Professional Documents
Culture Documents
Bioprocess Technology
Yuan Kun Lee
Department of Microbiology, National University of Singapore
5 Science Drive 2, Singapore 117597
Email: micleeyk@nus.edu.sg
When all nutrients required for cell growth are present in non-growth
limiting quantity, i.e. at sufficiently high concentrations (so that minor
changes do not significantly affect the reaction rate) and the culture
environment is favorable, most unicellular microorganisms reproduce
asexually. The size and biomass of individual cell increase with time,
resulting in biomass growth. Eventually, the DNA content doubles in
quantity and cell division occurs upon complete division of a cell into
two progenies of equal genome and approximately identical size, thereby
increasing the population number. This increase in the population number
of cells in a culture is known as population growth (Fig. 2.1).
23
Box 2.1
What is Biomass?
Biomass is defined as the materials essential for the structure and reproduction
of a living organism. Thus, storage products such as starch and glycogen are
not true biomass and should be subtracted from the weight of the biological
material measured, to give the true biomass. The biomass is often expressed
as a concentration, e.g. g-biomass/L-culture.
Total biomass
Mass Storage products
(g/L) True biomass
Time
B io m ass g ro w th P o p u latio n g ro w th
Fig. 2.1. Bacterial cell growth and division.
Growth kinetics deal with the rate of cell growth and how it is affected
by various chemical and physical conditions. During the course of growth,
the heterogeneous mixture of young and old cells is continuously changing
and adapting itself in the media environment, which is also continuously
changing in physical and chemical conditions.
There are distinct growth phases in the growth curve of a microbial
culture. A typical growth curve includes the following phases: (1) lag phase,
(2) growth phase (accelerating, exponential and decelerating growth
phase), and (3) stationary phase and death phase as depicted in Fig. 2.2.
C e ll
A cc eleratin g g ro w th p h a se
D eceleratin g g ro w th p h ase
N u m b er
S tatio n ary p h ase
D ea th p h ase
L ag p h ase
T im e
Fig. 2.2. Growth curve of a bacterial culture.
Box 2.2
Why is it Necessary to Understand Microbial Growth Kinetics?
Microbiologists should understand microbial growth kinetics for two basic
reasons:
(1) For appropriate interpretation of growth data
Take for example the case of studying the pH profile of a newly isolated
bacterium in a batch culture system. The new bacterial culture previously
cultured at pH 7.0 was used as the inoculum for the same medium adjusted
to pH 5.5 and 7.5. The cultures were incubated at the same temperature for a
period of time. Thereafter either the final cell number was enumerated or
biomass was measured and the results are as depicted in the following figure.
pH 7.5
Cells (cells/L)
or biomass con-
centration (g/L)
pH 5.5
1 2 3 4
Time
The interpretation of the outcome would depend largely on when the final
sample was taken. If the cultures were sampled at time 1, it appears that at
pH 5.5 growth of the culture was completely inhibited. In fact, the inoculum
used was previously cultured at pH 7.0, was still in the lag phase at time 1,
when the cells were adapting to the new pH condition.
If the cultures were sampled at time 2, it is easy to conclude that
the culture could grow at both pH 5.5 and 7.5, and the bacterium grew faster
at pH 7.5.
When the cultures were sampled at time 3, it may appear that pH 5.5 is a
favorable pH condition for faster growth. The truth is that the mineral nutrients
in the medium are more soluble in this acidic solution, rendering them more
available for bacterial growth, hence a higher biomass concentration.
to minimize or control the duration of the lag phase. Base on its mechanism,
growth lag could be differentiated into apparent lag and true lag.
Immediately after inoculation, a portion of the seed culture
population may grow at the maximum rate while the rest fail to grow,
thus resulted in an apparent lag phase. For example, if half of the cells in
the inoculum are not viable, and the other half of the cells grow at the
maximum rate, it may appear that the culture is growing at half of the
maximum rate.
