Aspergillus Tamarii Growth

Chp 4 16 January 2012 v4
Page 1 of 36

CHAPTER 4
Energetics of Growth of Aspergillus tamarii in a Biological Real Time Reaction Calorimeter

4.1 Introduction
Filamentous fungi are widely used in the production of homologous and heterologous
proteins due to their desirable growth characteristics, limited space required for their cultivation
and ease of downstream processing (Srinubabu et al. 2007). Fungi which exhibit literal
glycosylation/post transitional modification of proteins are classified as GRAS (generally
regarded as safe) by the FDA (Food and Drug Administration) and can be grown on a wide
variety of inexpensive substrates (Li et al. 2000). Growth can be defined as the increases in cell
size and number that take place during the life history of an organism (Encyclopedia-Britannica,
2010). Growth and product formation from the cultured microorganisms are governed by a wide
range of parameters such as culture medium, pH, temperature, shear stress and fungal
morphology. Fungal fermentation is a complicated multi-phase, multi-component process (Wang
et al. 2003). For an effective fungal fermentation process, knowledge of the influence of
operation parameters and morphology of fungi is needed. General methodologies adopted in
studying the fungal growth are colony diameter, hypal elongation rate, biomass, ergosterol
content and heat production rate (Li et al. 2011). Each parameter has a significant role in
quantifying the different aspects of fungal growth process.
Recently, biocalorimetry has been found to have potential for real-time bioprocess
monitoring due to its non-invasive and instantaneous mode of operation (Surianarayanan et al.
2011). Calorimetry also plays an important role in quantitative engineering and in bioreactor
control. Indeed, heat signals during a bioprocess provide a global insight into metabolic activity
of living cells. In some of the reported studies (von Stockar and Marison 1989; Voisard et al.
2002; Surianarayanan and Senthilkumar 2009), the metabolic heat production has been well
correlated to microbial growth, oxygen uptake and product formation. Improvements in
sensitivity of bench scale calorimeters have made it possible to undertake real-time monitoring
of several bioprocesses (Marison et al. 1998). Heat yield values and stoichiometric yields were
also established in literature for different microbes (Sandler and Orbey 1991). As the metabolism
is a coupled effect of both anabolic and catabolic reactions, a thorough and detailed study is
required to distinguish the individual activities of microbes. Both anabolic and catabolic
Page 2 of 36

activities of a cell culture contribute to the total enthalpy change of a biological reaction. In some
cases, catabolic process appears to be dominantly exothermic as compared to anabolic process
(Surianarayanan and Senthilkumar 2008). Heat production rates depend on the nature of
catabolism. Calorimetrically analyzing the catabolic reactions can give insight into the energy
changes involved in a growth process.
Isothermal calorimetry has the advantage of being a non-destructive technique and offers
a continuous recording of the thermal power as a function of time. The features of isothermal
calorimetry method in measuring the activity of fungi are
It is a general and unspecific technique that can be used for any types of substrate
and organisms.
During a calorimetric measurement, the thermal power is continuously measured.
One can thus monitor processes in detail while they take place.
It is a non-destructive technique.
As heat flows through all materials, one can monitor processes taking places
inside opaque materials and packages.
It is often a sensitive technique.
Biomass and thermal power are two quite different measures of growth. In an external
aspect (biomass) and the other one relates to its internal activity (thermal power). However,
during the active growth period of an organism, the biomass accumulation is closely related to its
anabolic and catabolic activities since the biomass is the result of organisms anabolismthe
conversion of carbon substrate into biomass coupled to the transformation of ADP to ATP.
Anabolism is driven by catabolism-the combustion of substrate carbon with oxygen to give
carbon dioxide, which generates the energy carrier ATP from ADP. From this perspective
biomass and thermal power are related; the thermal power comes from the catabolism that drives
the anabolism that results in new biomass. The thermal power from an organism can be
measured and used as an indicator of the organisms activity level. The measurement of thermal
power has therefore been used in studying the growth and production of filamentous fungi. Thus,
thermo kinetics is one of the vital information required for consistent yield, successful scale-up
and design of bioprocesses.
Assessing the enthalpy balances for the fermentation process is important for process
calculations. Enthalpy balance for a fermentation processes can be understood from the enthalpy
Page 3 of 36

content of major substrates, products and biomass. The enthalpy of dried biomass depends upon
origin and classification, hence data reported in the literature for the same generic species may
vary 1.5 times (Ishikawa and Shoda 1983) and it may be determined experimentally by burning
samples in a bomb calorimeter. In our study four models (Corider et al. 1987) that relate the heat
of combustion of organic matter to its elemental composition are compared with experimentally
determined value for A. tamarii.
The change of mycelia morphology of filamentous microorganisms in BioRTCal will
have remarkable effect on the primary or secondary metabolites production and also on enzyme
production. In submerged fermentation, filamentous fungal morphology is classified as either
dispersed or pelleted, and the dispersed form can be further divided as freely dispersed and
clumps. Many studies have discussed the advantages and disadvantages of growth morphologies
in terms of targeting different type of products (Liao et al. 2007). It has been concluded that the
fungal growth in pellet form was a favorable alternative to benefit the most of fungal
fermentations because it not only made fungal biomass reuse possible but also significantly
improved the culture rheology, resulting in better mass and oxygen transfer into the biomass, and
lower energy consumption for aeration and agitation (Papagianni and Moo-Young, 2002;
Dobson et al. 2008). Agitation rate, initial glucose concentration and DO will have significant
effect on morphology (Wang et al. 2003). In cold mode experiments, the pellet size are
controlled by growth more than by mechanical force and the pellets are stable for several hours
(Cui et al. 1997).
Aspergillus tamarii MTCC5152, a typical filamentous fungus was selected as a model
system to study the feasibility of monitoring growth in BioRTCal based on metabolic heat flux.
Coupling of hydrodynamics and bioreaction highlighted the complex relationship between
energy dissipation, substrate uptake rate and fungal physiology. An attempt was made to relate
the morphological changes during the fungal growth process to the heat flux. The enthalpy of the
dried biomass depends on the cellular composition and can be characterized by elemental
analysis are discussed in detail.

4.2 Materials and methods
The details about the fungal strain A. tamarii, growth media composition, analysis,
chemicals and reagents employed here were the same as in Chapter 3.
Page 4 of 36

Growth of A. tamarii
The growth of A. tamarii was monitored by gravimetric method. Samples taken from the
calorimeter were centrifuged at 10000 g, for 15 min, at 4C (Sigma 3-18k, Germany). The
supernatant was decanted and cells were separated. The harvested cells were freed from soluble
salts, nutrients and waste products by washing thrice with sterile water and dried at 80C to a
constant weight.

