A study of the growth for the microalga Chlorella vulgaris by photo-bio-calorimetry and other...

ARTICLE

A Study of the Growth for the Microalga Chlorellavulgaris by Photo-Bio-Calorimetry and OtherOn-line and Off-Line Techniques

Rodrigo Patino,1 Marcel Janssen,2 Urs von Stockar3

1Departamento de Fısica Aplicada, CINVESTAV–Unidad Merida, Apartado Postal 73,

Cordemex, 97310 Merida, Yucatan, Mexico; telephone: þ52(999)1242138;

fax: þ52(999)9812917; e-mail: [email protected] of Agrotechnology and Food Sciences, Food and Bioprocess Engineering

Group, Wageningen University, Wageningen, The Netherlands3Laboratoire de Genie Chimique et Biologique, Ecole Polytechnique Federale de Lausanne,

Lausanne, Switzerland

Received 10 April 2006; accepted 14 August 2006

Published online 1 September 2006 in Wiley InterScience (www.interscience.wiley.co
m). DOI 10.1002/bit.21182
ABSTRACT: Calorimetry and other on-line techniques areused for the first time as complement to the traditional off-line methods in order to follow the growth of the greenChlorella vulgaris microalgae. A 2-L photo-bio-reactor wasadapted from a commercial calorimeter used previously tostudy heterotrophic microbial growth. An external source oflight was added to favor the photosynthesis of the auto-trophic cells. Heterotrophic growth was also tested withexternal glucose in the broth. A third mode, mixotrophic,allowed faster autotrophic plus heterotrophic growth.Calorimetric measurements were performed consideringthe corresponding calibrations in order to consider onlythe energy involved during the microalgal growth. The threedifferent modes of Chlorella cultures were energeticallycharacterized. Besides calorimetry, the weight of dilutednitric acid added to maintain the pH of the culture wascorrelated with the cellular growth and the nitrogen com-position of the algae. Additionally, the on-line infraredspectroscopy proved to be an efficient technique to followthe composition of the broth in glucose, nitrates, andphosphates. These results were compared and complemen-ted with some classic off-line techniques used to track thiskind of cultures.

Biotechnol. Bioeng. 2007;96: 757–767.

� 2006 Wiley Periodicals, Inc.

KEYWORDS: Chlorella vulgaris; calorimetry; on-line mon-itoring; photo-bio-reactor; mixotrophic mode; microalgae

Correspondence to: R. Patino

Contract grant sponsor: FNS-Switzerland

� 2006 Wiley Periodicals, Inc.

Introduction

Photosynthesis is the most important process in nature.Radiant energy from the Sun is used through photosynthesisby plants, algae, and some other microorganisms tosynthesize carbohydrates (Berg et al., 2002). Carbohydratesare known as the chemical source of energy for biologicsystems. They are used, directly or indirectly, all over theworld by living organisms for anabolic reactions. In anextrapolation, solar energy may be seen as the originalsource of fossil combustible coming from the degradation ofbiosynthetic products (Dukes, 2003).

The natural process starts when photons are captured bychlorophyll. This green substance is commonly found aschlorophyll a or chlorophyll b in chloroplasts—the specialmembrane organelles in autotrophic cells. This photonicenergy excites specific chlorophyll molecules. A fraction ofthe decay energy is utilized to induce a cascade of reactionsin which molecular oxygen is obtained from water with theformation of two important biochemical compounds usedto carry energy during metabolism: NADPH (reducednicotinamide adenine dinucleotide phosphate) and ATP(adenosine triphosphate) (Govindjee and Krogmann, 2004).This first process of photosynthesis is named the light phasesince the principal former is precisely light.

The energetic molecules produced during the light phaseare then used in a second cascade of reactions known asthe dark phase. In this part, the global process involves theproduction of carbohydrate molecules from gaseous CO2.This second process is performed out of the photosynthetic

Biotechnology and Bioengineering, Vol. 96, No. 4, March 1, 2007 757

membrane and does not depend directly on light. Indeed, itmay happen simultaneously with the light phase.

The elucidation of the mechanisms in photosynthesis hasbeen extensively studied during the last century. Despite itsimportance, only some few efforts have been reported inrelation to the energy exchanges during the process.Calorimetry has been used in few cases to study thiscomplex process. Microalgae were thus used to obtain thequantum yield of photosynthesis (Magee et al., 1939): only0.08 mol of carbon dioxide been transformed by 1mol of theincident photons for a variety of light intensities andconcentrations of microalgae Chlorella vulgaris. Spinachleaves were also studied by calorimetric experiments(Johansson and Wadso, 1997) with a maximum value near

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

13% for the energy uptake during photosynthesis and aGibbs energyDG8¼ 478� 17 kJ �mol�1 for the formation ofglucose.

