Photo-biocalorimetry and micro-algae growth

Abstract

An overview is given of the first results on the growth of micro-algae Chlorella vulgaris in the dark and under light illumination. The difference in the heat exchanged during both growths can be used to deduce the energy involved during the photosynthetic process. The analysis of photosynthetic efficiency is complemented by intermittent measuring of the fluorescence of the biomas exposed to a short light pulse.

Full text

ARTICLE
A Study of the Growth for the Microalga Chlorella
vulgaris by Photo-Bio-Calorimetry and Other
On-line and Off-Line Techniques
Rodrigo Patin˜o,
1
Marcel Janssen,
2
Urs von Stockar
3
1
Departamento de Fı
´sica Aplicada, CINVESTAV–Unidad Me
´rida, Apartado Postal 73,
Cordemex, 97310 Me
´rida, Yucata
´n, Mexico; telephone: þ52(999)1242138;
fax: þ52(999)9812917; e-mail: rtarkus@mda.cinvestav.mx
2
Department of Agrotechnology and Food Sciences, Food and Bioprocess Engineering
Group, Wageningen University, Wageningen, The Netherlands
3
Laboratoire de Ge
´nie Chimique et Biologique, E
´cole Polytechnique Fe
´de
´rale de Lausanne,
Lausanne, Switzerland
Received 10 April 2006; accepted 14 August 2006
Published online 1 September 2006 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.21182
ABSTRACT: Calorimetry and other on-line techniques are
used for the first time as complement to the traditional off-
line methods in order to follow the growth of the green
Chlorella vulgaris microalgae. A 2-L photo-bio-reactor was
adapted from a commercial calorimeter used previously to
study heterotrophic microbial growth. An external source of
light was added to favor the photosynthesis of the auto-
trophic cells. Heterotrophic growth was also tested with
external glucose in the broth. A third mode, mixotrophic,
allowed faster autotrophic plus heterotrophic growth.
Calorimetric measurements were performed considering
the corresponding calibrations in order to consider only
the energy involved during the microalgal growth. The three
different modes of Chlorella cultures were energetically
characterized. Besides calorimetry, the weight of diluted
nitric acid added to maintain the pH of the culture was
correlated with the cellular growth and the nitrogen com-
position of the algae. Additionally, the on-line infrared
spectroscopy proved to be an efficient technique to follow
the composition of the broth in glucose, nitrates, and
phosphates. These results were compared and complemen-
ted with some classic off-line techniques used to track this
kind of cultures.
Biotechnol. Bioeng. 2007;96: 757–767.
ß2006 Wiley Periodicals, Inc.
KEYWORDS: Chlorella vulgaris; calorimetry; on-line mon-
itoring; photo-bio-reactor; mixotrophic mode; microalgae
Introduction
Photosynthesis is the most important process in nature.
Radiant energy from the Sun is used through photosynthesis
by plants, algae, and some other microorganisms to
synthesize carbohydrates (Berg et al., 2002). Carbohydrates
are known as the chemical source of energy for biologic
systems. They are used, directly or indirectly, all over the
world by living organisms for anabolic reactions. In an
extrapolation, solar energy may be seen as the original
source of fossil combustible coming from the degradation of
biosynthetic products (Dukes, 2003).
The natural process starts when photons are captured by
chlorophyll. This green substance is commonly found as
chlorophyll aor chlorophyll bin chloroplasts — the special
membrane organelles in autotrophic cells. This photonic
energy excites specific chlorophyll molecules. A fraction of
the decay energy is utilized to induce a cascade of reactions
in which molecular oxygen is obtained from water with the
formation of two important biochemical compounds used
to carry energy during metabolism: NADPH (reduced
nicotinamide adenine dinucleotide phosphate) and ATP
(adenosine triphosphate) (Govindjee and Krogmann, 2004).
This first process of photosynthesis is named the light phase
since the principal former is precisely light.
The energetic molecules produced during the light phase
are then used in a second cascade of reactions known as
the dark phase. In this part, the global process involves the
production of carbohydrate molecules from gaseous CO
2
.
This second process is performed out of the photosynthetic
Correspondence to: R. Patin
˜o
Contract grant sponsor: FNS-Switzerland
ß2006 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 96, No. 4, March 1, 2007 757

membrane and does not depend directly on light. Indeed, it
may happen simultaneously with the light phase.
The elucidation of the mechanisms in photosynthesis has
been extensively studied during the last century. Despite its
importance, only some few efforts have been reported in
relation to the energy exchanges during the process.
Calorimetry has been used in few cases to study this
complex process. Microalgae were thus used to obtain the
quantum yield of photosynthesis (Magee et al., 1939): only
0.08 mol of carbon dioxide been transformed by 1 mol of the
incident photons for a variety of light intensities and
concentrations of microalgae Chlorella vulgaris. Spinach
leaves were also studied by calorimetric experiments
(Johansson and Wadso
¨, 1997) with a maximum value near
13% for the energy uptake during photosynthesis and a
Gibbs energy DG8¼478 17 kJ mol
1
for the formation of
glucose.
Searching a better comprehension of the energy ex-
changes during the photosynthetic process, a photo-bio-
calorimeter is proposed here for the study of phototrophic
microorganisms. During previous years, the calorimetric
study of microbial growth has been an axis in our research.