Metabolic heat evaluation in a Biological Real Time Reaction Calorimeter (BioRTCal)
The BioRTCal is a heat-flux bench-scale calorimeter of Mettler-Toledo (Switzerland)
shown in Figure 4.1(a). The system was equipped with pH probe, turbidity probe, and DO probe
(Mettler-Toledo, Switzerland). The inbuilt DO probe was used to monitor the variations in
dissolved oxygen (DO) value. Heat generated from the BioRTCal was monitored and controlled
by iControl RC1e 4.0 software. The calorimeter shown in Figure 4.1 was a 2 liter jacketed glass
reactor that can operate in isothermal, adiabatic, or isoperibolic mode. For biological reactions,
the calorimeter was operated in isothermal mode to avoid excessive accumulation of heat inside
the reactor that might affect the growth of cell culture. In isothermal mode, the temperature of
the reactor (
r
T ) was held constant by proper control of the jacket temperature (
j
T ) by
circulating low-viscosity silicone oil at a high rate through the reactor jacket. Blending oils from
hot and cold oil circuits, through an electronically controlled metering valve, controlled the
jacket temperature. Agitation was achieved through a 4-blade pitched turbine impeller for
effective mixing. The inlet air for aeration was controlled by a Rotameter, sterilized through a
membrane filter (0.2m) and sparged through the bottom of the reactor and exited through a
second membrane filter (0.2m). Thus, a process dissipating or taking up heat resulted in a
decrease or increase in
j
T , leading to a temperature gradient across the reactor wall directly
proportional to the thermal flux liberated or absorbed by the process (
r
q ) as per
) (
j r r
T T UA q = (4.1)
where, UA was the overall heat transfer coefficient,
r
T the reactor temperature (C) and
j
T the
jacket temperature (C). The heat flow,
RTC
q
, was estimated online by the vertical and horizontal
inbuilt sensors bands shown in Figure 4.1(b). Heat flux sensors attached to the outer wall of the
Page 5 of 36

reaction vessel measured the specific heat flow through the horizontal sensor band. The fill level
was determined by the vertical sensor band. This allowed the heat flow through the reactor wall
to be calculated using the equation (4.2)
SO RTC
q A q . =
(4.2)
where
RTC
q was heat flow through the part of the reaction vessel wall that is wetted by its
contents (W), A is the effective heat exchange area, determined by the sensors of the vertical
band (m
2
), and
SO
q
is the specific heat flow through the horizontal sensor band (W/m
2
). The
principle behind, heat determinations from the calorimeter were well documented (Turker, 2004
and Senthilkumar et al. 2008).
The metabolic heat (heat flux) measured from microbial growth (
r
q ) given by Equation
(4.1) was corrected for the constant baseline heat production. Baseline heat was the sum of
external heat loss (loss due to aeration, environmental heat loss) and heat input in to the system
(stirring power, heat accumulation, and dosing heat). This baseline heat value (
b
q
) was carefully
eliminated from the heat evolved due to biological processes. The heat flow balance in
BioRTCal is depicted in Figure 4.1(c). The value of metabolic heat flux during the bioprocess (
q ) was determined by deducting the measured
r
q (from Equation 4.1) from that of the base-line
signal (
b
q
):
b r
q q q =
(4.3)
Power-time curve was obtained by plotting heat flow rate ( q ) against the corresponding
time. Heat-time curves were obtained by plotting cumulative heat values (calculated from power-
time curve by integrating for specified time intervals) against the corresponding time. Scatter and
errors in the heat evolution rate measurements were effectively smoothed by integration of the
signal. All experiments were performed with a working volume of 1.2 L and the reaction
temperature was maintained constant at 28C. Agitation rate and aeration was maintained at 350
rpm, 1vvm (volume of air per volume of medium per minute) throughout the experiment.
Cultures of A. tamarii were grown in batch mode until depletion of the carbon source. Dissolved
oxygen (DO) sensor in BioRTCal was initially calibrated by purging with pure oxygen and
nitrogen. A minimum DO value of 4 ppm was always maintained to ensure aerobic conditions
Page 6 of 36

throughout the reaction. Oxygen uptake rate (OUR) was calculated by the dynamic method
(Garcia-Ochoa et al. 2010).
The total heat liberated depended on the nature of the carbon and energy source,
particularly to the degree of reduction, the biomass yield coefficient and physical conditions such
as temperature, pH and the aerobicity of the culture, as well as the specific chemical composition
of the medium. For these reasons, the use of calorimetry for the finger-printing of strains had
to be undertaken in strictly controlled conditions.

Heat-yield coefficients
Microbial heat evolution data would be used indirectly to determine biomass
concentration (von Stockar & Marison 1989) and OUR (Garcia-Ochoa et al. 2010). The heat
yields might vary during the growth process, and also, during the production of secondary
metabolites in non-growth associated processes. The heat production rate is an important process
variable, since the heat yields are dependent on the other process yields such as biomass,
substrate and oxygen.

Experimental determination of Oxygen Uptake Rate (OUR) & volumetric mass transfer
coefficient ( a k
L
)
Dissolved oxygen (DO) sensor in BioRTCal was initially calibrated by purging with pure
oxygen and nitrogen. A minimum DO value of 4 ppm was always maintained to ensure aerobic
conditions throughout the reaction. The macroscopic mass balance for the dissolved oxygen in
the well mixed liquid phase could be written as
X O L L L
L
C q C C a k
dt
dC
. ) .(
2
*
=
(4.4)

where dt dC
L
is the accumulation of oxygen in the liquid phase, the first term on the right hand
side in Equation (4.4) is the oxygen transfer rate (OTR) and the second term is the oxygen uptake
rate (OUR). Rearranging of Equation (4.4) yielded
Page 7 of 36

a K
dt dC C q
C C
L
L X O
L L
) ( .
2 *
+
=
(4.5)

OUR was calculated by the dynamic method. It is based on the respiratory activity of
microorganisms [Garcia-Ochoa et al. 2010] actively growing in the BioRTCal. The air flow to
the broth is interrupted for a few minutes as the DO concentration decreases, values are recorded
by the iControl 4.0 software interfaced with the DO probe. The slope of the linear decrease in
L
C value with time, yields OUR. When the aeration is turned on again, the DO concentration
increases and reaches a steady-state concentration. The slope of the response curve at a given
point is measured to get dt dC
L
, and from Eq. (4.5) on plotting
L
C against [ ) ( .
2
dt dC C q
L X O
+ ],
slope becomes equal to the reciprocal of a k
L
.
The procedure was repeated several times during
the production process [Potumarthi et al. 2007]. Microbial heat evolution data would be used
indirectly to determine biomass concentration and OUR.

Energy dissipation per circulation function (EDCF) and Aeration Number in BioRTCal
Power was measured directly from electrical consumption of the motor, and a correction
was applied to compensate for power drawn due to friction in the gearbox and fittings. Power,
agitator speed and airflow rate were reported as energy dissipation/circulation function (EDCF)
and aeration number (
Ae
N ) as described here. EDCF (W/m
3
.s), was found to correlate better with
morphological and fragmentation data than power per unit liquid volume (
L g
V P / ) [Amanullah et
al., 1999; Justen et al., 1998 and Li et al. 2000]. In high-viscosity system the gas-filled cavities
behind the impeller blades were quite stable, and their size was almost independent of gassing
rate [Nienow, 1990]. From the reported information, it was evident that the turbulent regime
ungassed power numbers (
P
N ) were similar for shear thinning fluids (e.g., filamentous fungal
broth) and for water [Nienow et al., 1983]. Thus, to determine the power number in BioRTCal, a
previously worked out strategy was adopted [Senthilkumar et al. 2008],
P
N for the pitched blade
turbine was approximately 1.25 [Houcine et al, 2000]. To characterize mixing for the agitation
and aeration conditions, Reynolds number (
Re
N ) was employed. The ratio of gassed to ungassed
power (
o g
P P / ) was determined as per Doran [1995]. Thus, to arrive at EDCF for fungal
Page 8 of 36

fermentation in the BioRTCal, total gassed power was taken as proportional to
5
i
D . In addition to
power distribution, geometric constant, k (Eq. 4), and ungassed flow number,
Fl
N (Eq. 4.11),
were determined as previously reported (Smith et al., 1990). Gassed flow number
g Fl
N
,
(Eq.
4.10) and gassed circulation time,
c
t (Eq. 4.9) were also determined as previously reported
(Justen et al., 1998). Agitation and aeration rates were reported in terms of aeration number (
Ae
N
), determined according to Equation 4.12, below.
c i
g
t kD
P
EDCF
3
=
(4.6)