Searching a better comprehension of the energy ex-changes during the photosynthetic process, a photo-bio-calorimeter is proposed here for the study of phototrophicmicroorganisms. During previous years, the calorimetricstudy 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 describedifferences in the metabolism of a number of microorgan-isms (von Stockar and Liu, 1999). Some of the highlights ofthis bio-calorimeter involve the improvement of the thermalmeasurements, the use of a torque-meter to consider theheat from stirring, and a diminishing in the interchange ofheat with the room (Garcıa-Payo et al., 2002). At the sametime, the search for useful tools to monitor complementarykey parameters during microbial metabolism has been aconstant in this laboratory (von Stockar et al., 2003).

Being the study of photosynthetic microorganisms a lackin our experience for calorimetry and other monitoringtechniques, we propose here a photo-bio-calorimeter andsome additional techniques that may help to elucidate thecomplex behavior of the microalgae Chlorella vulgaris. Thisvariety of algae has been widely studied and numerousreports can be found in literature. Chlorella is often cited as areference photosynthetic microbial organism. Moreover, ithas been proposed for the treatment of wastewaters(Gonzalez et al., 1997) as well as for feeding (Gouveiaet al., 2002).

In order to understand the photosynthesis of this speciesduring its linear phase of growth, a recent study has beenperformed in our laboratory (Janssen et al., 2005). Never-theless, the strain is very interesting since it can grow notonly 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 foundbetween the three growth regimes.

Materials and Methods

Description of the Photo-Bio-Calorimeter

A commercial reaction calorimeter (Mettler-Toledo, RC1) isthe base of the one presented here. During previous years, alist of modifications has been proposed to improve both themicrobial growth and the sensibility of the calorimetricmeasurement (Garcıa-Payo et al., 2002; Marison et al.,1998). In Figure 1, it is shown the picture of the vessel withsome of the original and adapted parts. From the originalcalorimeter are the glass reactor, an oil temperaturecontroller, an electric heater, the motor and stirrer, internalbaffles and a pH probe. The adapted parts of the (photo)bio-calorimeter include oil and reactor thermistors to improvethe calorimetric signal, a torque-meter to obtain the stirring

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energy, a system for gas input and output, inoculation andsampling ports, a light diode and space for any additionalon-line probe. The reactor is a 2-L glass vessel surroundedwith a glass jacket (Mettler-Toledo, AP01). Silicon oil in thejacket circulates at a controlled temperature Tj measuredwith a thermistor.

A Plexiglas insulating housing around the reactor vessel isused to diminish the influence of the ambient temperature.Inside this box, a copper heat exchanger with water ismaintained at 298.15 K with an external thermostatic bath.The housing is covered with aluminum foil to avoid theinterference coming from external light and to increase thereflection of light inside the housing. Inside the reactor,where around 1.7 L of liquid can be added, the temperatureTr is measured with a glass-covered thermistor andcontrolled through heat exchange with the oil jacket. Boththermistors—in the reactor and the oil bath—have asensibility of 0.001 K each. The difference of temperaturebetween the reactor and the jacket, DT¼Tr�Tj, can bedirectly related to the heat interchange through the reactorwall.

A constant agitation guarantees homogeneity in compo-sition and temperature of the liquid inside the reactor, thisliquid is stirred with a stainless-steel double propel. Amechanical motor out of the reactor is used for rotation. Inorder to measure the energetic contribution due to thestirring inside the reactor, a torque-meter has beenconnected between this propel and the motor. A systemof metallic baffles is used to favor the microbial growth byimproving the aeration of the medium. The thermostat andcirculation of the jacket oil, as well as the thermostat andstirring of the liquid inside the reactor, are done by theoriginal Mettler-Toledo system through the correspondingsoftware manipulated from a personal computer.

The vessel is covered on the top with a metallic tap insidewhich water circulates at temperature T¼ 300.15 K. The tapis attached to the vessel by using five bolts with nuts. An o-ring and some lubricant grease are in between. Through thetap, a variety of connections can be done: the thermistor formeasuring and controlling the temperature inside thereactor, a heater for thermal calibration of the calorimeter,the liquid and gas feeding in the medium, a drain for gasoutgoing, a system for collection of samples, a light diodeand pH, pO2, dielectric, infrared or any other probe.Unfortunately, due to the limited capacity for eightconnections, not all of them are possible at the same time.Combining them in different ways performed the alternateexperiments presented here. The heater for calibrationprovides a constant power of 1.97 W into the mediumduring a defined period of time. In this way, the electricalwork applied is related to the exchange of heat trough thewall coming from the value of DT to keep Tr constant.