A 2-L reaction calorimeter has been adapted for this purpose,
and very interesting results have been found to describe
differences in the metabolism of a number of microorgan-
isms (von Stockar and Liu, 1999). Some of the highlights of
this bio-calorimeter involve the improvement of the thermal
measurements, the use of a torque-meter to consider the
heat from stirring, and a diminishing in the interchange of
heat with the room (Garcı
´a-Payo et al., 2002). At the same
time, the search for useful tools to monitor complementary
key parameters during microbial metabolism has been a
constant in this laboratory (von Stockar et al., 2003).
Being the study of photosynthetic microorganisms a lack
in our experience for calorimetry and other monitoring
techniques, we propose here a photo-bio-calorimeter and
some additional techniques that may help to elucidate the
complex behavior of the microalgae Chlorella vulgaris. This
variety of algae has been widely studied and numerous
reports can be found in literature. Chlorella is often cited as a
reference photosynthetic microbial organism. Moreover, it
has been proposed for the treatment of wastewaters
(Gonza
´lez et al., 1997) as well as for feeding (Gouveia
et al., 2002).
In order to understand the photosynthesis of this species
during its linear phase of growth, a recent study has been
performed in our laboratory (Janssen et al., 2005). Never-
theless, the strain is very interesting since it can grow not
only in autotrophic conditions, but also in heterotrophic
(glucose or acetate as carbon source) and in mixotrophic
(auto- plus heterotrophic) conditions (Ogawa and Aiba,
1981). In this study, some energetic differences were found
between the three growth regimes.
Materials and Methods
Description of the Photo-Bio-Calorimeter
A commercial reaction calorimeter (Mettler-Toledo, RC1) is
the base of the one presented here. During previous years, a
list of modifications has been proposed to improve both the
microbial growth and the sensibility of the calorimetric
measurement (Garcı
´a-Payo et al., 2002; Marison et al.,
1998). In Figure 1, it is shown the picture of the vessel with
some of the original and adapted parts. From the original
calorimeter are the glass reactor, an oil temperature
controller, an electric heater, the motor and stirrer, internal
baffles and a pH probe. The adapted parts of the (photo)bio-
calorimeter include oil and reactor thermistors to improve
the calorimetric signal, a torque-meter to obtain the stirring
Figure 1. Two pictures of the photo-bio-reactor/calorimeter: (a) is a picture with
the medium and (b) is a picture with the Chlorella culture growing. In the center, it is
possible to observe the light from the Xe lamp. [Color figure can be seen in the online
version of this article, available at www.interscience.wiley.com.]
758 Biotechnology and Bioengineering, Vol. 96, No. 4, March 1, 2007
DOI 10.1002/bit

energy, a system for gas input and output, inoculation and
sampling ports, a light diode and space for any additional
on-line probe. The reactor is a 2-L glass vessel surrounded
with a glass jacket (Mettler-Toledo, AP01). Silicon oil in the
jacket circulates at a controlled temperature T
j
measured
with a thermistor.
A Plexiglas insulating housing around the reactor vessel is
used to diminish the influence of the ambient temperature.
Inside this box, a copper heat exchanger with water is
maintained at 298.15 K with an external thermostatic bath.
The housing is covered with aluminum foil to avoid the
interference coming from external light and to increase the
reflection of light inside the housing. Inside the reactor,
where around 1.7 L of liquid can be added, the temperature
T
r
is measured with a glass-covered thermistor and
controlled through heat exchange with the oil jacket. Both
thermistors—in the reactor and the oil bath — have a
sensibility of 0.001 K each. The difference of temperature
between the reactor and the jacket, DT¼T
r
T
j
, can be
directly related to the heat interchange through the reactor
wall.
A constant agitation guarantees homogeneity in compo-
sition and temperature of the liquid inside the reactor, this
liquid is stirred with a stainless-steel double propel. A
mechanical motor out of the reactor is used for rotation. In
order to measure the energetic contribution due to the
stirring inside the reactor, a torque-meter has been
connected between this propel and the motor. A system
of metallic baffles is used to favor the microbial growth by
improving the aeration of the medium. The thermostat and
circulation of the jacket oil, as well as the thermostat and
stirring of the liquid inside the reactor, are done by the
original Mettler-Toledo system through the corresponding
software manipulated from a personal computer.
The vessel is covered on the top with a metallic tap inside
which water circulates at temperature T¼300.15 K. The tap
is attached to the vessel by using five bolts with nuts. An o-
ring and some lubricant grease are in between. Through the
tap, a variety of connections can be done: the thermistor for
measuring and controlling the temperature inside the
reactor, a heater for thermal calibration of the calorimeter,
the liquid and gas feeding in the medium, a drain for gas
outgoing, a system for collection of samples, a light diode
and pH, pO
2
, dielectric, infrared or any other probe.
Unfortunately, due to the limited capacity for eight
connections, not all of them are possible at the same time.
Combining them in different ways performed the alternate
experiments presented here. The heater for calibration
provides a constant power of 1.97 W into the medium
during a defined period of time. In this way, the electrical
work applied is related to the exchange of heat trough the
wall coming from the value of DTto keep T
r
constant.