2 . 0
3 / 2
4
25 . 0
1 . 0

(
=
L
i
L
g
o
g
gWV
ND
NV
F
P
P
(4.7)

i
D
W
k
4
t
=
(4.8)

3
, i g Fl
L
c
ND N
V
t =
(4.9)

(
=
P
g P
Fl g Fl
N
N
N N
,
,
(4.10)
5 . 0
91 . 0
(
=
i
P Fl
D
W
N N
(4.11)
3
i
g
Ae
ND
F
N =
(4.12)

Stoichiometry of fungal growth process
If heat flux calorimetry is to play a more important role in quantitative engineering
related studies and in bioprocess monitoring, the black box model for cell growth process need
to be revealed. The quantitative relationship of the heat evolution rate with other relevant process
variables, such as biomass concentration, growth rate and so on must be elucidated. Heat flow
Page 9 of 36

has often been considered as non specific information, which may account for some prejudices in
the field of biocalorimetry. However, it has been shown that specific, quantitative information
on the above mentioned parameters may readily be deduced from heat evolution measurements
on analysing them in terms of combined enthalpic and elemental balances of microbial growth
(von Stockar and Marison, 1989). The stoichiometry of a general growth process under pure
aerobic conditions (no side product) may be described in terms of a chemical equation as
follows:
O H Y CO Y N O CH Y NH Y O Y N O CH
S W S C X X X S X S N S O S S S 2
'
/ 2
'
/ 3 2 1
'
/ 3
'
/ 2
'
/ 3 2 1
+ + + + (4.13)
This equation has been formulated in terms of C-moles, which means that each chemical formula
has been reduced to the basis of one carbon atom. In this notation, the letters appearing in the
subscript stand for substrate (S), biomass (X), O
2
(O), NH
3
(N), CO
2
(C) or H
2
O (W), whereas
the numbers designate the elements H (1), O (2), and N (3). In order to use the combined
elemental and enthalpy balances the chemical formula for all compounds listed in Equation
(4.13) as well as data on their heats of combustion is required.
Elemental composition and heat of combustion of A. tamarii
Estimation of the enthalpic content of dried microbial biomass from the literature is
difficult due to difference in isolation nature of microorganism, growth pattern and medium
composition.
Preparation of A. tamarii sample for elemental analysis
Colonies of A. tamarii were harvested in the exponential phase, in a vessel cooled by ice
water to prevent lysis. Washing was performed thrice with ice-cold water to remove cell debris
and the cells were resuspended in water, poured into petri dishes as thin layer, frozen overnight
at -20C and lyophilized. The lyophilized and dried cells obtained in powder form were sealed in
a sample bottle used for combustion and elemental analysis.
Combustion calorimetry
The enthalpy content of dried biomass can be estimated experimentally by combustion
calorimetry, but the measurements are tedious and time consuming. An isoperibol Bomb
Calorimeter (Parr, USA, Model: 6200) was used for the determination of heat of combustion.
The calorimeter controlled at constant temperature using a circulating water bath, enables the
control section of the calorimeter to determine the heat leak corrections and apply them in real
Page 10 of 36

time. This combination makes it possible to operate the calorimeter in dynamic mode for rapid
testing without a detectable difference in the precision of the test.
The bomb features an automatic inlet check valve and an adjustable needle valve for
controlled release of residual gasses following combustion. They are intended for samples
ranging from 0.6 to 1.2 g with a maximum energy release of 8000 calories. The removable
bucket has been designed to hold the bomb, stirrer and thermistor with a minimum volume of
water and to provide an effective circulating system which will bring the calorimeter to rapid
thermal equilibrium both before and after firing. The bomb is made of a high-strength, high
nickel stainless steel designed to resist the corrosive acids produced. The calorimeter can be
operated on an open system where tap water is used to cool the jacket and to fill the bucket. The
heat capacity of the calorimeter is determined by burning a specified mass of benzoic acid in
oxygen. A comparable amount of the analysis sample is burned under the same condition in the
calorimeter as per the ASTM D 5865-02 protocol. The calorific value of the analysis sample is
computed by multiplying the corrected temperature rise, adjusted for extraneous heat effects, by
the heat capacity and dividing by the mass of sample.

Elemental analysis
The general stoichiometric equation for the combustion of ill-defined biomass is
represented as
2 2 2 2 2 2 2
1
4
1
) ( N O H CO x O x x x N O H C
N H
N O H C
x x
C O H C x x x x
+ + + + (4.14)
where
O N H C
x x x x , , ,
are the stoichiometric indices of C, H, N and O respectively. The energy
content of fungal biomass ultimately depends on the cellular composition which can be
characterized by elemental analysis. It is an indirect determination of the heat of combustion and
is less time consuming. 5 mg of the lyophilized fungal sample was used for elemental analysis
(C, H and N) (Perkin-Elmer, Model PE2400, Perkin Elmer Co., Norwalk, CT, USA). The
stoichiometric indices can be computed from elemental analysis as follows
12
CT
C
f
x = (4.15)
HT H
f x = (4.16)
14
NT
N
f
x =
(4.17)
Page 11 of 36

16
OT
O
f
x =
(4.18)
where
OT NT HT CT
f f f f , , ,
represents mass fraction of the content of C, H, N and O corrected for
ash and residual water. From the analysis results the H was corrected for the contribution of
residual water as shown in Equation (4.19)
O H app H H
f f f
2
18
2
,
= (4.19)
where
O H app H H
f f f
2
, ,
,
is the true content of H, the content of H is indicated by the elemental
analyser, the content of residual water respectively. The fraction of O was computed as follows
) ( 1
2
O H ash N H C O
f f f f f f + + + + = (4.20)
where
O H ash N H C
f f f f f
2
, , , , are mass fraction of the contents of C, H, N, ash and residual water.
The number of moles of oxygen consumed during combustion of organic compound is found
from the stoichiometric Equation (4.14).
Four models were taken from the literature that relates the heat of combustion of organic
matter to its elemental composition.

Thornton Model
The molar heat of combustion of many organic compounds is directly proportional to the
number of oxygen atoms during combustion, represented by Equation (4.21)
C O
x Q H = A
(4.21)
C
O H C
x
x x x ) ( 4
2
1
4
1
+
=
(4.22)

whereQ(assigned as 110.88 kJ/mol) is the heat evolved per number of available electron
equivalents transferred to one gram atom of oxygen during combustion; is degree of
reductance, defined as the number of electrons transferred to oxygen per atom of carbon defined
by Equation (4.22). The enthalpy content of many organic compounds can be predicted
accurately using Equation (4.21) and thus could also be used to predict the heat of combustion of
various types of ill-defined organic mixtures (fungal biomass) at high degree of accuracy.

Page 12 of 36

Giese Model
Giese proposed a procedure for estimating the energy content of microbial biomass based
on reduction level (RL). The RL is one fourth of the reductance degree () as defined by
Equation (4.23)

C O
x Q H = A RL 4
(4.23)
C
O H C
x
x x x
2
5 . 0 2
RL
+
=
(4.24)

where Qassigned as 115.06 kJ/mol and the RL indicate the number of molecules of oxygen
utilized per carbon atom during the combustion.

Ho and Payne Model
The calculation procedure proposed by Ho and Payne is also based on the oxygen
consumption during combustion as obtained from the combustion stoichiometric Equation
(4.14).
CT O
f H = A 852 . 44
(4.25)

Dulong Model
The energy content of fuels was estimated based on the correlation
|
.
|
\
|
+ = A
8
05 . 144 76 . 33
OT
HT CT O
f
f f H
(4.26)

The coefficients in Equation (4.26) are derived from the enthalpy contents of carbon and
hydrogen in their elemental states.