True lag occurs when the culture is not able to grow at its maximum
rate initially due to one of the following factors:
Box 2.3
Why is the Inoculum Size Usually 10% v/v of the Culture Volume?
Inoculum is often taken from a culture in the late exponential or early
stationary growth phase. The desired size is usually 10% of the culture volume.
An inoculum size that is too large would dilute the rate-limiting substrate
concentration in the medium, resulted in a lower specific growth rate. On the
other hand, a low inoculum size would result in a long lag phase. In a culture
of alga Chlorella, glycolate in the culture supernatant needs to be accumulated
to a critical concentration before cell growth is possible (see also Inoculum
Effect on True Lag, Sec. 2.1.1(1)).
Box 2.4
When does the Lag Phase in a Growth Curve End?
Let us plot the graph of ln X (X = biomass) versus incubation time, T, as
shown in the figure below, and extrapolate the exponential growth line to
meet the original biomass concentration. The time when the two lines intercept
is defined as the end of the lag phase.
ln x
Exponential phase
= viable cell
Cell number = non-viable cell
Fig. 2.3. Substrate accelerated cell death results from the transfer of nutritionally starved
cells to a nutritionally rich medium.
It can be seen from Table 2.1 that the doubling time of bacterial cells
measured at their optimal growth temperature varies widely. Some
organisms take slightly longer than others to double their population. In
general, the higher the optimal growth temperature, as in the thermophiles,
the shorter is the doubling time. Furthermore, small, structurally simple
bacteria such as the spherical and rod-shape bacteria double faster than
large, structurally complex helical and filamentous bacteria.
The number of doubling (n) at a time interval t, is determined by the
relation t/td. Thus, the number of cells (Nt ) in an exponentially growing
culture after incubated for some time, t, can be estimated as:
N t = N 0 2n
= N 0 2 t/t d
N t / N 0 = 2 t/t d
ln( N t / N 0 ) = (ln 2) t/t d (2.1)
Table 2.1. Doubling time of various bacteria measured at their optimal growth temperature.
During the exponential growth phase, the growth rate of the cells is
proportional to the biomass of cells. Since biomass generally can be
measured more accurately than the number of cells, the basic microbial
growth equations are very often expressed in terms of mass. A biomass
component such as protein may be used as an alternative to direct
weighing of biomass. Hence, Eq. (2.1) can be modified by assuming the
biomass concentration at time 0 (initial) and time t as X0 and Xt
respectively:
ln(X t / X 0) /t = 0.693/t d
d(ln X) /dt = 0.693/t d
d(ln X ) /dX dX /dt = 0.693/t d
1/ X dX /dt = 0.693/t d
= 0.693/t d
Box 2.5
What is a Stringent Response?
Some bacteria (e.g. Enterobacteriaceae), on experiencing a depletion of
nutrition, especially nitrogen source (amino acid starvation), can initiate a
stringent response to reduce the rates of protein and other macromolecular
synthesis, by decreasing the synthesis of ribosomal RNA. Using this
mechanism of transcription control, the cells are able to shut down a number
of energy-draining activities as a survival mechanism under poor growth
conditions.
Box 2.6
What are Ntr and Pho Systems?
The activity of Ntr system is activated in response to nitrogen starvation,
when ammonia is the growth limiting substrate. The bacteria are able to turn
on transcription of genes for glutamine synthetase. The system enables the
bacteria (e.g. Enterobacteriaceae) to assimilate very low levels of ammonia
by catalyzing the assimilation of ammonia into glutamine in an ATP-dependent
reaction. It also activates additional operons (e.g. nac operon) that are involved
in the utilization of organic nitrogen sources. This permits the cells to use
alternate sources of nitrogen.
A phosphate utilization network (Pho system) is activated on inorganic
phosphate starvation (in Enterobacteriaceae), resulting in the production of a
high concentration of alkaline phosphatase, so that phosphate can be obtained
from organic sources.