The source of light was a 150-W xenon lamp into a special
housing (Oriel, 60100), with a cooling system and an easy
orientation using an F/4.4 ellipsoidal AlMgF
2
reflector. The
system is controlled by an arc lamp power supply (Oriel,
68806). The well-known radiation spectrum for a xenon
lamp includes infrared, visible, and ultraviolet emissions.
Some infrared and ultraviolet radiations are filtered with a
0.2 M NaCrO
2
aqueous solution and a dichroic filter. The
rest of the light is condensed on the extreme of a liquid
visible-light guide. The other extreme of the guide is
connected to a quartz rod inside the calorimeter. A previous
hydrofluoric acid treatment of the rod allows a rough
surface to improve dispersion of the light going out into the
reactor.
A fluorescent flat lamp (Osram, Planon 10,400 /868 6) was
also used for one of the experiments to be compared with the
Xenon lamp illumination. During this experiment, the lamp
is fixed directly from outside on one of the transparent walls
of the reactor housing to avoid extreme heating of the
system.
Microbiological Strain and Culture Medium
The Chlorella vulgaris (211/11B) micro-alga was obtained
from the Culture Collection of Algae and Protozoa, UK. A
defined inorganic culture medium to maintain the strain in
stock and to perform the calorimetric experiments is
suggested here based on the one proposed by Schlo
¨sser
(1994) for unicellular green algae and by Mandalam and
Palsson (1998) for Chlorella cultures. Some flask tests were
performed to get a medium without limitation of the basic
biomass elements (nitrogen, phosphorus, magnesium)
obtaining a growth at relatively high cell concentrations.
The composition of the inorganic medium was: KNO
3
30
mM, NaH
2
PO
4
9 mM, Na
2
HPO
4
1 mM, MgSO
4
1 mM,
CaCl
2
0.1 mM, FeNaEDTA 0.02 mM, H
3
BO
3
1mM, MnSO
4
1mM, ZnSO
4
1mM, 3(NH
4
)
2
O7MoO
3
0.05 mM, and
CuSO
4
0.01 mM.
Micro-algal cultures were kept under sterile conditions in
Erlenmeyer flasks at room temperature, on a plate to stir at
100 rpm and with a cotton top for gas exchange with
ambiance air. Fluorescent lamps were used in 12 h on/off
cycles to maintain photosynthesis active. Because hetero-
trophic growth is also possible for this strain, sometimes
glucose was added as carbon source. A concentration
between 5 and 20 g of glucose per liter was used in some
experiments for mixotrophic or heterotrophic experiments.
Analysis
Biomass Concentration
Two complementary methods were used to obtain off-line
biomass concentration: optical density measurements and
dry weight determination. For optical density, a double-
beam spectrophotometer (Perkin Elmer 550) was used.
Three milliliter acrylic disposable cells were used, and
inorganic medium was used as a blank and to dilute the
samples. A wavelength of 550 nm was chosen to avoid
interferences of chlorophyll absorbance. An optical density
between 0.1 and 0.3 was always guaranteed with the
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˜o et al.: On-Line Monitoring of Microbial Chlorella Growth 759
Biotechnology and Bioengineering. DOI 10.1002/bit

corresponding dilution. This method is very sensitive for
low biomass concentrations, but some important errors can
be found for dilutions over 100 times.
The dry weight determination was done using 0.2 mm
membrane filters (Pall, HT-200). The filters were dried by
heating them during 15 min at 150 W in a microwave oven,
leaving them in a desiccator to get cold. For filtration, a
stainless steel pressure holder (Sartorius, SM 16249) was
used. Volumes between 1 and 10 mL of sample were filtered
and washed with the same water quantity. The weight of the
dry filters was compared before and after filtration. This
method is very reproducible when biomass concentration is
not less than 1 g per liter. A linear correlation between dry
weight (DW) and optical density (OD) has been obtained
for this strain:
DWðg=LÞ¼ð0:1272ÞðODÞ0:0209ðr2¼0:9968Þ(1)
Some experiments were performed to test the on-line
determination of biomass by dielectric spectroscopy using
the method of Cannizzaro et al. (2003). A logarithmic
scanning for frequencies from 10 to 0.1 MHz was used to
measure the conductance of the medium broth every hour.
The differences of the conductance at two frequencies were
correlated with the off-line biomass determination.
Metabolites Concentration
The evolution of the concentrations of glucose, nitrate, and
phosphate in the culture medium could be followed off-line
by high performance liquid chromatography (HPLC). An
automatic system (Agilent, series 1100) for multiple samples
was used. A 5 mM H
2
SO
4
aqueous solution was used as the
eluent phase at a rate of 0.5 mL/min (P¼5 MPa) in an ion-
exclusion column (Supelco, Supelcogel H 300 mm) with a
guard column (Supelco, Supelguard C610H), both at 308C.
The detector compares the refraction index of the liquid
going out with that of the eluent during a program of 30
min. The retention times of nitrate, phosphate, and glucose
were 7.8, 10.4, and 12.2 min, respectively. The areas of the
peaks in the chromatogram were related to the concentra-
tion of the compounds by comparison with standard
solutions previously prepared. Two other secondary
metabolites were detected in the last phase of the experi-
ments with glucose, with retention times of 7.5 and
17.4 min; these metabolites have not been identified, but
they disappeared after glucose was totally consumed.
