industrial fermentations
• Especially members of the glutamate and aspartate families are • Oxygen supply
detrimentally affected by DOC levels below Ccrit • Determination of KLa values
• Factors affecting KLa values in fermentation vessels

0,55 0,60 0,85 187 188

Oxygen supply Oxygen supply

• Cheapest available source of oxygen: AIR • Cheapest available source of oxygen: AIR
• Method of air supply varies with the scale of the process • Method of air supply varies with the scale of the process
o Lab-scale fermentors: aerated by shake-flask technique o Pilot- and industrial-scale fermentors: stirred, aerated vessels

189 190

10 10

Oxygen supply Oxygen supply

• Transfer of oxygen from air to the cell occurs in different Suppose: low viscosity fermentation
steps
o Transfer of oxygen from an air bubble into the solution
Transfer of the dissolved oxygen through the medium to the Step 1
o Air bubble Step 2
microbial cell with O2 O2 Step 3
o Uptake of the dissolved oxygen by the cell O2 aerobic
microorganism

Aeration in medium: production of air bubbles

Step 1: Transfer of oxygen from air bubbles to medium: Rate limiting! Slow step!
Step 2: Transfer throughout the medium (increased by agitation)
Step 3: Uptake of dissolved O2 by the cell

! Steps 2 and 3: also rate limiting for high viscosity media !

191 192

10
 Rate of oxygen transfer (OTR) from air to medium Oxygen Transfer Rate (OTR) from air to medium
(step 1) (step 1)
O2 saturation
𝒅𝑪𝑳 ∗
𝑶𝑻𝑹 = = 𝑲𝑳 ∗ 𝒂 ∗ 𝑪 −𝑪𝑳 (eq. 9.1)
𝒅𝒕
𝒅𝑪
Slope = 𝑳 = OTR CL: the concentration of dissolved oxygen in the fermentation broth
𝒅𝒕
High in the beginning (mmol/l)
t : time (h)
Becomes 0 when saturated
KL : the mass transfer coefficient (cm/h)
with O2 a : the gas/liquid interface area per liquid volume (cm2/cm3)
C* : the saturated dissolved oxygen concentration (mmol/l)
Degassed
solution

Aeration in function of time

193 194

Oxygen Transfer Rate (OTR) from air to medium Rate of oxygen transfer (OTR) from air to
(step 1) medium (step 1)
𝒅𝑪𝑳 ∗
𝑶𝑻𝑹 = = 𝑲𝑳 ∗ 𝒂 ∗ 𝑪 −𝑪𝑳 (eq. 9.1) 𝑶𝑻𝑹 =
𝒅𝑪𝑳 ∗
= 𝑲𝑳 ∗ 𝒂 ∗ 𝑪 −𝑪𝑳 (eq. 9.1)
𝒅𝒕
𝒅𝒕

CL: the concentration of dissolved oxygen in the fermentation broth CL: the concentration of dissolved oxygen in the fermentation broth
(mmol/l) (mmol/l)
t : time (h) t : time (h)
KL : the mass transfer coefficient (cm/h) KL : the mass transfer coefficient (cm/h)
a : the gas/liquid interface area per liquid volume (cm2/cm3) a : the gas/liquid interface area per liquid volume (cm2/cm3)
C* : the saturated dissovled oxygen concentration (mmol/l) C* : the saturated dissovled oxygen concentration (mmol/l)
C*- CL → driving force for O2 transfer 𝒅𝑪𝑳
= 0, when C*= CL is measured with Clark oxygen electrode, measure CL in
𝒅𝒕
= max, when CL=0 functionof time

195 196
Oxygen Transfer Rate (OTR) from air to medium Oxygen Transfer Rate (OTR) from air to medium
(step 1) (step 1)
𝒅𝑪𝑳 ∗ 𝒅𝑪𝑳 ∗
𝑶𝑻𝑹 = = 𝑲𝑳 ∗ 𝒂 ∗ 𝑪 −𝑪𝑳 (eq. 9.1) 𝑶𝑻𝑹 = = 𝑲𝑳 ∗ 𝒂 ∗ 𝑪 −𝑪𝑳 (eq. 9.1)
𝒅𝒕 𝒅𝒕

CL: the concentration of dissolved oxygen in the fermentation broth
(mmol/l)
t : time (h)
KL : the mass transfer coefficient (cm/h)
a : the gas/liquid interface area per liquid volume (cm2/cm3)
C* : the saturated dissovled oxygen concentration (mmol/l)
CL: the concentration of dissolved oxygen in the fermentation broth
(mmol/l)
t : time (h)
KL : the mass transfer coefficient (cm/h)
a : the gas/liquid interface area per liquid volume (cm2/cm3)
C* : the saturated dissovled oxygen concentration (mmol/l)

Difficult to measure both KL and a separately, both terms are KLa-value (h-1)
combined into KLa-value (h-1) = volumetric mass transfer coefficient or oxygen transfer coefficient
= volumetric mass transfer coefficient or oxygen transfer coefficient → Describes the efficiency with which oxygen can be delivered to a
bioreactor under given operation conditions