The death phase (or decline phase) is the result of the inability of
the bacteria to carry out further reproduction as condition in the medium
become less and less supportive of cell division. Eventually the number
of viable bacterial cells begins to decline at an exponential rate. Industrial
fermentation is usually interrupted at the end of the exponential growth
phase or before the death phase begins.
At the stationary phase, the maximum concentration of biomass that
can be achieved in a given medium is influenced by the following factors:
dX = kX dt
ln X = ln X0 + ( k)t. (2.2)
resulting onset of decline (death) phase. The onset of the decline phase is
dependent on the type of organism and the culture conditions.
Deviations from the norm in the growth kinetics described above were
reported. These kinetic variations provide useful insights to the nutrient
metabolism and physiological status of the cultures, which will be
discussed in the following sections.
Substrate
1
m
4
1
m m
2
Biomass
Time
Fig. 2.4. Bacterial growth curve with a rate-limiting substrate that is conserved within
the cells.
B io m ass
C ell nu m b er
Ln X
or
Ln N A syn ch ro n y in cell d iv isio n
T im e
Fig. 2.5. Synchrony in cell division in a bacterial culture. N = cell number; X = biomass
concentration.
Fig. 2.6. Diauxic growth on two substrates, glucose and lactose in a Escherichia
coli culture.
XT = X + Xs (2.3)
X s/Xs = Ks (2.4)
q = qm Xs/XT (2.5)
q = qm s/Ks + s. (2.6)
q = /Y
where the yield constant Y (see Sec. 2.1.4) is the growth yield = dX/ds
(g-cell/g-substrate).
Thus Eq. (2.6) can be re-written as:
= m s/(Ks + s) (2.7)
Box 2.7
How to Determine the Saturation Constant of a
Growth-Limiting Substrate?
The saturation constant could be determined from the plots of specific growth
rate, versus substrate concentration, s or 1/ versus 1/s,
m
0.5m
Ks
Ks
1 m
1
m
1
s
= 0.5 m
s = Ks
specific growth rate could be obtained from the plot of 1/ versus 1/s
(Box 2.7).
A = B A
A > B
B
B > A
Sc
s
Fig. 2.7. Growth curves of two bacteria, A and B, in a mixed culture competing for the
same rate-limiting substrate. = specific growth rate; s = rate-limiting substrate
concentration.
It has been observed frequently that the amount of cell mass produced is
proportional to the amount of a limiting substrate consumed.
Growth yield (YX/S, g-cells/g-substrate) is defined as the amount of
biomass produced (dX) through the consumption of unit quantity of a
substrate (ds), i.e.
YX/S = dX/ds.
Respiration
O2 + methanol CO2 + H2O
H = 14.4 Kcal/g-methanol
Biosynthesis Biomass
H = 4.7 Kcal/g-biomass
Thus, the lower the growth yield on the energy substrate, the larger
amount of the chemical energy stored in the substrate would be released as
heat through respiration process. In order to maintain the optimal culture
temperature in the fermentation system, energy supply is needed for the
circulation of cooling water through the system.
(2) Substrates
(a) Carbon source
The C-substrate is also the energy source for heterotrophic bacterial
culture. The energy content of different carbon substrates varies, which are
as shown in Table 2.4.
The salt of organic acid oxalic acid has the lowest energy content
(2.4 Kcal/g-carbon) among the C-substrates listed, and the maximum
growth yield of microbial cultures reported in the literature is also the
lowest (0.14 g-biomass/g-carbon). The maximum growth yield seems
to increase with increasing energy content of the various C-substrates,
down the list from oxalate to glycerol. However, higher energy content in
methanol and methane does not support a higher growth yield.
The data imply that the microbial cultures were energy-limited in
media containing the first four C-substrates (oxalate to glucose). Thus,
higher growth yield was obtained with C-substrate of higher energy content.
