Chapter 4

How Cells Grow

Ir. Normadyzah Ahmad

Semester Sept 2017 – Jan 2018
SYNOPSIS
This course imparts in-depth knowledge of design and
scale-up of bioreactors along with transport phenomena in
bioreactors.
It also imparts the knowledge of sterilisation of bioreactor
systems.
It also imparts the knowledge of alternate bioreactor
configurations for microbial, plant, and animal cells.
After successfully completing this course, students will be
able to choose, design, scale-up and analyse bioreactors for
various applications.
CBE654: BIOREACTOR ENGINEERING
3.0 OBJECTIVES
Upon completion of this course, students should be able
to:
1) Do design and analysis of Bioreactor for free microbial
cell, plant and animal cells.
2) Explain the transport processes in stirred tank
bioreactors
3) Explain the alternatives of bioreactor configurations
 Where do these come from?

 Antibiotics
 Liquor (wine, beer)
 Yogurt
 Fermented Food (Tempeh, Tapai, Kimchi)
 Enzymes (protease, amylase for detergents)

Answers: Bioreactions
Take a step back…….
QUESTION 1: Why do we grow microbial cells, plant cells, and animal
cells?
ANSWER: Because we want them to convert some feedstock (input) into
useful products (output) , for us to sell.

QUESTION 2: If we want them to convert some feedstock to useful

products, what information do we need?
ANSWER: We need information on the stoichiometric equation of the
conversion bioreaction, we need the rate of the conversion.

QUESTION 3: If we want to design the bioreactor in which this

bioreaction will happen, what further information do we need?
ANSWER: We need information on what variables affect the rate, and
how.
The opportunities…..
If you know what bioreaction convert what feedstock to what
useful new products which are NOT available but are in demand
in the market, you may have a viable business proposal! (new
product, new process).

If you know the stoichiometric equation of the conversion

bioreaction, if you know the rate of the conversion bioreaction,
and if you know what variables affect the rate of the conversion
bioreaction and in what way, you can design the bioreactor.

Hence the ability of students to: (1) Design and analyse Bioreactor
for free microbial cell, plant and animal cells, (2) Explain the
transport processes in stirred tank bioreactors, and (3) Explain the
alternatives of bioreactor configurations, as in the OBJECTIVES.
WHAT PRODUCTS?
TYPES OF PRODUCT:
Product 1: FREE MICROBIAL CELLS. eg: yeast, single cell protein
(SCP), algae
Product 2: ENZYMES. eg: proteases and amylases (for
detergents)
Product 3: PRIMARY METABOLITES: eg: ethanol and lactic acid
Product 4: SECONDARY METABOLITES. eg: penicillin
Product 5: BIOTRANSFORMATION PRODUCTS. eg: gluconic acid
from glucose
Product 6: PRODUCT OF SYNTHESIS BY RECOMBINANT GENE. eg:
proinsulin
WHAT BIOREACTIONS?
WHAT BIOREACTIONS?
Do you know what type of bioreactions are involved in the
production of each of the above products?
Do you know the stoichiometric equation of each bioreaction?
Do you know the rate of each bioreaction?
Do you know what variables affect the rate of each
bioreaction?
WHAT IS A BIOREACTOR?
If you know the stoichiometric equation of the conversion bioreaction, if
you know the rate of the conversion bioreaction, and if you know what
variables affect the rate of the conversion bioreaction and in what way,
you can design a bioreactor system.

Bioreactor – a vessel in which is carried out a chemical process which

involves organisms or biochemically active substances derived from such
organisms (eg. Enzymes) to produce bioproducts. Example: yeast
(organisms) + glucose (chemical substances) to produce ethanol
(bioproduct)

Types of bioreactor:
• Batch bioreactor (batch culture)
• Continuous Stirred Tank Bioreactor or Chemostat (continuous culture)
• Plug Flow Bioreactor (continuous culture)
How Cell Grow?
What bioreactions are involved in the production of free microbial cells?:
Growth

Substrates + cells → extracellular products + more cells

∑S+X → ∑ P + nX

The rate of growth is directly related to cell concentration.