Some experiments were performed to use the infrared
spectroscopy for on-line quantification of metabolite
concentrations in the medium, accordingly with the method
of Kornmann et al. (2003). A previous calibration was
necessary, and 50 standard independent compositions of the
medium were prepared within a range of concentrations for
glucose, nitrate, and phosphate. The spectra for each
standard mixture were measured in a screening from 2,000
to 400 cm
1
and a matrix of the absorbance with wavelength
was constructed by correlation with metabolite concentra-
tions. During heterotrophic experiments with algae, a
spectrum of the medium broth was registered every hour,
and the profiles for each of the three metabolites could be
obtained and compared with the off-line measurements.
Calorimetric Characterization of the Reactor
One and seven tenths liter of the inorganic medium was
poured into the photo-bio-calorimeter. A temperature
T
r
¼298.15 K was fixed to keep the temperature inside the
reactor constant by changing the temperature of the jacket T
j
when necessary. The temperature of the metallic plate-head
of the reactor was controlled at 300.15 K and the
temperature of the heat exchanger for the thermostatic
housing was maintained at 298.15 K. A stirring speed was
also kept fixed. If the experimental conditions were not
changed, the difference between the temperatures in the
reactor and the jacket, DT(K) ¼T
r
T
j
, remained constant
with time in a baseline.
When the calibration heater was turned on, an electrical
current heated the corresponding resistance with a power of
q
cal
¼1.97 W. This power was related to the change of the
DTin the baseline to obtain the global heat transfer
coefficient UA (W K
1
) of the calorimeter:
qcalðWÞ¼UA ðTrTjÞ(2)
A series of three calibrations was made before and after each
different experiment.
The ambient temperature T
lab
showed an influence in this
baseline, but a correction could be done with the on-line
values of the housing temperature T
box
. The correction with
T
box
in the heat-flow rate exchanged between the reactor
jacket and the culture broth, q
f
, was done by the next
equation:
qfðWÞ¼UA ½ðTrTjÞkTbox(3)
Because T
lab
changed slowly during the journey, the value of
the constant k could be calculated from a short period of
time (around 3 h) considering q
f
¼0 for a stable horizontal
baseline.
The heat-flow rate caused by agitation of the broth, q
s
,
could be determined in two ways. Maintaining the other
experimental conditions constant, a series of stirring speeds
R(rpm) was tested and correlated with the corresponding q
f
value. An extrapolation of the differences of the q
f
from the
differences in Rcould give the absolute value for q
s
at each
R. However, during microbial growth, some changes in the
physical-chemical properties of the medium may modify
the stirring heat even if the value of Rremains constant. The
torque or moment of torsion, t, can then be another more
accurate measurement way of this contribution, according
with the next equation:
qsðWÞ¼ð2p=60ÞRt(4)
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DOI 10.1002/bit

With a torque master (Vibro-Meter, 205), and a display and
control unit (Vibro-Meter, DCU285), the torque value
was obtained on-line to have the variations of the q
s
contribution.
The illumination from the lamp also caused a heat-flow
rate q
l
in the reactor. Two series of experiments were made in
which an estimation of this magnitude could be determined.
Light was turned on and off in cycles of 1 or 2 h, the
differences in the baseline corresponded to this q
l
with the
culture medium. In a second phase, around 20 mL of black
ink was added to the medium in order to guarantee that all
light provided in the reactor was absorbed by the dark
solution. The on/off-light cycle was repeated to evaluate the
maximal heat absorption of light by the liquid inside the
reactor.
The heat-flow rate due to the addition of any liquid or gas
in the reactor was not determined experimentally. Pressur-
ized air from the ambient was used to bubble in the medium.
Besides the small proportion of carbon dioxide in the air, an
additional quantity was sometimes mixed with the air. The
flux of the gases was of 2 L per minute, and a trap with water
was used to humidify it before bubbling in the reactor.
Water-evaporation from the reactor (and the corresponding
heat of evaporation) was compensated with this humidified
gas.
The nitrogen and inert gases were dissolved in the
medium and then desorbed; with a constant temperature
and flux of gases, the rates of inert gases dissolved and
desorbed were the same, and the heat contribution to the
system was also eliminated. The oxygen and carbon dioxide
were the two gases metabolized by the algal cells, and the
rates for dissolution and desorbing of these gases are not
always the same. Actually, they continuously change during
the microbial growth and contribute to the total heat
exchange measured by calorimetry.
Therefore, the heat flux with time due to the microbial
growth, q
bio
, is computed accordingly with the next
equation:
qbioðWÞ¼qfqsqlð5Þ
where the contribution of the stirring and light is subtracted.
Furthermore, it is possible to calculate the total biological
heat exchange, Q
bio
(J), from the integration of the curve of
q
bio
against time. When the biomass yield is considered, it is
possible to calculate the molar heat yield.