In those C-substrates, which have an energy content of higher than
11.0 Kcal/g-carbon, the microbial cultures were probably carbon-limited.
There is no difference in the growth yield with increasing energy content
of the substrate.
This brings us to the question of whether it is possible to utilize the
excess energy from an energy-rich substrate, such as alcohol and sugar
alcohol, in the metabolism of the carbon in a carbon-rich substrate, such
as organic acid and sugar. Such co-metabolism of the two substrates
would achieve a growth yield that is higher than any one of the substrate
alone. Table 2.5 presents the growth yield of single substrates and substrate
mixtures.
In the case of Candida utilis, the energy from one part of ethanol
was sufficient to bring up the growth yield from 13.8 parts of sucrose
to 0.68 g-biomass/g-sucrose, which represents a 36% increase from
0.50 g-biomass/g-sucrose. When formate and mannitol were mixed in a
molar ratio of 4 to 1, the growth yield from the mixture was 10 times that
of formate and 1.3 times that of mannitol.
The enhancement of growth yield in mixed substrate fermentation can
only be observed in microbial cultures where the substrates are metabolized
(d) Environment
Oscillation or fluctuation in environmental conditions, such as pH,
dissolved O2 and supply of carbon substrate, may cause uncoupling of
substrate oxidation from biosynthesis. The growth yield of Pseudomonas
methylotropha on methanol was found to decrease with increasing interval
of addition of methanol into the culture, and corresponding increase in
the yield of carbon dioxide.
The phenomenon of uncoupling of substrate oxidation from bio-
synthesis has been exploited in the design of bioreactor for wastewater
treatment. In ICIs deep shaft process and Union Carbides Unox process,
oscillation in the temperature, oxygen concentration and substrate level are
allowed in large volume (tower-type) bioreactors, giving rise to a high rate
of substrate oxidation and corresponding low biomass growth yield. Thus,
biodegradable carbon in wastewater is transformed into gaseous CO2, and
relatively little solid mass is left.
Box 2.8
Uncoupling
The separation (uncoupling) of the two processes: formation of the
protonmotive force and its utilization to generate ATP, leading to dissipation
of energy without ATP formation.
X/YE = X/YG + mX
qE X = X/YG + m X
qE = /YG + m (2.9)
1
YE
m
1
YG
Fig. 2.8. A plot of 1/YE versus 1/ according to the equation 1/YE = 1/YG + m/, where
YE is the overall growth yield, YG is the true growth yield; m, the maintenance coefficient;
the specific growth rate.
qE
YG
mYG
Fig. 2.9. Plot of qE versus according to the equation qE = /YG + m, where qE is the
specific rate of energy uptake; the specific growth rate; YG, the true growth yield and
m, the maintenance coefficient.
There are several factors that could affect the maintenance energy
requirement of a culture.
Environment
Environmental parameters, which fall outside the optimal range for growth,
may have an effect on the maintenance energy requirement of microbial
cultures. The energy presumably is consumed for maintaining a favorable
intracellular environment for optimal metabolic activities and growth.
It appears that the ionic strength of media has an effect on maintenance
energy requirement. For example, in the culture of Saccharomyces
cerevisiae cited in Table 2.7, the incorporation of 1M salt resulted in a
10-fold increase in maintenance energy for maintaining an osmotic balance
across the cell membrane. Whereas N2-fixing Azotobacter vinelandii spent
mYG
Fig. 2.10. The relationship between specific growth rate, and substrate concentration, s
in a microbial culture with measurable maintenance energy requirement.
Table 2.7. Maintenance energy requirement of some protists (eukaryotes other than plants,
animals or fungi) with glucose as the energy source.
Metabolite leakage
Microbial cells are known to release polysaccharides, glycolic acid,
amines, amides and vitamins into the culture environment. These
extracellular metabolites may play a role in their survival and reproduction,
however, as they do not contribute directly to cell growth they are
considered maintenance requirements.
dp = Yp/x dX
where
i.e.
qp = Yp/x . (2.11)
qp = Yp/x + (2.12)
The first term on the right hand side of the equation represents the
growth-linked component, whereas is a constant specific rate of product
formation independent of the specific growth rate. Formation of end
products of energy metabolism, e.g. lactic acid, follows this relation, where
includes lactic acid formation, which results from either the maintenance
energy requirement or uncoupling of ATP.