Pellet size, apparent density and pellet porosity
The particle characteristics of the A. tamarii fungal pellets were studied by randomly
choosing 20 pellets from the sample. The sizes of the pellets were measured under a microscope
on a hemocytometer. The average diameter (D) was calculated as
=
i
d
n
D
1
(4.27)
Page 13 of 36

where n is the number of pellets and d
i
is the diameter of each pellet. The volume related specific
surface (
v
S ) was determined by assuming that all the pellets have the same spherical shape
defined as
i
v
d
S
6
=
(4.28)
The same samples were used to measure the apparent density of the pellets. The terminal
settling velocity ( u ) of the fungal pellets were determined by dropping the pellets into a long
narrow tube of water at 25C and allowed to settle freely. Using a stopwatch, the time taken for a
pellet to pass 20cm length at a uniform velocity was recorded. The apparent density of the wet-
pellets (
ap
) was calculated using Stokes Law (Bird et al. 1960).
( )

18
2
w ap
gD
u

= , for 1 Re 10
4
s s
(4.29)
or
( )
6 . 0
2
Re 27 . 0
w
w ap
gD
u

= for
3
10 Re 1 s s (4.30)
where
ap
was the apparent wet pellet density,
w
is the water density (997 kg/m

3
at 25C) and
g is acceleration due to gravity. From published sources wet hypae density would be assumed as
1100 kg/m
3
(Cui et al. 1997). Thus, from the apparent density of wet pellets calculated above, the
porosity of the pellets ) (c was estimated as
c c
p p ap
+ = ) 1 ( (4.31)
where
p
was the wet hyphae skeleton density. The calculated pellet porosity represented the
comprehensive features of a pellets structure (dense or loose) and surface conditions (smooth or
fluffy, hairs being long or short). Loose and fluffy pellets would have higher porosity values. The
data then could be related to the fermentation conditions. The culture morphology was analyzed
by 2D photographs using a microscope (Nikon Eclipse 80i, USA ).

Batch kinetics (unstructured growth model)
The metabolism of growth of fungi A. tamarii was analyzed in single substrate (glucose)
growth medium using metabolic heat obtained from BioRTCal. For designing a fermentor, it is
Page 14 of 36

necessary to consider the bioreactor performance and the microbial kinetics. Description of the
bioreactor performance involves modeling of mass transfer effects and flow pattern in gas and
liquid phases. Microbial kinetics deals with the individual cell level and the level of the entire
cell population. Single substrate driven growth (Bailey and Ollis, 1987) of the biomass is
governed by the following equations
X
dt
dX
= ,
o
X X t = = ; 0
(4.32)
S X
Y
X
dt
dS
/
=
,
o
S S t = = ; 0 (4.33)
where Xis the biomass concentration, S is the substrate concentration,
o
X
is the initial biomass
concentration,
o
S
is the initial substrate concentration, is the specific growth rate and
S / X
Y
is
the yield coefficient. Essential parameters for developing correlation were obtained from
BioRTCal experimental results.

Results and discussion
Measurement of A. tamarii growth in BioRTCal
The heat flux due to cultivation of A. tamarii in a BioRTCal for different glucose
concentrations is presented in Figure 4.2. At 2.5 g/L glucose concentration the heat release rates
are less when compared with that of 5 g/L and 7.5 g/L. The power time curve exhibits three
distinct regions of growth irrespective of the glucose concentration. The initial lag phase extends
up to 1100 min for 5 g/L and 7.5 g/L glucose concentration, while for 2.5 g/L glucose
concentration it is 1400 min. The culture A. tamarii obviously needs more time for adaptation
and relaxation before it enters the exponential growth phase. In the exponential phase, the heat
release rate peaks as a result of both cell multiplication and glucose consumption. In the case of
7.5 g/L glucose concentration experiment, the exponential phase extends for a long time before
the culture returns to decay or endogenous state. In 5 g/L glucose concentration experiments the
exponential phase ends sharply at 1700 min, the shape and trend of the power time curve are
comparable to the previously reported studies with different types of microorganisms. The
details of pellet characteristics and other growth data are summarized in Table 4.1. A close
Page 15 of 36

examination of the data indicates that 5 g/L glucose concentration is the limiting concentration
for maximum growth.
In Figures 4.3-4.5, a comparative plot of power time curve, biomass growth-cell dry
weight (CDW), glucose consumption and OUR is presented for 2.5, 5 and 7.5 g/L initial glucose
concentrations to correlate growth to heat release profiles. In Figure 4.4 glucose consumption
begins right from the addition of inoculum of A. tamarii, to the reactor. Maximum consumption
(80%) occurs within the first lag phase (0-1100 min). However it may be noticed that during this
phase, the biomass growth is not appreciable. The sudden increase of heat flow rate shows that
the fungal cells enter the log phase and this is validated by the increase in CDW in Figure 4.4.
Interestingly growth or cell multiplication (marked by biomass increase) rapidly occurs during
the later part of the exponential phase. After reaching maximum growth rate, both biomass and
OUR remained almost flat. While the heat profile is not gradually decreasing, it exhibited a
short dip at the end of exponential phase and was fluctuating around 0.25 W/L, suggesting
stationary behavior. Thus the heat signal finger printed well all phases of growth.
The power time curve is quite characteristic of A. tamarii; biomass concentration
increases exponentially until the carbon source glucose becomes limiting. The heat generation
rate, q, follows the growth curve until the end of the exponential growth phase 1560 min. Heat
signal falls rapidly during the endogenous metabolism of the cells at 1740 min. This power-time
curve has a distinct shape that can be used for the identification of specific strains.

Biomass heat yield
In order to determine the energy efficient process in a fungal metabolism (anabolic or
catabolic), to observe the substrate shifts and for validating stoichiometry, it was necessary to
estimate the yield coefficients. Results published (Senthilkumar et al. 2008) reveals that the
amount of heat evolved by a microbial culture is directly related to growth and can be
represented by the heat yield coefficient with respect to biomass,
X Q
Y
/
(kJ heat evolved per g of
CDW). Heat yield coefficient can be determined from a plot between the total heat evolved by
the culture (kJ/L) and the biomass concentration (g/L) or from the rate of heat evolution by the
culture (W/L) and the biomass production rate (g/L.hr). Figure 4.6 shows the values obtained for
an aerobic batch culture of the fungi A. tamarii grown on a glucose-limited mineral salt medium.
At low biomass concentrations, i.e. the period immediately after inoculation, the rate of heat
Page 16 of 36