197 198

Oxygen Transfer Rate (OTR) from air to medium Rate of oxygen transfer (OTR) from air to medium
(step 1) (step 1)
𝒅𝑪𝑳 ∗ If we suppose that dissolved O2 (DOC) provided by aeration and
𝑶𝑻𝑹 = = 𝑲𝑳 ∗ 𝒂 ∗ 𝑪 −𝑪𝑳 (eq. 9.1)
𝒅𝒕
agitation is immediately removed from the solution due to uptake by
culture, then:
KLa-value (h-1) 𝒅𝑪𝑳
= volumetric mass transfer coefficient or oxygen transfer coefficient 𝑶𝑻𝑹 = = 𝑸𝑶𝟐 * x = (Oxygen Uptake Rate)
𝒅𝒕
→ Describes the efficiency with which oxygen can be delivered to a
bioreactor under given operation conditions OTR : oxygen transfer rate (mmol O2 l-1h-1)
o Depends on the design and operating conditions x :biomass concentration (g DW l-1)
o Affected by variables such as aeration rate, agitation rate 𝑄𝑂2 :specific oxygen uptake rate (mmol O2 g-1 DW h-1).
and impeller design The specific oxygen uptake rate shows maximum values
for each organism in a specific medium.

199 200
Table of contents Determination of KLa values
• Introduction
• Determination of the KLa value of a fermentor is essential to
• Oxygen requirements of industrial fermentations calculate its aeration efficiency and to quantify the effects of
• Oxygen supply operating variables on the provision of oxygen
• Determination of KLa values • Determination of KLa values
• Factors affecting KLa values in fermentation vessels o The sulphite oxidation technique
o Gassing-out techniques
• Static method of gassing out
• Dynamic method of gassing out
o The oxygen-balance technique

Static method of gassing out

201 202

10

Dynamic method of gassing out Dynamic method of gassing out

• Based on the respiratory activity of a growing culture in a real

fermentor
• Measured using a probe to monitor change in DOC
• Gives a realistic assessment of the fermentor’s aeration
efficiency A → B, Gas off
• KLa = 0
• Procedure 𝑑𝐶𝐿
o Stop supply of air to the fermentation :
• = − 𝑄𝑂2 ∗ 𝑥
𝑑𝑡
• results in a linear decline in DOC due to the respiration of the • OTR = -OUR
culture
→ the slope is a measure of the respiration rate of the culture
o Start aeration again :
• DOC increases until it reaches the maximum concentration
again
203 204

10 10
Dynamic method of gassing out Dynamic method of gassing out
Over the period BC, the observed rearrange
increase in DOC is the difference
between the transfer of oxygen into 𝑑𝐶𝐿 ∗
−1 𝑑𝐶𝐿 ∗
solution and the uptake of oxygen by = 𝐾𝐿𝑎(𝐶 −𝐶𝐿) − 𝑄𝑂2 ∗ 𝑥 (𝑒𝑞. 9.4) 𝐶𝐿 = + 𝑄𝑂2 𝑥 + 𝐶 (𝑒𝑞. 9.5)
𝑑𝑡 𝐾𝐿𝑎 𝑑𝑡
the respiring culture

B → C, Air sparging resumed

𝑑𝐶𝐿
• = 𝑂𝑇𝑅 − 𝑂𝑈𝑅
𝑑𝑡

𝑑𝐶𝐿 ∗
• = 𝐾𝐿𝑎(𝐶 −𝐶𝐿) − 𝑄𝑂2 ∗ 𝑥 (𝑒𝑞. 9.4)
𝑑𝑡

205 206

Table of contents Factors affecting KLa values

• Introduction
• The effect of air-flow rate on KLa
• Oxygen requirements of industrial fermentations
• Oxygen supply
• Determination of KLa values Rarely falls outside
• Factors affecting KLa values in fermentation vessels the range of 0,5-
1,5, and tends to
be maintained
constant on scale-
up

207 208

10
Factors affecting KLa values
• The effect of viscosity on KLa
• The effect of agitation degree on KLa
o Agitation increases the area available for oxygen transfer by dispersing
the air in the culture fluid in the form of small bubbles
o Agitation delays the escape of air bubbles from the liquid
o Agitation prevents coalescence of air bubbles
o Agitation decreases the thickness of the liquid film at the gas-liquid
interface by creating turbulence in the culture fluid
• The degree of agitation may be measured by the amount of power
consumed in stirring the vessel content
Factors affecting KLa values
o Rheology of the broth is influenced by
• Composition of the original medium and its modification by the growing culture
o Starch containing medium is degraded by microorganism → lower viscosity
• Concentration and morphology of biomass
o Bacterial and yeast fermentations: low viscosity
o Fungal and streptomycete fermentations: high viscosity
o Pellets: low viscosity / Hyphae: high viscosity
• Concentration and rheological properties of microbial products
o Production of bacterial polysaccharides (pseudoplastic!) → higher viscosity
Increasing viscosity µ → decreasing KLa

209 210

10 10

Factors affecting KLa values Factors affecting KLa values

• Fermentation products may change KLa • The effect of mycelium concentration on KLa

increasing viscosity → decreasing KLa

increasing viscosity
212 → decreasing KLa
211
Factors affecting KLa values

• The effect of foam and antifoam on oxygen transfer

o Foam may overflow via the air outlet or sample line, resulting in loss of
medium/product as well as increasing the risk of contamination
o May also have adverse effect on oxygen transfer rate:
• Presence of foam may increase the residence of bubbles and therefore
make air bubbles depleted of oxygen
• Foam in the impeller region prevents adequate mixing
o Therefore, desirable to break down the foam, e.g. with mechanical
foam breakers of chemical antifoams
o Antifoams are surfactants, resulting in
• Collapse of bubbles in foam
• Decrease oxygen transfer rate
• Favours coalescence of bubbles (surface area )

213

10