The source of light was a 150-W xenon lamp into a specialhousing (Oriel, 60100), with a cooling system and an easyorientation using an F/4.4 ellipsoidal AlMgF2 reflector. Thesystem 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 a0.2 M NaCrO2 aqueous solution and a dichroic filter. Therest of the light is condensed on the extreme of a liquidvisible-light guide. The other extreme of the guide isconnected to a quartz rod inside the calorimeter. A previoushydrofluoric acid treatment of the rod allows a roughsurface to improve dispersion of the light going out into thereactor.

A fluorescent flat lamp (Osram, Planon 10,400/868 6) wasalso used for one of the experiments to be compared with theXenon lamp illumination. During this experiment, the lampis fixed directly from outside on one of the transparent wallsof the reactor housing to avoid extreme heating of thesystem.

Microbiological Strain and Culture Medium

The Chlorella vulgaris (211/11B) micro-alga was obtainedfrom the Culture Collection of Algae and Protozoa, UK. Adefined inorganic culture medium to maintain the strain instock and to perform the calorimetric experiments issuggested here based on the one proposed by Schlosser(1994) for unicellular green algae and by Mandalam andPalsson (1998) for Chlorella cultures. Some flask tests wereperformed to get a medium without limitation of the basicbiomass elements (nitrogen, phosphorus, magnesium)obtaining a growth at relatively high cell concentrations.The composition of the inorganic medium was: KNO3 30mM, NaH2PO4 9 mM, Na2HPO4 1 mM, MgSO4 1 mM,CaCl2 0.1 mM, FeNaEDTA 0.02 mM, H3BO3 1 mM, MnSO4

1 mM, ZnSO4 1 mM, 3(NH4)2O � 7MoO3 0.05 mM, andCuSO4 0.01 mM.

Micro-algal cultures were kept under sterile conditions inErlenmeyer flasks at room temperature, on a plate to stir at100 rpm and with a cotton top for gas exchange withambiance air. Fluorescent lamps were used in 12 h on/offcycles to maintain photosynthesis active. Because hetero-trophic growth is also possible for this strain, sometimesglucose was added as carbon source. A concentrationbetween 5 and 20 g of glucose per liter was used in someexperiments for mixotrophic or heterotrophic experiments.

Analysis

Biomass Concentration

Two complementary methods were used to obtain off-linebiomass concentration: optical density measurements anddry weight determination. For optical density, a double-beam spectrophotometer (Perkin Elmer 550) was used.Three milliliter acrylic disposable cells were used, andinorganic medium was used as a blank and to dilute thesamples. A wavelength of 550 nm was chosen to avoidinterferences of chlorophyll absorbance. An optical densitybetween 0.1 and 0.3 was always guaranteed with the

Patino et al.: On-Line Monitoring of Microbial Chlorella Growth 759

Biotechnology and Bioengineering. DOI 10.1002/bit

corresponding dilution. This method is very sensitive forlow biomass concentrations, but some important errors canbe found for dilutions over 100 times.

The dry weight determination was done using 0.2 mmmembrane filters (Pall, HT-200). The filters were dried byheating them during 15 min at 150 W in a microwave oven,leaving them in a desiccator to get cold. For filtration, astainless steel pressure holder (Sartorius, SM 16249) wasused. Volumes between 1 and 10 mL of sample were filteredand washed with the same water quantity. The weight of thedry filters was compared before and after filtration. Thismethod is very reproducible when biomass concentration isnot less than 1 g per liter. A linear correlation between dryweight (DW) and optical density (OD) has been obtainedfor this strain:

DWðg=LÞ ¼ ð0:1272ÞðODÞ � 0:0209ðr2 ¼ 0:9968Þ (1)

Some experiments were performed to test the on-linedetermination of biomass by dielectric spectroscopy usingthe method of Cannizzaro et al. (2003). A logarithmicscanning for frequencies from 10 to 0.1 MHz was used tomeasure the conductance of the medium broth every hour.The differences of the conductance at two frequencies werecorrelated with the off-line biomass determination.