evolution is very small and close to the limits of calorimetric detection, whereas at the end of the
exponential growth phase, q varies as a function of the limiting substrate concentration.
The biomass yield coefficient
G X
Y
/
(deduced from slope of CDW and residual glucose
concentration) had a pronounced effect on
X Q
Y
/
. The effects of
G X
Y
/
on
X Q
Y
/
for three different
glucose concentrations (2.5, 5 and 7.5 g/L) are shown in Figure 4.7-4.9. The first growth phase
was characterized by high substrate consumption per unit biomass formed, and thus a low value
of
G X
Y
/
(0.182 g of CDW/g of glucose). The second phase was characterized by lower substrate
consumption and thus a higher value of
G X
Y
/
(1.611 g of CDW/g of glucose). Similar behavior
was observed for 2.5 g/L and 7.5 g/L glucose levels (0.255 g/g to 1.257 g/g & 0.177 g/g to 0.594
g/g) from Figure 4.8 & 4.9 respectively. This showed that the culture utilizes maximum amount
of glucose in medium at phase I and reproduces at very slow rate. The energy available in the
reduced substrate (Glucose) was effectively used by the culture for adaptation on medium during
the growth in Phase I.
The total heat evolved was dependent on the biomass formation (Figure 4.6), this plot
also, exhibited three distinct phases. The first phase corresponded to a lower level of heat
dissipation per unit biomass and was clearly influenced by the low value of
G X
Y
/
. The second
phase corresponded to a higher level of heat dissipation (
X Q
Y
/
) resulting from a higher biomass
yield (
G X
Y
/
). The third phase in Figure 4.6 was due to the non-growth related oxidation of by-
product. Consequently,
G X
Y
/
was zero and
X Q
Y
/
tends to infinity.
The fungal metabolism appeared to behave quite differently from that of any other
microorganism (Rao et al. 2006), which needs substrate during exponential growth. The
organism A. tamarii appeared to build-up a strong cell structure in contrast to other
microorganisms such as Pseudomonas aeruginosa, Staphylococcus lentus, Bacillus badius
reported earlier. The OUR profile followed the heat profile very closely in all the three phases of
the growth as evidenced with other types of microorganisms (Karthikeyan et al. 2011).

Influence of agitation intensity and aeration on protease production in BioRTCal
Page 17 of 36

Figure 4.10 depicts the influence of agitation intensities on total metabolic heat, a k
L
and
CDW. Higher agitation intensity resulted in lower growth and lower metabolic heat. Although
a k
L
continued to increase with increase in agitation intensity, both fungal growth and metabolic
heat decreased beyond 350 rpm. This was because the fungal hypae got severely damaged with
increasing impeller shear resulting in poor biomass development.
The heat flux profile during growth of A. tamarii for different agitation intensities (250,
350 & 450rpm) are presented in Figures 4.11, 4.4 & 4.12. In Figure 4.11, at 250 rpm agitation
the metabolic heat release rate was higher than that of 350 & 450 rpm respectively. At 250 rpm
the fungal hypae remained stable and the metabolism lasted longer, i.e., at lower agitation
intensities growth related processes were dominating. At 350 rpm, A. tamarii growth, substrate
heat yield and biomass heat yield were maximum. When the agitation intensity was raised
beyond 350 rpm, a decrease in fungal growth and heat yield was observed. It was concluded that
450 rpm agitation intensity was not favorable for promoting growth. Thus, 350 rpm agitation rate
was found to be the optimal rate for A. tamarii growth in BioRTCal. A growing fungal pellet was
to be carefully nurtured in a bioreactor so that the natural morphology did not get disturbed and
at the same time oxygen mass transfer promoted by agitation had to be in its the optimum range;
for example 350 rpm in the present study.
The growth of A. tamarii in BioRTCal as a function of aeration rate is presented in Table
4.2 and time course of parameters is shown in Fig 13, Fig 4 & Fig 14. The aeration rates varied
from 0.5 to 2 vvm. During fermentation, the DO levels decreased rapidly during the first 24 hr in
BioRTCal due to high viscosity (0.091 kg/m.s) of the broth as shown in Fig 15. On initiation of
pellet formation, the viscosity of the broth reduced. Later, on complete pellet formation, the DO
level went up slowly to 26 ppm. The change in morphology from free filamentous form to pellets
helped in reducing the viscosity observed initially. Again, a comparison of the data presented in
Table 4.2 showed that 1 vvm aeration was the optimum for maximum fungal growth. Decrease
and increase of aeration supply rates affected the fungal growth levels marginally. The data in
Table 4.2 clearly indicated that variation in aeration rates was not as influential as the agitation
rates for maximum growth. The power requirement increased with increase in impeller speed.
At optimal conditions, 350 rpm and 1 vvm, the gassed power required was 0.0899 W. An
examination of Table 4.2 also revealed that for each set of variation in air flow and agitation rate,
Page 18 of 36

growth of A. tamarii was different, indicating that these parameters could act as metabolic
regulating parameters.
DO tension affects productivity, cell autolysis, the rigidness of the cell wall, and many
other features in fungal fermentation [Cui et al 1997]. Wang et al. [2003] reported that oxygen
enrichment in the gas supply resulted in a higher percent of active lengths in the hyphal
elements and glucoamylase activity. In this study, A. tamarii growth was found affected by the
DO level, as higher DO concentration resulted in higher growth. Therefore, optimum balance
between aeration (oxygen tension) and agitation intensity (shear), for maximum fungal growth,
was achieved in BioRTCal.

Morphology
In order to explore relationship between morphology and heat release pattern, the surface
structures of A. tamarii at various time intervals (Figure 4.16) was superimposed on the
metabolic heat curve. It is essential to understand physiological phenomena such as the breakage
of hypae and its relationship to mechanical stress. The spore started to germinate at about 480
min after inoculation resulting in the formation of clumps. Small pellets were found formed after
1440 min of inoculation. The dense, hairy pellets kept growing larger until the stationary phase.
Since the pellet was subjected to impeller shear stress for a longer period, after 2160 min, the
hypae gets weakened and the hairs continued to be shaved off and the pellet surface became
smoother than they were at 1800 min. Bristles were observed in the broth; their mass fraction
was small compared to total biomass. Sample pellets were collected at the end of exponential
phase for the measurement of pellet size, apparent density and porosity. Fungal pellets with
diameter of 1 to 4 mm were formed, no hollow and broken pellets were observed in our
experimental conditions. The porosity of the pellets varied from 79.2 % to 92.6 %. At low
agitation rate (250 rpm) the medium became more viscous and poor mixing was observed.
Agitation intensity and DO tension also affected the pellet porosity significantly. On increasing
the substrate concentration the spores were found to aggregate together and fluffier and loose
pellets were formed.
A close examination of Figure 4.16 suggests that under lag and exponential phase, the
growth process involves, germination, small pellet formation followed by fully matured dense
Page 19 of 36

hairy pellets. After the peak exponential growth at 1700 min, depletion of carbon source and
longer shear stress give rise to weakness of the hypae and it starts to wear off to form smooth
pellets. No quantitative comments at this stage can be made, but it is certain that the heat release
pattern does reflect the changes in morphology during the formation of A. tamarii. This will
allow better understanding of production process and help to establish process control schemes
on a true physiological basis.
In submerged fermentation, morphology of filamentous microorganisms varies between
pellet and filamentous forms and the exact morphology was found to depend on the culture
conditions and genotype of the strain (Thomas, 1992). Fungal morphology was influenced by
inoculum level, pH and agitation. Filamentous growth is common in industrial fermentation.
However, reduced extracellular protease secretion was found in pelleted growth and it is
beneficial for heterologous protein production (Xu et al. 2000). Pelleted growth could result in
reduced cell mass due to substrate and oxygen limitation in the dense core of the pellet when the
pellets exceeded critical radius (Cui et al. 1997). In the center of pellets, oxygen depletion
would cause autolysis of the cells and eventually leads to the formation of a hollow center. Pellet
morphology was a preferred one because of decrease in the viscosity of the culture fluid,
resulting in improved mixing and mass transfer properties (Teng et al. 2009).
Fungal growth in pellet form can be monitored calorimetrically and reliable information
on the growth dynamics of the organism achieved. Growth in terms of fungal biomass profile
closely follows the metabolic heat profile. Respirogram too follows the powertime curve in all
the phases of growth and a linear correlation between respirometric and calorimetric data is
visible. The heat yields estimated due to fungi biomass growth, oxygen uptake, and biomass
yield, help to understand the energetics of the organism under study. The oxycalorific coefficient
agrees well with results published suggesting that the process is aerobic. Heat of combustion
determination from the four models seems to lie quite close together, but differs significantly
from experimental values. Hence, for fungal cultures exact determination of heat of combustion
can be found experimentally in a bomb calorimeter. A quantitative relationship between
morphology and heat release pattern is discussed. Further work in necessary to establish process
control strategies based on morphology. This study reveals that both growth and non-growth
related reactions involved in this cell culture metabolism can be monitored efficiently by
calorimeter and the heat yield values used for better design and scale up of fermentor.
Page 20 of 36