C H N O P S
Biomass
Bacteria 53.0 7.3 12.0 19.0 CH1.67N0.20O0.27
Yeast 44.7 6.2 8.5 31.2 1.1 0.6 CH1.64N0.16O0.52P0.01S0.005
Fungus 43.0 6.9 8.0 35.0 CH1.93N0.16O0.61
Antibiotics
Erythromycin C37H67NO13
Penicillin G C16H18N2O4S
Organic acids
Citric acid C6H8O7
Lactic acid C3H6O3
Alcohol
Butanol C4H10O
Ethanol C2H6O3
% w/w
Maltose 52
Hexoses (glucose, fructose) 19
Sucrose 2
Dextrin 15
Other carbohydrates 4
Total nitrogen 5
Ash 1.5
Water 2
Total nitrogen 13 9
Amino nitrogen 7 2
Carbohydrate 29
NaCl 3 6
Ash 6 14
Water 5 4
Alanine 16.8 3.3
Arginine 30.2 1.1
Aspartic acid 8.7 2.4
Glutamic acid 38.6 5.6
Glycine 4.4 2.3
Histidine 13.3 1.1
Isoleucine 29.9 2.8
Leucine 71.9 9.4
Lysine 61.1 7.9
Methionine 22.7 3.3
Phenylanine 33.0 3.7
Proline 7.4 0.8
Serine 28.7 4.8
Threonine 21.5 1.9
Tryptophan 8.6 1.6
Tyrosine 14.0 0.7
Valine 36.4 3.8
% w/w
Protein 50
Carbohydrate 30
(Sucrose, stachyose, raffinose, arabinoglucan,
arabinan, acidic polysaccharides)
Fat 1
Lecithin 1.8
Inoculum
Culture medium
V dX = V X dt FX dt
dX/dt = ( D) X
F1 F2
pump pump
Medium Harvest
If there is a steady state whereby the rate of growth equal the rate of
cell removal, the biomass concentration remains constant, i.e. dX/dt = 0.
Then, ( D) = 0 or = D.
Steady state in chemostat was experimentally established in bacterial,
fungal and algal cultures. The theory indicates that it is possible to fix the
of a culture at any value from zero to the maximum by adjusting the
value of D.
A chemostat culture system can be used for the following purposes:
(i) To vary the specific growth rate (or metabolic rate) of a culture
without changing the environmental condition, other than the concen-
tration of growth-limiting substrate. This application is particularly
useful in the understanding of the relationship between growth rate and
product formation or a physiological function of the microbial culture.
(ii) To fix the specific growth rate while varying a culture condition,
i.e. converse of (i). The application is useful in the study of the effect
of the culture environment without interfering the growth rate.
(iii) To maintain substrate limited growth with a constant specific growth
rate. This could not be achieved in a batch culture, as the growth
rate is determined by the concentration of the substrate in the culture
medium.
(iv) To obtain time-independent balanced growth (in steady state), with
phenotype fully adjusted to environment, i.e. the culture is history
independent, unlike cells in batch culture which performance is often
where
D = Dc sr/(sr + Ks)
The critical dilution rate (Dc), which equals the maximum specific
growth rate, is the highest possible dilution rate that a steady state could be
established. Above which, complete washout (see (2)(i) below) of cells is
observed (Fig. 2.13).
The primary effect of varying the dilution rate (D) is to change the
residual concentration of the growth-limiting substrate, thereby affecting a
change in , i.e. of a chemostat culture at steady state is determined by
sr only, independent of sm.