Metabolites Concentration

The evolution of the concentrations of glucose, nitrate, andphosphate in the culture medium could be followed off-lineby high performance liquid chromatography (HPLC). Anautomatic system (Agilent, series 1100) for multiple sampleswas used. A 5 mM H2SO4 aqueous solution was used as theeluent phase at a rate of 0.5 mL/min (P¼ 5 MPa) in an ion-exclusion column (Supelco, Supelcogel H 300 mm) with aguard column (Supelco, Supelguard C610H), both at 308C.The detector compares the refraction index of the liquidgoing out with that of the eluent during a program of 30min. The retention times of nitrate, phosphate, and glucosewere 7.8, 10.4, and 12.2 min, respectively. The areas of thepeaks in the chromatogram were related to the concentra-tion of the compounds by comparison with standardsolutions previously prepared. Two other secondarymetabolites were detected in the last phase of the experi-ments with glucose, with retention times of 7.5 and17.4 min; these metabolites have not been identified, butthey disappeared after glucose was totally consumed.

Some experiments were performed to use the infraredspectroscopy for on-line quantification of metaboliteconcentrations in the medium, accordingly with the methodof Kornmann et al. (2003). A previous calibration wasnecessary, and 50 standard independent compositions of themedium were prepared within a range of concentrations forglucose, nitrate, and phosphate. The spectra for eachstandard mixture were measured in a screening from 2,000to 400 cm�1 and a matrix of the absorbance with wavelength


was constructed by correlation with metabolite concentra-tions. During heterotrophic experiments with algae, aspectrum of the medium broth was registered every hour,and the profiles for each of the three metabolites could beobtained and compared with the off-line measurements.

Calorimetric Characterization of the Reactor

One and seven tenths liter of the inorganic medium waspoured into the photo-bio-calorimeter. A temperatureTr¼ 298.15 K was fixed to keep the temperature inside thereactor constant by changing the temperature of the jacket Tj

when necessary. The temperature of the metallic plate-headof the reactor was controlled at 300.15 K and thetemperature of the heat exchanger for the thermostatichousing was maintained at 298.15 K. A stirring speed wasalso kept fixed. If the experimental conditions were notchanged, the difference between the temperatures in thereactor and the jacket, DT (K)¼Tr�Tj, remained constantwith time in a baseline.

When the calibration heater was turned on, an electricalcurrent heated the corresponding resistance with a power ofqcal¼ 1.97 W. This power was related to the change of theDT in the baseline to obtain the global heat transfercoefficient UA (W �K�1) of the calorimeter:

qcalðWÞ ¼ UA � ðTr � TjÞ (2)

A series of three calibrations was made before and after eachdifferent experiment.

The ambient temperature Tlab showed an influence in thisbaseline, but a correction could be done with the on-linevalues of the housing temperature Tbox. The correction withTbox in the heat-flow rate exchanged between the reactorjacket and the culture broth, qf, was done by the nextequation:

qf ðWÞ ¼ UA � ½ðTr � TjÞ � k Tbox� (3)

Because Tlab changed slowly during the journey, the value ofthe constant k could be calculated from a short period oftime (around 3 h) considering qf¼ 0 for a stable horizontalbaseline.

The heat-flow rate caused by agitation of the broth, qs,could be determined in two ways. Maintaining the otherexperimental conditions constant, a series of stirring speedsR (rpm) was tested and correlated with the corresponding qfvalue. An extrapolation of the differences of the qf from thedifferences in R could give the absolute value for qs at eachR. However, during microbial growth, some changes in thephysical-chemical properties of the medium may modifythe stirring heat even if the value of R remains constant. Thetorque or moment of torsion, t, can then be another moreaccurate measurement way of this contribution, accordingwith the next equation:

qsðWÞ ¼ ð2p=60Þ � R � t (4)

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With a torque master (Vibro-Meter, 205), and a display andcontrol unit (Vibro-Meter, DCU285), the torque valuewas obtained on-line to have the variations of the qscontribution.

The illumination from the lamp also caused a heat-flowrate ql in the reactor. Two series of experiments were made inwhich an estimation of this magnitude could be determined.Light was turned on and off in cycles of 1 or 2 h, thedifferences in the baseline corresponded to this ql with theculture medium. In a second phase, around 20 mL of blackink was added to the medium in order to guarantee that alllight provided in the reactor was absorbed by the darksolution. The on/off-light cycle was repeated to evaluate themaximal heat absorption of light by the liquid inside thereactor.