Elemental and Enthalpy Balance for Analyzing Microbial Growth Processes
The composition of A. tamarii obtained from elemental analysis and the stoichiometric
index is shown in Table 4.3 & 4.4. Residual water and oxygen contents were calculated
according to the reported procedure (Dermoun, Z 1980). Ash content was determined by burning
1 g of the dried cells in a muffle furnace at 500C to a constant weight. Based on the elemental
analysis, the molecular formula for the fungal strain A. tamarii was deduced as
129 . 0 878 . 0 693 . 2
N O CH
. The molecular weight of this culture was found to be 30.547 g/gmol. Stoichiometric equation
for pure aerobic growth of A. tamarii was obtained as follows:
2 0042 . 0 0288 . 0 0881 . 0 0327 . 0 3 2 6 12 6
69 . 5 493 . 9 0399 . 0 558 . 5 CO N O H C NaNO O O H C + + +

+
+ + Na O H 0399 . 0 582 . 5
2
(4.34)
Several models are available to predict the heat of combustion based on its elemental
composition. Ho, Dulong, Thornton and Giese models (Corider et al. 1987) are found to correlate
well with experimentally predicted heat of combustion values. The values are shown in Table 4.5
and the best fit between predicted and observed was obtained using Gieses model followed by
Thornton; Ho and Payne, the worst fit was obtained for Dulong model. On comparison of the
model equations it differs only with respect to the numerical values assigned to the various
coefficients. For precise measurements the combustion of fungal cells is reliable because the
predicted values are significantly different from the experimentally observed ones. The number
of available electrons to be transferred to oxygen upon complete oxidation of whole cells was
4.94 electrons/(C mol) according to the following combustion reaction:
2 2 2 2 129 . 0 878 . 0 693 . 2
0021 . 0 0441 . 0 0327 . 0 04 . 0 N O H CO O N O CH + + +
(4.35)
Table 4.6 shows the comparison of theoretical and experimental heat yields (BioRTCal)
for growth of A. tamarii in a glucose-limited mineral salt medium. A good agreement was
observed between these values. This proves the efficiency of heat-flux calorimetry (BioRTCal)
in monitoring metabolic activity of A. tamarii.
Coupling biokinetics and bioenergetics may help to understand microbial growth
processes deeply. The discussion of growth stoichiometry is limited to a simple equation (with
one or two by-products apart from new biomass). Since metabolic pathway of organisms
comprises a large number of parallel reactions, it is impossible to provide a well defined
Page 21 of 36

stoichiometry for all reaction steps. Heat evolved from living systems is considered as overall
output of all metabolic actions i.e. all parallel reactions; it is justifiable to approximate growth
stoichiometry to a single overall reaction since most other reactions are either endothermic or
non contributors to overall heat.

Determination of rate of biomass generation and substrate consumption based on
metabolic heat
The relationship between the metabolic heat generated due to biomass growth and
substrate consumption at time (t)

is obtained as follows: let,
*
S
H A ,
*
X
H A and
*
P
H A be the
heat liberated on burning a unit mass of substrate, fungal biomass, and products respectively. By
Hesss law,
}
+ A A = A
t
0
met o
*
P o
*
X o
*
S
dt ) t ( q ) P P ( H ) X X ( H ) S S ( H
(4.36)
where
met
q
is the rate at which metabolic heat is liberated per unit volume of the reactor, and
P , X , S is concentrations of substrate, biomass, and product, respectively. In this study A. tamarii
was cultivated in pure aerobic respiration mode and hence there is no byproduct formation.
Hence Equation (4.36) becomes
}
+ A = A
t
0
met o
*
X o
*
S
dt ) t ( q ) X X ( H ) S S ( H
(4.37)
On differentiating Equation (4.37) with respect to time:
met
*
X
*
S
q
dt
dX
H
dt
dS
H + A = A (4.38)
Substituting for
dt
dS
and
dt
dX
from Equation (4.32) and (4.33) in Equation (4.38)
met
*
X
S / X
*
S
q X H
Y
X
H + A =
A
(4.39)
Solving Equation (4.39) for
met
q

Page 22 of 36

|
|
.
|
\
| A A
=
S / X
*
X S / X
*
S
met
Y
) H ( Y H
X q
(4.40)
Biomass growth rate can be represented in terms of heat rate by substituting for X from
Equation (4.40) in Equation (4.32) as follows:
( )
S / X
*
X S / X
*
S
met
X
Y
) H ( Y H
q
dt
dX
r
A A
= = (4.41)
where g kJ H
S
/ 9 . 15
*
= A and g kJ H
X
/ 95 . 18
*
= A (Corider et al. 1987). The value of biomass
yield (
S / X
Y
) was obtained experimentally as 1.611 g/g, details shown in Figure 4.8. From
Equation (4.41), all the parameters on the right side except the metabolic heat (
met
q
) can be
estimated. The rate of biomass generation was determined by substituting the experimental
values of
met
q
in Equation (4.41). In Figure 4.17 a linear correlation between the rate of biomass
generation and metabolic heat was observed, which further substantiates the importance of the
relationship developed to estimate growth rate instantaneously from the powertime profile.
Similarly a relationship for substrate (glucose) consumption was developed for the growth of A.
tamarii. From Equation (4.33), the rate of substrate consumption (
S
r
) can be represented by
dt
dX
Y
1
dt
dS
r
S / X
S
= =
(4.42)
On substituting Equation (4.41) in (4.42) and solving
( )
S / X
*
X S / X
*
S
met
S / X
S
Y
) H ( Y H
q
Y
1
r
A A
= (4.43)
A linear plot is obtained between estimated values of substrate consumption rate by Equation
(4.43) and experimental metabolic heat values (Figure 4.18). Since the substrate is consumed
during the course of the biological reaction by the metabolic activity of the cell culture, a straight
line with a negative slope is observed. Figure 4.6 shows the linear plot between experimental
values of cumulative metabolic heat (Q) and biomass concentration (cell dry weight) for growth
of A. tamarii in glucose-limited (5 g/L) growth media under optimized conditions in BioRTCal.
Page 23 of 36