The steady state biomass concentration can be defined and calculated
by the following equation:
Box 2.9
Comparison of the Output Rate of Batch and that of Chemostat Culture
The biomass output rate (R = X) can be compared quantitatively as follows:
tc = 1/m ln(Xm/X0) + ta
Rbatch = Yx/s sm/tc
= m Yx/s sm/(ln[Xm/X0] + m ta)
Rchemotat = m Yx/s sm
Rchemostat/Rbatch = ln(Xm/X0) + m ta
= ln(Xm/X0) + 0.693ta/td
X s
D
Dc, Critical dilution rate
Fig. 2.13. The steady state biomass (X) and rate-limiting substrate concentrations (s) at
various dilution rates (D) in a chemostat culture.
Harvest pump
Medium
controller
light
light sensor
Fig. 2.14. Turbidostat culture system.
(2) Turbidostat
Turbidostat is a continuous flow culture in which the biomass con-
centration in the culture vessel is maintained at constant by monitoring
the turbidity of the culture through the use of a photoelectric cell (an
optical sensing device), and regulating the feeding rate of nutrient solution.
Thus, the addition of fresh medium to the system is activated when the
turbidity of the culture exceeds the set level. The specific growth rate of
the culture is therefore allowed to adjust itself in respond to the growth-
limiting parameter. Figure 2.14 is the schematic of a turbidostat.
(i) To fix biomass concentration and allow the specific growth rate to
adjust itself. This application is particularly useful for slow growing
microorganisms and those with complex cell cycles. In the latter case,
the m of a culture may be very different at various stages in the cell
cycle, resulting in complete washout (total removal of cells from the
culture system through excessive dilution) of the culture in a fixed
dilution rate chemostat.
(ii) To maintain a constant biomass concentration under unstable
environment conditions, such as for the treatment of wastewater
containing variable concentration of toxic compounds. Temporary
suspension of specific growth rate by toxic compounds would result
in washout in a simple chemostat with fixed dilution rate.
(iii) To select for fast growing microbial strains, e.g. resistance to an
inhibitor. The dilution rate of a turbidostat is determined by the growth
rate of the culture, thus evolution of a resistant strain would cause an
increase in the dilution rate and washout of the slow growing cells
(e.g. those could not tolerate the inhibitor) from the culture system.
1st cycle 2nd cycle 3rd cycle 4th cycle 5th cycle 6th cycle
Time
Fig. 2.15. Growth kinetics in a cyclic variable volume fed batch culture. V = volume of
the culture, X = biomass concentration, = specific growth rate, S = rate-limiting substrate
concentration.
Further Readings
About Biotech
http://outcast.gene.com/ae/AB/
Access Excellence
http://www.gene.com.ae
Biotechnology Industry Organization
http://www.accessexcellence.com/AB/BC/Bioprcess Technology.html
Biotechnology Information Center
http://www.inform.umed.edu:8080/EdRes/Topic/AgrEnv/Biotech
Encyclopedia of Bioprocess Technology
http://www.cbs.umn.edu/bpti/MCF/ebt.html
Genetic Engineering News
http://www.genengnews.com
Microbiology Laboratory Simulation Programme
http://www.leeds.ac.uk/bionet/compend/bnt05pst.html
What could be the possible reasons that the yeast culture in the
medium containing molasses was found to achieve a higher biomass
growth yield?
5. Explain with examples, the importance of growth yield in fermentation
processes.
6. Two batch cultures of yeast, containing an initial glucose concentration
of 0.5 and 2 g/L respectively, were compared. It was observed that the
specific growth rate and final biomass concentration of the culture
containing 2 g/L glucose were lower than the one containing 0.5 g/L
glucose. The final concentration of alcohol was, however, comparable.
Give possible explanations.
7. Compare and contrast closed and open microbial culture methods.
8. If you are working for a dairy company, how would you convince your
company that you could achieve a higher output rate for the production
of fermented milk in a continuous flow fermentation process than the
currently used batch culture system?