Batch and Fed-Batch Experiments
For calorimetric measurements, 1.6 L of the inorganic
medium enriched with glucose (between 27 and 111 mM)
was used to perform the algae culture. The medium was
sterilized in situ at 394.15 K and 0.2 MPa for 20 min or by
filtering with a 0.2 mm membrane before pouring it in the
sterilized reactor. The temperature of the calorimeter was
maintained at 298.15 K. The temperature of the plate head
was again 300.15 K and the temperature of the heat ex-
changer in the housing was 298.15 K. A stirring of 300 or 400
rpm was fixed, with a constant flux of gas (2 L per minute).
Sampling of the culture medium was done periodically by
using an automatic lab-made Biosampler: with a pump for
liquids, an air valve and a mechanic arm, sampling was
automatically controlled with a LabView program in a
personal computer. The samples were kept at 0.58C until
they were prepared for quantification of the concentrations
of biomass and metabolites.
A pH probe (Mettler-Toledo) and sometimes a pO
2
probe
(Ingold) in the reactor were used to follow the evolution of
the medium conditions. As well as the housing for the pH
probe, the display and control unit for pH and pO
2
was from
Bioengineering AG. During the first experiments, the pH
was only monitored, but for the next experiments, adding a
diluted aqueous solution of nitric acid also controlled it. The
mass of acid added to the culture was followed with the on-
line weight of a bottle containing the solution, measured
with an electronic balance (Mettler-Toledo, PG5001-S).
The gas going out from the reactor was dried and heated
to 408C before going to a gas analyzer. A single infrared
beam (Servomex, 402) was used to quantify carbon dioxide,
and a paramagnetic transducer (Servomex, 1100) was used
to quantify oxygen. A calculation of the oxygen and carbon
dioxide exchanged by the microalgae was performed from
on-line measurements.
All the signals are read on-line through a board (National
Instruments, CB-68LP) connected to a PC, or through a
FieldPoint interface (National Instruments) using a Lab-
View program developed previously in our laboratory for
automatic data acquisition. After all signals were stable for
at least 2 h, inoculation was made with a volume around
100 mL of the cultures conserved in stock. Just before and
after inoculation, and during all the experiment, samples
from the reactor were taken to analyze the biomass, glucose,
nitrate, and phosphate concentrations.
A number of different experiments were performed to
improve the conditions of algal growth and the correspond-
ing calorimetric measurements. The three metabolic modes
were tested: heterotrophic, autotrophic, and mixotrophic.
The heterotrophic and mixotrophic conditions were
chosen to accelerate the biomass growth by using glucose in
the medium. A second addition of medium with glucose
(fed-batch) was sometimes tested in order to obtain bigger
cell concentrations. The autotrophic growth, much slower,
was only tested just after one of the other modes, when
glucose was over and the biomass concentration was high
enough to increase the possibilities to detect any photo-
synthetic activity.
Results
Calorimetric Characterization of the Calorimeter
According to Equation (2), the value of the global heat
transfer coefficient UA was determined before and after each
experiment. For similar conditions, the UA values remained
Patin
˜o et al.: On-Line Monitoring of Microbial Chlorella Growth 761
Biotechnology and Bioengineering. DOI 10.1002/bit

constant around an average of UA ranging between 6 and
7WK
1
.
A typical baseline of DTas a function of time is presented
in the graph of Figure 2a. The temperatures in the housing
T
box
and in the laboratory T
lab
were also graphed. From a
graphic of DTas a function of T
lab
, a linear correlation could
be obtained to have the constant k, which was applied as
correction for the q
f
(Eq. 3) as showed in Figure 2b for the
same data. After applying a moving average over every 100
points, a smoothing of the signal could be obtained with a
final deviation of less than 20 mW L
1
during the period
of the experiment.
The values of the heat-flux related to stirring, q
s
, were
calculated at different angular velocities of the stirrer,
assuming q
s
¼0 for null agitation. Figure 3 represents these
values for two independent series of experiments. For every
angular velocity, there was also a constant torque value
related to q
s
in Equation (4).
On the other hand, the use of the two optical filters for the
xenon light to avoid infrared and ultraviolet radiations
before coming into the reactor diminished the heating
significantly due to the absorption of light. The heat-flux by
illumination q
l
using these two filters was calculated from
differences in DTwith the lamp turned on and off. The
obtained values from experiments were q
l
¼(5.1 4.1)
mW L
1
with medium and q
l
¼(9.9 2.1) mW L
1
with a
black ink aqueous solution. The latter with black ink
represented the maximum possible value of light absorbance
for the system, with a negligible contribution to the total
heat-flux when the xenon lamp was used. On the contrary,
the use of the fluorescent lamp represented a much bigger
contribution, although in a very constant value:
q
l
¼(139 15) mW L
1
with medium and q
l
¼(276 9)
mW L
1
with black ink solution. In both cases, with xenon
and fluorescent lamps, q
l
for the medium was near 50% of q
l
for the dark ink solution.
Batch and Fed-Batch Experiments
More than 10 experiments were performed. Since every
experiment was different from the others, it is not possible to
compare the results directly. However, some trends could be
observed, principally to distinguish among the different
metabolic modes. Typical behavior of Chlorella growth is
showed with some examples in next lines, and comparative
tables are also presented at the end of this section.