Let the denominator in Equation (4.41) can be represented as D. The values of all the parameters
in the denominator D are known. By integrating both sides of the Equation (4.41):
}
=
t
0
met o
dt ) t ( q
D
1
X ) t ( X
(4.44)
Let the total metabolic heat liberated until a given time t be ) t ( Q
met
as shown below:
}
=
t
0
met met
dt ) t ( q ) t ( Q
(4.45)
On substituting Equation (4.45) in (4.44)
D
) t ( Q
X ) t ( X
met
o
= (4.46)
On rearranging Equation (4.46)
D
) t ( Q
X ) t ( X
met
o
+ = (4.47)
On substituting Equation (4.47) in (4.40)
|
|
.
|
\
| A A
|
.
|
\
|
+ =
S X
X S X S met
o met
Y
H Y H
D
t Q
X q
/
*
/
*
) ( ) (
(4.48)
Equation (4.48) can be simplified to
)) ( ( t Q DX q
met o met
+ =
(4.49)
Figure 4.12 shows a linear relationship between
met
q
and
)) ( ( t Q DX
met o
+
, with the slope being
the specific growth rate ( ). Specific growth rate for the A. tamarii cultivated in glucose-limited
(5 g/L) growth media under optimized conditions was observed to be 0.0029 1/min. The linearity
of the profile (R
2
= 0.983) shown in Figure 4.19 further proves the feasibility of applying
calorimetric results for estimation of biokinetic parameters. Dielectric spectroscopy can be used
to estimate instantaneous biomass concentration values for determination of growth rates.
However, its applicability in practical situations has some limitations, such as generation of
electronic noise degrading the quality of output signal, modifications in dielectric properties of
cell culture due to enzyme blockage (Maskow et al. 2008). Soley et al. (2005) used impedance
spectroscopy as an in situ probe during yeast fermentation studies and found erroneous signals at
higher aeration and agitation rates. Mid-IR and fluorescence spectroscopic probes were used to
monitor online metabolites and byproducts formation in a bioprocess. However, they suffer from
Page 24 of 36

problems of drifts in the measured signal and also limited application to different bioprocess
systems. Research groups are presently involved in developing suitable calibration models to
resolve the measured signal from drifts and various interferences (Schenk et al. 2007 and Schenk
et al. 2008). Taking into account these problems with using different online probes,
biocalorimetry is a promising tool for bioprocess monitoring due to its non-invasive mode of
operation. As heat generation is a product of anabolic and catabolic reactions, the measured heat
signal, could be interpreted to determine the instantaneous growth rate, substrate consumption
and product formation.

Conclusion
- Fungal growth in pellet form can be monitored calorimetrically and reliable information
on the growth dynamics of the organism achieved.
- Growth in terms of fungal biomass profile closely follows the metabolic heat profile.
- Respirogram too follows the powertime curve in all the phases of growth and a linear
correlation between respirometric and calorimetric data is visible.
- The heat yields estimated due to oxygen uptake and biomass growth, help to understand
the energetics of the organism under study.
- The oxycalorific coefficient agrees well with results published suggesting that the process
is aerobic.
- A quantitative relationship between morphology and heat release pattern is discussed.
- Ho & Payne and Dulong model will directly determine the enthalpy content. Whereas
Thornton and Giese model require knowledge of the dry biomass chemical formulae
for the evaluation of heat of combustion.
- Heat of combustion determination from the four models seems to lie quite close together,
but differs significantly from experimental values. Hence, for microbial cultures exact
determination of heat of combustion can be found experimentally in a bomb calorimeter.
- Using Hesss law the relationship between the online metabolic heat variable and the
offline bioprocess variables, i.e. biomass generation, substrate consumption were
explored and validated.
Page 25 of 36

- A linear relationship was observed between metabolic heat and other offline variables.
This finding proved the feasibility of deducing the values of biokinetic variables
indirectly from calorimetric results.
- This study reveals that both growth and non-growth related reactions involved in this cell
culture metabolism can be monitored efficiently by calorimeter and the heat yield values
used for better design and scale up of fermentor.

Table 4.1 Comparison of heat yield coefficients and heat flux of A. tamarii grown at varying
glucose concentrations
Initial Glucose
Concentration, g/L
Pellet
diameter, mm
Apparent wet pellet
density, kg/m
3

Pellet
porosity, %
v
S
,
1/mm
2.5 1 1018.4 79.2 6
5 2 1012.5 84.9 3
7.5 4 1004.6 92.6 1.5
Table 4.2 Effect of aeration and agitation intensity on growth of A. tamarii in BioRTCal
Aeration,
vvm
0.5 1 2
Agitation,
rpm
250 350 450 250 350 450 250 350 450
CDW,
g/L
1.761 1.618 1.324 1.955 1.74 1.427 2.181 2.006 1.792
a k
L
, 1/hr
27.774 35.062 45.825 38.031 47.130 58.207 51.954 63.109 72.009
EDCF,
W/m
3
.s
309.1 401.0 580.6 380.6 476.9 620.9 466.6 563.1 632.0
o
P
, W
0.062 0.082 0.113 0.080 0.103 0.132 0.103 0.129 0.150
g
P
, W
0.063 0.085 0.119 0.069 0.090 0.117 0.074 0.095 0.112
c
t
, s
3.880 4.002 3.863 3.417 3.565 3.565 3.013 3.184 3.345
Ae
N

0.012 0.009 0.007 0.025 0.018 0.014 0.049 0.035 0.027

Table 4.3 Elemental analysis (in wt %) of A. tamarii
Substrate Residual water Ash
Elemental analysis
C
f

H
f
N
f

O
f

Glucose 1.6 0.05 0.25 0.01 38.55 0.15 8.65 0.09 5.8 0.06 45.14 0.21

Table 4.4 Stoichiometric index calculated from elemental analysis of A. tamarii

Substrate
Stoichiometric index (x 100)

'
x
M
C
x

H
x
N
x

O
x

Glucose 3.27 0.01 8.81 0.08 0.42 0.01 2.88 0.06 4.94 0.05 30.55 0.12
Page 26 of 36

Table 4.5 Experimentally determined and model predicted heat of combustion of A. tamarii
Substrate
Heat of combustion of cells,
O
H A
, kJ/g
Ho Dulong Giese Thornton Experimental
Glucose 17.62 0.1 17.67 0.12 18.59 0.14 17.9 0.09 18.95 0.06

Table 4.6 Comparison of predicted and experimental yield coefficients of aerobic strain
A. tamarii cultivated in glucose limited growth media
Initial Glucose
Concentration,
g/L
G X
Y
/
,
g of DCW/
g of glucose
'
/ O Q
Y ,
kJ/mol
X Q
Y
/
,
kJ/g
Theoretical Experimental Theoretical Experimental
2.5 1.257
460
479
26
24.97
5 1.611 486 29.84
10 0.594 494 26.95

4.1 (a)

Page 27 of 36

4.1 (b) 4.1 (c)

Figure 4.1 (a) Principle setup of BioRTCal (b) Measurement principle (c) Heat flow
balance

0 500 1000 1500 2000 2500 3000
0.0
0.1
0.2
0.3
0.4
0.5
III
III
III
II
II
I
7.5 g/L
2.5 g/L
q
,

W
/
L
Time, min
5 g/L
I

Figure 4.2 Comparative BioRTCal heat flux profiles for the growth of A. tamarii at various
glucose concentrations

Page 28 of 36

0 500 1000 1500 2000
0.0
0.1
0.2
C
D
W
,

g
/
L
Time, min
Q
,

k
J
/
L
q
,

W
/
L
0
2
4
6
8

0 10 20 30 40
0.00
0.25
0.50
0.75
1.00
1.25
0.0
0.5
1.0
1.5
2.0
2.5
G
l
u
c
o
s
e

c
o
n
c
e
n
t
r
a
t
i
o
n
,

g
/
L
0.0000
0.0001
0.0002
0.0003
0.0004
O
U
R
,

m
g
/
L
.
m
i
n

Figure 4.3 Growth of A. tamarii in a glucose-limited medium (2.5 g/L): () cell dry weight
CDW, () oxygen uptake rate OUR, (+) residual glucose concentration,(--) total
heat evolved Q, () heat evolution rate q.