The rate of microbial growth is characterized by the net specific growth rate μ, defined as:
dX
= μnet .X, by rearranging this equation, it becomes,
dt
μnet = 1 dX
X dt
………… (1)
 μnet = 1 dX ……………… (1)
X dt
μnet = μg − k d …..………… (2)

where X is cell mass concentration (g/l)

t is time (h)
μnet is net specific growth rate (h−1 )
μg is gross specific growth rate (h−1 )
kd is rate of loss of cell mass due to cell death or
endogeneous metabolism (h−1 )
Microbial growth can also be described in terms of cell number,
N, as well as X.

μR = 1 dN
N dt
…………… (3)

where μR is the net specific replication rate (h−1 )

If we ignore cell death, k d , then we use the symbol μ′R ;

and in cases where cell death is negligible, μR = μ′R
Fig 1: Typical growth curve for a bacterial population (cell no. vs time)
 A typical batch growth curve includes the following phases:

Phase Description Specific

Growth Rate
Lag Cells adapt to the new 0
environment; no or very little
growth
Acceleration Growth starts  < max
Growth Growth achieve its maximum   max
rate
Decline/Decelera Growth slows due to nutrient  < max
tion phase exhaustion or build-up of
inhibitory products
Stationary Growth ceases (reduces) =0
Death Cells lose viability and lyse <0
The exponential growth phase
 Cells have adjusted to their new environment.
 Cell mass, X & cell number density, N increases exponentially with time.
 Balanced growth, in which all components of cell grow at the same rate.
 Since the nutrient concentrations are large in this phase, the growth rate is
independent of nutrient concentration.
The exponential growth rate is first order:

𝑑𝑋
𝑑𝑡
= μ𝑛𝑒𝑡 . 𝑋, X = 𝑋𝑜 𝑎𝑡 𝑡 = 0 ………………... (4)
Integration of eq. 4 yields:

ln 𝑋
𝑋𝑜
= μ𝑛𝑒𝑡 . 𝑡, or
X=𝑋𝑜 . 𝑒 μ𝑛𝑒𝑡 .𝑡 ……………..(5)

where X and 𝑋𝑜 are cell concentrations at time t and at time zero.

The time required to double the microbial mass is given by eq. 5. The exponential
growth is characterised by a straight line on a semi-log plot of ln X vs time:

τ𝑑 = 𝑙𝑛2
μ𝑛𝑒𝑡
= 0.693
μ𝑛𝑒𝑡
…………….. (6)
where τ𝑑 is the doubling time of cell mass.
Similiarly, we can calculate a doubling time based on cell numbers
and the net specific rate of replication:
′
τ𝑑 = ln 2
μ𝑅
………. (7)

where τ′𝑑 is the doubling time based on the replication rate.

During balanced growth τ𝑑 = τ′𝑑 since the average cell composition &
size will not change with time.
 The decline/deceleration growth phase
 Occurs after the exponential growth phase.
 Growth decelerates due to either depletion of one or more
essential nutrients or the accumulation of toxic by-products.
 Results in unbalanced growth.
 Cell composition and size change and τ𝑑  τ′𝑑 .
 The stresses induced by nutrient depletion or waste accumulation
causes a restructuring of the cell to increase the prospect of
survival in a hostile environment.
  < max
 Stationary phase
 occurs after the deceleration growth phase, when the net = 0, ie no cell division,
or growth rate is equal to death rate.
 But cells are still metabolically active and produce secondary (non-growth
related) metabolites.
 The production of certain metabolites (eg antibiotics) is enhanced during the
stationary phase, due to metabolite deregulation.
During the course of the Stationary Phase, the following phenomena may take place:
1. Total cell mass concentration may stay constant, but the number of viable cells may
decrease.
2. Cell lysis may occur and the number of viable cells may drop. A second growth phase
may occur and cells may grow on lysis products of lysed cells (cryptic growth).
3. Cells may not be growing but may have active metabolism to produce secondary
metabolites.
Cell lysis – breaking down of the membrane of the cell.
During stationary phase, the cell catabolises cellular
reserves for new building-blocks and for energy-producing
monomers (endogeneous metabolism).