The heat-flow rate due to the addition of any liquid or gasin 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, anadditional quantity was sometimes mixed with the air. Theflux of the gases was of 2 L per minute, and a trap with waterwas used to humidify it before bubbling in the reactor.Water-evaporation from the reactor (and the correspondingheat of evaporation) was compensated with this humidifiedgas.

The nitrogen and inert gases were dissolved in themedium and then desorbed; with a constant temperatureand flux of gases, the rates of inert gases dissolved anddesorbed were the same, and the heat contribution to thesystem was also eliminated. The oxygen and carbon dioxidewere the two gases metabolized by the algal cells, and therates for dissolution and desorbing of these gases are notalways the same. Actually, they continuously change duringthe microbial growth and contribute to the total heatexchange measured by calorimetry.

Therefore, the heat flux with time due to the microbialgrowth, qbio, is computed accordingly with the nextequation:

qbioðWÞ ¼ qf � qs � ql ð5Þ

where the contribution of the stirring and light is subtracted.Furthermore, it is possible to calculate the total biologicalheat exchange, Qbio (J), from the integration of the curve ofqbio against time. When the biomass yield is considered, it ispossible to calculate the molar heat yield.

Batch and Fed-Batch Experiments

For calorimetric measurements, 1.6 L of the inorganicmedium enriched with glucose (between 27 and 111 mM)was used to perform the algae culture. The medium wassterilized in situ at 394.15 K and 0.2 MPa for 20 min or byfiltering with a 0.2 mm membrane before pouring it in thesterilized reactor. The temperature of the calorimeter wasmaintained at 298.15 K. The temperature of the plate headwas again 300.15 K and the temperature of the heat ex-

changer in the housing was 298.15 K. A stirring of 300 or 400rpm was fixed, with a constant flux of gas (2 L per minute).

Sampling of the culture medium was done periodically byusing an automatic lab-made Biosampler: with a pump forliquids, an air valve and a mechanic arm, sampling wasautomatically controlled with a LabView program in apersonal computer. The samples were kept at 0.58C untilthey were prepared for quantification of the concentrationsof biomass and metabolites.

A pH probe (Mettler-Toledo) and sometimes a pO2 probe(Ingold) in the reactor were used to follow the evolution ofthe medium conditions. As well as the housing for the pHprobe, the display and control unit for pH and pO2 was fromBioengineering AG. During the first experiments, the pHwas only monitored, but for the next experiments, adding adiluted aqueous solution of nitric acid also controlled it. Themass of acid added to the culture was followed with the on-line weight of a bottle containing the solution, measuredwith an electronic balance (Mettler-Toledo, PG5001-S).

The gas going out from the reactor was dried and heatedto 408C before going to a gas analyzer. A single infraredbeam (Servomex, 402) was used to quantify carbon dioxide,and a paramagnetic transducer (Servomex, 1100) was usedto quantify oxygen. A calculation of the oxygen and carbondioxide exchanged by the microalgae was performed fromon-line measurements.

All the signals are read on-line through a board (NationalInstruments, CB-68LP) connected to a PC, or through aFieldPoint interface (National Instruments) using a Lab-View program developed previously in our laboratory forautomatic data acquisition. After all signals were stable forat least 2 h, inoculation was made with a volume around100 mL of the cultures conserved in stock. Just before andafter inoculation, and during all the experiment, samplesfrom the reactor were taken to analyze the biomass, glucose,nitrate, and phosphate concentrations.

A number of different experiments were performed toimprove the conditions of algal growth and the correspond-ing calorimetric measurements. The three metabolic modeswere tested: heterotrophic, autotrophic, and mixotrophic.

The heterotrophic and mixotrophic conditions werechosen to accelerate the biomass growth by using glucose inthe medium. A second addition of medium with glucose(fed-batch) was sometimes tested in order to obtain biggercell concentrations. The autotrophic growth, much slower,was only tested just after one of the other modes, whenglucose was over and the biomass concentration was highenough 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 heattransfer coefficient UA was determined before and after eachexperiment. For similar conditions, the UA values remained



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 Tbox

(thin line), and in the laboratory Tlab (dashed line). The influence of Tlab can be

observed on Tbox and DT. b: It shows the corrected heat-flow signal qf (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 qf¼ 0.

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.

constant around an average of UA ranging between 6 and7 W �K�1.

A typical baseline of DT as a function of time is presentedin the graph of Figure 2a. The temperatures in the housingTbox and in the laboratory Tlab were also graphed. From agraphic of DT as a function of Tlab, a linear correlation couldbe obtained to have the constant k, which was applied ascorrection for the qf (Eq. 3) as showed in Figure 2b for thesame data. After applying a moving average over every 100points, a smoothing of the signal could be obtained with afinal deviation of less than �20 mW � L�1 during the periodof the experiment.