For the mixotrophic and heterotrophic growths, a typical
evolution of biomass and glucose concentrations with time
could be followed. Figure 4 shows an example with
heterotrophic conditions. A logarithmic-growth region
was observed, and it was possible to determine the specific
growth rate of the strain. The yield of biomass per glucose
amount could also be computed from the total fed glucose.
In some cases, when the glucose was totally consumed, as
checked by HPLC, new fresh medium was immediately
added to continue with the growth. After the mixotrophic
growth, a slight autotrophic growth of biomass could
sometimes be detected without addition of the glucose. It is
important to note that a long lag time was detected many
Figure 2. Typical results for the calorimetric signal. a: It shows the evolution
with time of temperature changes in the calorimeter DT(thick line), in the housing T
box
(thin line), and in the laboratory T
lab
(dashed line). The influence of T
lab
can be
observed on T
box
and DT.b: It shows the corrected heat-flow signal q
f
(baseline) for
the same data. The points represent the experimental data; the solid line is a moving
average over every 100 points. The total average is on the axe with q
f
¼0.
Figure 3. The heat-flux from stirring at different angular velocities. Two inde-
pendent experimental series were performed: the circles are for the first series and
the stars represent the second.
762 Biotechnology and Bioengineering, Vol. 96, No. 4, March 1, 2007
DOI 10.1002/bit

times before reaching a significant cell growth. This lag time
was related to the adaptation between different metabolic
conditions, since it was noted that the time could be reduced
at the beginning of the experiments with previous
adaptation to the corresponding metabolic mode just
before inoculation.
As seen in Figure 4, the evolution of the pH during the
algae growth was very characteristic. Contrary to the typical
pH drop during growth of heterotrophic microorganisms,
here, the pH increased along the biomass growth for the
three possible metabolisms. In fact, a typical correlation of
biomass formation with the pH rise was found, as can be
seen in Figure 5, for the heterotrophic and mixotrophic
growths.
In both heterotrophic and mixotrophic conditions,
besides the pH increase, there was a significant diminution
of the nitrate concentration in the medium, as seen by the
HPLC analysis. The phosphate concentration was also
reduced, but in a smaller proportion. When the glucose
concentration was almost over, two non-identified meta-
bolites appeared, as detected by the HPLC analysis. These
metabolites were consumed once the glucose in the medium
was over.
In order to fix the pH value as well as to avoid the lack of
nitrate in the medium, an aqueous solution of nitric acid was
added automatically to the reactor. As it can be seen in
Figure 6 for mixotrophic conditions, a correlation was also
found between the biomass concentration and the quantity
of nitric acid added to the medium. Moreover, the yield of
biomass per nitrogen consumption can be computed from a
linear regression.
The oxygen and carbon dioxide exchanges were calculated
during the growth process. Because gas going into the
reactor was previously humidified, water content was the
same for the gas going out of the reactor. It would be
possible to obtain the corresponding yield from the oxygen
uptake and the carbon dioxide expel rates. For the
heterotrophic growth, the carbon dioxide expel was evident,
although the oxygen uptake rate was not easily quantified,
since the small measured variations during the growth are
mixed up at the level of the corresponding noise. For the
mixotrophic growth, the tendencies for the oxygen uptake
and the carbon dioxide extent were not clearly defined. After
glucose oxidation, some mixotrophic conditions were
changed to just autotrophic growth. In this case, a slight
carbon dioxide uptake was detected.
The pO
2
signal was useful to follow the metabolic activity
in all the culture modes. Consumption of glucose is related
with a diminishing of oxygen dissolved in the medium. After
glucose extinction, the level of pO
2
returned around the
basal value. In addition, for the autotrophic growth, a faint
production of oxygen could be detected with a continuous
increase in the oxygen dissolved in the medium.
Figure 4. Biomass (crosses), glucose concentration (circles), and pH (line) with
time for heterotrophic growth of Chlorella.
Figure 5. Change of the pH in the culture medium as a function of the biomass
growth for heterotrophic (squares) and mixotrophic (solid triangles) conditions.
Figure 6. Determina tion of the yield of biomass per nitrogen consumption for
heterotrophic growth with addition of diluted nitric acid for pH control around 5.6. The
experimental results (squares) are correlated in a straight line.
Patin
˜o et al.: On-Line Monitoring of Microbial Chlorella Growth 763
Biotechnology and Bioengineering. DOI 10.1002/bit

The heat-flux from metabolic activity q
bio
was also a
representative signal of the algal growth, as showed in
Figure 7 where the on-line measurement for q
bio
production
is in relation to the glucose consumption and the
corresponding pO
2
signal.
During the lag time, before a significant glucose uptake, a
slight drop of q
bio
was frequently observed. After the total
glucose consumption, in heterotrophic conditions, the total
heat production remained constant. However, when light
was on, now in autotrophic conditions, an immediate
consumption of heat could always be observed. Especially
with the fluorescent lamp, the consumption of q
bio
was
remarkable from lag time to total glucose uptake. A contrast
between heterotrophic and mixotrophic conditions is
showed in Figure 8 for the integrated valued of q
bio
, the
total biological heat exchange Q
bio
. With these results, the
heat yield per biomass formed can be calculated at different
stages of the cellular metabolism for every experiment.