This is so that the cell can always maintain an energised

membrane and transport of nutrients, and so that the
cell can always maintain essential metabolic functions
such as motility and repair of damage to cellular
structures.
This energy expenditure is called “maintenance
energy”. The conversion of cell mass into maintenance
energy, or the loss of cell mass due to cell lysis during
the Stationary Phase can be described thus:
𝒅𝑿
𝒅𝒕
= - 𝑘𝑑 . 𝑋 or X = 𝑋𝑠𝑜 . 𝑒 −𝑘𝑑 .𝑡 ………….. (8)

where 𝑘𝑑 = 1st-order rate constant for endogeneous metabolism,

𝑋𝑠𝑜 = cell mass concentration at the beginning of the
stationary phase.
 Death phase
 Follows the stationary phase.
 The rate of death usually follows first-order kinetics:

′
𝑑𝑁
𝑑𝑡
=− 𝑘𝑑′ . 𝑁 or N = 𝑁𝑠 . 𝑒 −𝑘𝑑 .𝑡
……….. (9)

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

𝑘𝑑′ is the first – order death rate constant.

A plot of ln N vs t yields a line of slope − 𝑘𝑑′ .

 Stoichiometric parameters in growth kinetics
 Yield coefficients - defined based on the amount of consumption of a
given material:

E.g. the growth yield is 𝑌𝑋ൗ𝑆 = - ∆𝑋

∆𝑆
……………. (10)

At the end of the batch growth period, we have an apparent growth yield (or
observed growth yield).
Substrate may be consumed as the following:
∆𝑆 = ∆𝑆 𝑎𝑠𝑠𝑖𝑚𝑖𝑙𝑎𝑡𝑖𝑜𝑛 + ∆𝑆𝑎𝑠𝑠𝑖𝑚𝑖𝑙𝑎𝑡𝑒𝑑 𝑖𝑛𝑡𝑜 + ∆𝑆𝑔𝑟𝑜𝑤𝑡ℎ + ∆𝑆𝑚𝑎𝑖𝑛𝑡𝑒𝑛𝑎𝑛𝑐𝑒 ….(11)
𝑖𝑛𝑡𝑜 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 𝑎𝑛 𝑒𝑥𝑡𝑟𝑎𝑐𝑒𝑙𝑙𝑢𝑙𝑎𝑟 𝑒𝑛𝑒𝑟𝑔𝑦 𝑒𝑛𝑒𝑟𝑔𝑦
𝑝𝑟𝑜𝑑𝑢𝑐𝑡
 Because ∆𝑆 changes with growth condition, YX/S is not a constant.
 Yield coefficients based on other substrates or
product formations may be defined:
𝑌𝑋/𝑂2 = - ∆𝑋
∆𝑂2
……………………… (12)

𝑌𝑃/𝑆 = - ∆𝑃
∆𝑆
…………………… (13)

For organisms growing aerobically on glucose, 𝑌𝑋/𝑆 is typically 0.4 to 0.6

g/g for most yeast and bacteria, while 𝑌𝑋/𝑂2 is 0.9 to 1.4 g/g.

Anaerobic growth is less efficient, and the yield coefficient is smaller (see
Fig. 6.5)
Fig. 6.5 Aerobic and anaerobic growth yields of Streptococcus faecalis with
Glucose as substrate.
 Maintenance Coefficient

 Maintenance coefficient is used to describe the specific rate of substrate, S

uptake for cellular maintenance:
𝑑𝑆/𝑑𝑡 𝑚
𝑚=− 𝑋
…………………. (14)

However, during stationary phase where little external substrate is

available, endogeneous metabolism of biomass components is used
for maintenance energy.
Maintenance energy represents energy expenditures to repair
damaged cellular components, to transfer some nutrients and
products in and out of cells, for motility, and to adjust the osmolarity
of the cells’ interior volume.
Microbial Products

Growth-
associated
products.