0 500 1000 1500 2000 2500
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
C
D
W
,

g
/
L
Time, min
Q
,

k
J
/
L
q
,

W
/
L
-5
0
5
10
15
20
25

0 10 20 30 40
0.0
0.5
1.0
1.5
2.0
0
1
2
3
4
5
G
l
u
c
o
s
e

c
o
n
c
e
n
t
r
a
t
i
o
n
,

g
/
L
0.000
0.005
0.010
0.015
0.020
0.025
O
U
R
,

m
g
/
L
.
m
i
n

Figure 4.4 Growth of A. tamarii in a glucose-limited medium (5 g/L): () cell dry weight

Page 29 of 36

0 500 1000 1500 2000 2500
0.0
0.1
0.2
0.3
0.4
0.5
C
D
W
,

g
/
L
Time, min
Q
,

k
J
/
L
q
,

W
/
L
0
5
10
15
20
25
30
35

0 10 20 30 40
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
0
1
2
3
4
5
6
7
8
G
l
u
c
o
s
e

c
o
n
c
e
n
t
r
a
t
i
o
n
,

g
/
L
0.0000
0.0005
0.0010
0.0015
O
U
R
,

m
g
/
L
.
m
i
n

Figure 4.5 Growth of A. tamarii in a glucose-limited medium (7.5 g/L): () cell dry weight

Figure 4.6 Biomass heat yield calculation of A. tamarii in BioRTCal for 5 g/L of initial
glucose concentration.

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20
0
2
4
6
8
10
12
14
Y
Q/X
=10.42kJ/g
Q
,

k
J
/
L
X-X
O
, g/L
Y
Q/X
=29.84kJ/g
I
II
III
Page 30 of 36

0.0 0.5 1.0 1.5 2.0 2.5
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Y
X/G
= 1.257 g/g
C
D
W
,

g
/
L
Glucose, g/L
Y
X/G
= 0.255 g/g
I
II

Figure 4.7 Biomass yield due to glucose uptake for the growth of A. tamarii in BioRTCal for
2.5 g/L of initial glucose concentration.

5 g/L of initial glucose concentration.

Page 31 of 36

7.5 g/L of initial glucose concentration.
250 300 350 400 450
35
40
45
50
55
60
k
L
a
,

1
/
m
i
n
Stirrer speed, rpm
C
D
W
,

g
/
L
1.25
1.50
1.75
2.00
8
10
12
14
M
e
t
a
b
o
l
i
c

h
e
a
t
,

k
J
/
L

Figure 4.10 Influence of Impeller speed on total metabolic heat (), a k
L
() & CDW ()
Page 32 of 36

0 500 1000 1500 2000 2500
0.0
0.2
0.4
0.6
0.8
1.0
C
D
W
,

g
/
L

q
,

W
/
L

Time, min
0 10 20 30 40
0.0
0.5
1.0
1.5
2.0
0 10 20 30 40
0
2
4
6
G
l
u
c
o
s
e

c
o
n
c
e
n
t
r
a
t
i
o
n
,

g
/
L
0 10 20 30 40
0.000
0.002
0.004
0.006
0.008
0.010
O
U
R
,

m
g
/
L
.
m
i
n

Figure 4.11 BioRTCal batch responses for proteolytic activity of A. tamarii at 250 rpm
agitation rate: () CDW, () residual glucose concentration, () OUR, () heat evolution
rate q.

0 500 1000 1500 2000 2500
0.0
0.1
0.2
0.3
O
U
R
,

m
g
/
L
.
m
i
n
q
,

W
/
L
Time, min
G
l
u
c
o
s
e

c
o
n
c
e
n
t
r
a
t
i
o
n
,

g
/
L
0 10 20 30 40
0.0
0.5
1.0
1.5
0 10 20 30 40
0
1
2
3
4
5
6
C
D
W
,

g
/
L
0 10 20 30 40
0.00000
0.00025
0.00050
0.00075
0.00100

Figure 4.12 BioRTCal batch responses for proteolytic activity of A. tamarii at 450 rpm
agitation rate: () CDW, () residual glucose concentration, () OUR, () heat evolution
rate q.
Page 33 of 36

0 500 1000 1500 2000 2500
0.00
0.25
0.50
0.75
1.00
O
U
R
,

m
g
/
L
-
m
i
n
q
,

W
/
L
Time, min
G
l
u
c
o
s
e

c
o
n
c
e
n
t
r
a
t
i
o
n
,

g
/
L
0 10 20 30 40
0.0
0.5
1.0
1.5
0 10 20 30 40
0
1
2
3
4
5
6
C
D
W
,

g
/
L
0 10 20 30 40
-0.001
0.000
0.001
0.002
0.003
0.004
0.005

Figure 4.13 BioRTCal batch responses for proteolytic activity of A. tamarii at 0.5 vvm
aeration rate: () CDW, () residual glucose concentration, () OUR, () heat evolution
rate q.
0 500 1000 1500 2000 2500
0.0
0.5
1.0
1.5
2.0
q
,

W
/
L
Time, min
G
l
u
c
o
s
e

c
o
n
c
e
n
t
r
a
t
i
o
n
,

g
/
L
0 10 20 30 40
0.0
0.5
1.0
1.5
2.0
2.5
0 10 20 30 40
0
1
2
3
4
5
6
C
D
W
,

g
/
L
0 10 20 30 40
0.0000
0.0025
0.0050
0.0075
0.0100
O
U
R
,

m
g
/
L
-
m
i
n

Figure 4.14 BioRTCal batch responses for proteolytic activity of A. tamarii at 2 vvm
aeration rate: () CDW, () residual glucose concentration, () OUR, () heat evolution
rate q.

Page 34 of 36

0 10 20 30 40
430
440
450
460
470
480
V
i
s
c
o
s
i
t
y
,

k
g
/
m
.
s
P
g

&

P
o
,

W
a
t
t
E
D
C
F
,

W
/
m
3
.
s
Time, hr
0.080
0.085
0.090
0.095
0.100
0.105
0.082
0.084
0.086
0.088
0.090
0.092
0.094
0.096

Figure 4.15 EDCF as a function of time for the growth of A. tamarii in BioRTCal at
impeller speed of 350rpm and 1vvm aeration () viscosity, () EDCF, () Gassed Power,
() Ungassed Power

-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0 500 1000 1500 2000 2500 3000
q
,

W
/
L

Time, min
a
b
c
d
Page 35 of 36

Figure 4.16 Growth phases of A. tamarii in BioRTCal. a. small pellet with short hypae b.
pellet with long hypae c. pellets with hypae becoming shorter d. smoother pellet with short
hairs

0.000000 0.000005 0.000010 0.000015 0.000020 0.000025 0.000030
0.00
0.05
0.10
0.15
0.20
0.25
0.30
q
,

W
/
L
r
X
, g/L.s

Figure 4.17 Linear correlation between metabolic heat and rate of biomass growth of A.
tamarii in BioRTCal

-0.000016 -0.000012 -0.000008 -0.000004 0.000000
0.00
0.05
0.10
0.15
0.20
0.25
q
,

W
/
L
r
s
, g/L.s

Figure 4.18 Linear correlation between metabolic heat and rate of substrate consumption
of A. tamarii in BioRTCal

Page 36 of 36

0 1000 2000 3000 4000 5000 6000
0.00
0.05
0.10
0.15
0.20
0.25
0.30
q
,

W
/
L
DX
o
+Q
met
= 0.0029 1/min

Figure 4.19 Determination of specific growth rate from instantaneous heat release of A.
tamarii in BioRTCal.

Aspergillus Tamarii Growth

Enviado por

Dados do documento

Descrição original:

Título original

Direitos autorais

Formatos disponíveis

Compartilhar este documento

Compartilhar ou incorporar documento

Opções de compartilhamento

Você considera este documento útil?

Este conteúdo é inapropriado?

Direitos autorais:

Formatos disponíveis

Aspergillus Tamarii Growth

Enviado por

Direitos autorais:

Formatos disponíveis

Chp 4 16 January 2012 v4

is the water density (997 kg/m

Você também pode gostar