As a compilation of the different experiments, Table I
shows a variety of the conditions and results for each of the
batch and fed-batch experiments. Mean values were
computed in order to remark the differences among the
three growth modes and even between the two lamps. By
instance, the growth specific rate, m, is the smallest for the
autotrophic mode, but the maximum for the mixotrophic
mode. It is possible to observe that the biomass yield is not
favored when the fluorescent lamp was used. A discussion
about the differences in the heat yields is widely presented
later on.
Some test experiments were additionally performed in
order to check the utility of performing on-line measure-
ments with dielectric and infrared spectroscopies. For
dielectric measurements, the differences of the conductance
at two frequencies were correlated with the off-line biomass
determination. Figure 9 shows one of the best examples for
this experimental correlation. As it can be seen, a linear
correlation was found only at biomass concentrations below
1gL
1
. For higher biomass concentrations, the con-
ductance in the medium seems to remain constant.
From infrared experiments, the profiles of the most
important metabolites in the medium broth (glucose,
nitrate, and phosphate) were obtained during heterotrophic
growth of algae (Fig. 10). A good agreement of these profiles
was found with the values measured off-line by HPLC. It
should be noted, however, that for each experimental batch
a previous calibration with the matrix of standard solutions
is fundamental for the success of this analytical method.
Discussion
The total chemical process for the Chlorella vulgaris growth
could be regarded in a general simplified set of equations as
follows:
2:45 CH2Oþ0:09 NO3þ1:07 O2
¼CH1:76O0:35 N0:09 þ1:45CO2þ1:52H2O
þ0:09 OH(5)
2H
2Oþ2 NADPþ¼O2þ2 NADPH þ2H
þ(6)
CO2þ2 NADPH þ2H
þ
¼CH2Oþ2 NADPþþH2O (7)
Equation (5) represents normal metabolic growth as
generally described for heterotrophic organisms. An external
source of carbohydrates (CH
2
O) is used as the source of
chemical energy for biosynthetic reactions that lead to the
formation of new biomass. The process is not totally
efficient, and some of the energy is lost as heat and
Figure 7. A comparison of the heat-flux from metabolic activity, q
bio
(continuous
line), with the changes in glucose concentration (squares) and dissolved oxygen
(dashed line), during mixotrophic growth conditions.
Figure 8. Differences of the total biological heat exchange, Q
bio
, between four
experiments during heterotrophic growth (continuous line) and mixotrophic growth
(dashed lines). The mixotrophic growth with the fluorescent lamp is represented by the
line arriving near Q
bio
¼70 kJ L
1
.
764 Biotechnology and Bioengineering, Vol. 96, No. 4, March 1, 2007
DOI 10.1002/bit

production of carbon dioxide. In the case of Chlorella, the
chemical composition of biomass was taken from previous
reports for microalgae (Duboc et al., 1999). It should be
remarked that this consideration is approximate, since the
chemical composition of the microalgal cells may vary for
mixotrophic, heterotrophic, and autotrophic growth,
although the differences cannot be very large. Unfortunately,
the elemental analysis of the biomass produced in every
experiment was not determined. The stoichiometric
coefficient for carbohydrates was computed from the
average value for the batch experiments with the hetero-
trophic growth from the glucose. The nitrogen balance was
easily computed from the experiments where the pH was
fixed by addition of diluted nitric acid, and corresponds
perfectly to the value for the elemental composition of
microalgae. The other coefficients were added to complete
the total balance. As it was said before, the exchange of
oxygen and carbon dioxide during the growth was not very
clear and it was useless to include them in the balances.
Equation (5) also represents the experimental observation
for the consumption of nitrates and the rising of the pH in
the medium along the microbial growth. It should be noted
Table I. Principal results for a number of different experimental conditions growing the microalga Chlorella vulgaris.
Experiment
Mode
(1)
S(2)
(g L
1
)
X(3)
(g L
1
)
Stirring
(rpm) pH
m(4)
(s
1
)
Y(X/S) (5)
(mol mol
1
)
Y(X/N) (6)
(mol mol
1
)
Y(Q/X) (7)
(kJ mol
1
)
1 b, m 11.3 0.0–4.7 300 6.0–7.5 148 0.52 — 200.4
2 fb, m 5.3 2.9–6.8 300 7.0–7.4 130 1.07 — 265.5
3 fb, a — 6.8–7.5 300 7.4–7.5 7 — — 461.4
4 fb, h 19.4 5.9–8.7 300 7.2–7.5 36 0.20 — 523.8
5 b, h 8.4 0.0–3.6 300 6.0–6.5 111 0.59 — —
6 b, h 9.9 0.0–4.3 400 5.6–6.8 105 0.21 — 494.4
7 fb, h 15.0 4.0–9.2 400 6.8–7.3 61 0.30 — 465.9
8 b, m 11.9 0.0–5.5 400 5.5–7.0 121 0.54 — 150.2
9 fb, m 13.5 5.0–9.5 400 6.7–7.2 45 0.40 — 244.6
10 fb, a — 9.5–9.9 400 7.2–7.3 5 — — 300.1
11 b, h 12.9 0.0–4.1 400 5.6 113 0.42 13.1 —
12 b, m* 12.0 0.0–1.8 400 5.5 216 0.22 4.8 436.5
13 fb, m* 18.5 1.5–4.3 400 5.5 65 0.19 11.1 107.8
14 b, h 13.0 0.0–3.8 300 6.0 86 0.41 10.9 —
x
8
h8513 0.36 0.06 12.0 0.6 495 17
xa 61 — — 381 81
x m 111 23 0.63 0.15 — 215 26
xm* 140 0.20 0.02 8164
Notes: (1) b, batch; fb, fed-batch; m, mixotrophic with halogen lamp; a, autotrophic with halogen lamp; h, heterotrophic without light; m*, mixotrophic
with fluorescent lamp; (2) glucose substrate; (3) biomass product as dry weight; (4) growth specific rate; (5) molar yield of biomass from glucose; (6) molar
yield of biomass from nitrate; (7) heat yield per molar biomass; (8) mean values with uncertainties being the standard deviation of the mean.