Non-growth-
Microbial
associated
Products
products

Mixed-growth-
associated
products.
Growth-associated products.
Non-growth-associated products.
 Mixed-growth-associated product

The specific rate of product formation is given by:

𝑞𝑝 = α. μ𝑔 .β ……………
(17)
Examples of mixed-growth-associated products are lactic acid, xantan gum.
 Example 6.1
Time (h) Cell Concentration, X (g/l) Glucose concentration, S (g/l) Ln X

0 1.25 100 0.223

9 2.45 97 0.896

16 5.1 90.4 1.629

23 10.5 76.9 2.351

30 22 48.1 3.091

34 33 20.6 3.496

36 37.5 9.38 3.624

40 41 0.63 3.714
 Example 6.1
Ln X vs t
4.5

net = ln X 2  ln X 1  3.62  1.63  0.1h 1

4 3.624
3.5 t 2  t1 36  16
3
2.5
ln X

2 1.629
1.5
1
0.5
0
0 5 10 15 20 25 30 35 40 45

Time(h)
 Example 6.1
X 41  1.25
 Apparent growth yield, Y =    0.4 g cells/g substrate
S 0.63  100

 If S0 =150 g, then
Xmax = X0 + YS0 = 1.25 + 0.4(150) = 60.25 g cells/l

Now, do Prob. 6.1!

ENVIRONMENTAL FACTORS ON GROWTH KINETICS
 Effect of Temperature
 Product formation – may be different from cell growth.
 Yield coefficient – critical. Increasing temperature may result in a
decrease in yield coefficient because cell maintenance
requirement increase.
 rate-limiting step - At high temperatures, the rate of bioreaction
might become higher than the diffusion rate, and diffusion would
then become the rate-limiting step (eg in an immobilised cell
system).
Buffer is needed or active pH control system
0.15
Saturated dissolved oxygen (DO) concentration in
water at 25𝑜 𝐶 and 1 atm pressure is about 7ppm.

The critical oxygen concentration for bacteria and yeast is about 5% to 10% of
this saturated DO concentration.

The presence of dissolved salts and organics can alter the saturation value.

Increasingly high temperatures decreases the saturation value.

Oxygen is usually introduced to fermentation broth by
sparging air through the broth.
Oxygen transfer from gas bubbles to cells is usually limited by oxygen
transfer through the liquid film surrounding the bubbles.
The rate of oxygen transfer (OTR) from the gas to liquid phase is
given by:

𝑁𝑂2 = 𝑘𝐿 . 𝑎. 𝐶 ∗ − 𝐶𝐿 = 𝑂𝑇𝑅 ……………………………………… (15)

where 𝑘𝐿 is the oxygen transfer coefficient (cm/h)

𝑎 is the gas-liquid interfacial area (𝑐𝑚2 /𝑐𝑚3 )
𝑘𝐿 . 𝑎 is the volumetric oxygen transfer coefficient (ℎ−1 )
𝐶 ∗ is the saturated DO concentration (mg/l)
𝐶𝐿 is the actual DO concentration in the broth (mg/l)
𝑁𝑂2 is the rate of oxygen transfer (mg 𝑂2 /l.h)
The oxygen uptake rate (OUR) is given by:

OUR = 𝑞𝑂2 . 𝑋 =
μ𝑔 .𝑋
…………….. (16)
𝑌𝑋/𝑂
2

where 𝑞𝑂2 is the specific rate of oxygen consumption (mg𝑂2 /gdw

cells.h)
𝑌𝑋/𝑂2 is the yield coefficient on oxygen (g dw cells /g 𝑂2 )
X is cell concentration (g dw cell /l)
When oxygen transfer is the rate-limiting step, the rate of
oxygen consumption is equal to the rate of oxygen transfer
(OTR).
μ𝑔 .𝑋
= 𝑘𝐿 . 𝑎. 𝐶 ∗ − 𝐶𝐿 ………………. (17)
𝑌𝑋/𝑂
2

𝑑𝑋
= 𝑌𝑋/𝑂2 . 𝑘𝐿 . 𝑎. 𝐶 ∗ − 𝐶𝐿 ……………. (18)
𝑑𝑡

Growth rate varies nearly linearly with OTR under oxygen-

transfer limitations.