Figure 9. The differences of capacitance DCat two measurement frequencies
(0.21 and 5.75 MHz) correlated with the off-line measurements of biomass.
Figure 10. On-line concentrations of glucose, nitrate, and phosphate as com-
puted from infrared spectra in the broth for heterotrophic growth.
Patin
˜o et al.: On-Line Monitoring of Microbial Chlorella Growth 765
Biotechnology and Bioengineering. DOI 10.1002/bit

that potassium, phosphorous, and magnesium are also
important in the composition of biomass, but these
elements could be ignored in this work since they have a
small contribution compared with the others elements in the
formula (Mandalam and Palsson, 1998).
A simplified description of photosynthesis can be seen in
Equations (6) and (7). Equation (6) corresponds to a
process known as the light phase, in which photons from
light are absorbed to keep the corresponding energy as ATP
molecules, needed for the second process called dark phase,
and represented by Equation (7). In order to get the balances
of charge and hydrogen in both equations, the nicotine
adenine dinucleotide phosphate (NADPH) and the corre-
sponding oxidized molecule are presented. However, it is
easy to find that the addition of these two reactions gives a
simplified equation in which carbon dioxide and water
participate to be transformed in oxygen and carbohydrates.
It should be remarked that the production of ATP was
omitted in the first equation, as well as the consumption of
ATP in the second reaction. This is because the balance of
ATP is kept in the total photosynthetic process. Actually,
energy from light photons is not kept by ATP molecules, but
transformed to the so-called chemical energy stored as
carbohydrate molecules.
Nevertheless, as showed before, the glucose is not stored
but consumed during the Chlorella growth. The hetero-
trophic and autotrophic growth modes were always related
to external glucose transformation through exothermic
global processes, as expected from results of previous reports
for non-autotrophic microorganisms (von Stockar and Liu,
1999; von Stockar et al., 2006). A clear difference is observed
between these two modes when released heat is compared:
the heterotrophic growth has more energetic heat yields than
the mixotrophic growth. This can be reasonably understood
when the only two autotrophic growth experiments are
observed to be markedly endothermic. In this sense, the
heterotrophic growth, as represented by Equation (5),
releases heat, while the autotrophic growth, as represented
by Equations (5), (6), and 7), absorbs it. It is concluded that
Equations (6) and (7) together are clearly endothermic as
expected from previous thermodynamic results (Johansson
and Wadso
¨, 1997). Moreover, the mixotrophic growth has
intermediate values of energy between the heterotrophic and
the autotrophic growths, with a final value depending on
which of both modes is favored. By instance, when the
fluorescent lamp was used, the increment in light intensity
(compared with the Xe lamp illumination) allowed an
endothermic mixotrophic growth because photosynthesis
was significantly favored.
Even finer details can be obtained with the heat yields:
between the batch and fed-batch mixotrophic growth, the
second process releases more energy than the former. This
could be explained if an additional exothermic process is
considered in addition to the heterotrophic growth and the
photosynthesis. This process may be related to death of cells,
and it could be crudely represented as the combustion of
biomass with oxygen to produce carbon dioxide and water.
Evidently, this and the Equations (5)–(7) are over-
simplified since it is clear that every one of these ‘‘reactions’’
is composed of a number of enzymatic coupled reactions.
Moreover, a metabolic regulation exists which is connecting
all the processes in a unique cellular process, but the
simplified model used here helps to understand a link
between the biological process and the corresponding
measurements during the microalgae growth in the
bioreactor.
Although numerous works have been reported in relation
to the growth of Chlorella vulgaris and other microalgae,
very little is found about the metabolic differences between
the autotrophic, heterotrophic, and mixotrophic growth
modes. A wide variety of techniques were tested here in
order to follow and understand the algal growth, being
significantly useful. Traditional techniques were combined
with some others presented for the first time in a study of
microalgae, being meaningful to distinguish three different
metabolic modes: (i) the calorimetry, to detect when
photosynthesis is favored; (ii) the nitric acid pH regulation,
to follow the kinetics of growth; and (iii) the on-line infrared
spectroscopy, to follow the changes of nutrients in the broth.
Rodrigo Patin
˜o thanks Conacyt-Mexico and EPFL-Switzerland for the
stipendium as postdoctoral researcher.
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