The values of the heat-flux related to stirring, qs, werecalculated at different angular velocities of the stirrer,assuming qs¼ 0 for null agitation. Figure 3 represents thesevalues for two independent series of experiments. For everyangular velocity, there was also a constant torque valuerelated to qs in Equation (4).

On the other hand, the use of the two optical filters for thexenon light to avoid infrared and ultraviolet radiationsbefore coming into the reactor diminished the heatingsignificantly due to the absorption of light. The heat-flux byillumination ql using these two filters was calculated fromdifferences in DT with the lamp turned on and off. The


obtained values from experiments were ql¼ (5.1� 4.1)mW � L�1 with medium and ql¼ (9.9� 2.1) mW � L�1 with ablack ink aqueous solution. The latter with black inkrepresented the maximum possible value of light absorbancefor the system, with a negligible contribution to the totalheat-flux when the xenon lamp was used. On the contrary,the use of the fluorescent lamp represented a much biggercontribution, although in a very constant value:ql¼ (139� 15) mW � L�1 with medium and ql¼ (276� 9)mW � L�1 with black ink solution. In both cases, with xenonand fluorescent lamps, ql for the medium was near 50% of qlfor the dark ink solution.

Batch and Fed-Batch Experiments

More than 10 experiments were performed. Since everyexperiment was different from the others, it is not possible tocompare the results directly. However, some trends could beobserved, principally to distinguish among the differentmetabolic modes. Typical behavior of Chlorella growth isshowed with some examples in next lines, and comparativetables are also presented at the end of this section.

For the mixotrophic and heterotrophic growths, a typicalevolution of biomass and glucose concentrations with timecould be followed. Figure 4 shows an example withheterotrophic conditions. A logarithmic-growth regionwas observed, and it was possible to determine the specificgrowth rate of the strain. The yield of biomass per glucoseamount could also be computed from the total fed glucose.In some cases, when the glucose was totally consumed, aschecked by HPLC, new fresh medium was immediatelyadded to continue with the growth. After the mixotrophicgrowth, a slight autotrophic growth of biomass couldsometimes be detected without addition of the glucose. It isimportant to note that a long lag time was detected many

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Figure 4. Biomass (crosses), glucose concentration (circles), and pH (line) with

time for heterotrophic growth of Chlorella.

times before reaching a significant cell growth. This lag timewas related to the adaptation between different metabolicconditions, since it was noted that the time could be reducedat the beginning of the experiments with previousadaptation to the corresponding metabolic mode justbefore inoculation.

As seen in Figure 4, the evolution of the pH during thealgae growth was very characteristic. Contrary to the typicalpH drop during growth of heterotrophic microorganisms,here, the pH increased along the biomass growth for thethree possible metabolisms. In fact, a typical correlation ofbiomass formation with the pH rise was found, as can beseen in Figure 5, for the heterotrophic and mixotrophicgrowths.

In both heterotrophic and mixotrophic conditions,besides the pH increase, there was a significant diminution

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.

of the nitrate concentration in the medium, as seen by theHPLC analysis. The phosphate concentration was alsoreduced, but in a smaller proportion. When the glucoseconcentration was almost over, two non-identified meta-bolites appeared, as detected by the HPLC analysis. Thesemetabolites were consumed once the glucose in the mediumwas over.

In order to fix the pH value as well as to avoid the lack ofnitrate in themedium, an aqueous solution of nitric acid wasadded automatically to the reactor. As it can be seen inFigure 6 for mixotrophic conditions, a correlation was alsofound between the biomass concentration and the quantityof nitric acid added to the medium. Moreover, the yield ofbiomass per nitrogen consumption can be computed from alinear regression.

The oxygen and carbon dioxide exchanges were calculatedduring the growth process. Because gas going into thereactor was previously humidified, water content was thesame for the gas going out of the reactor. It would bepossible to obtain the corresponding yield from the oxygenuptake and the carbon dioxide expel rates. For theheterotrophic growth, the carbon dioxide expel was evident,although the oxygen uptake rate was not easily quantified,since the small measured variations during the growth aremixed up at the level of the corresponding noise. For themixotrophic growth, the tendencies for the oxygen uptakeand the carbon dioxide extent were not clearly defined. Afterglucose oxidation, some mixotrophic conditions werechanged to just autotrophic growth. In this case, a slightcarbon dioxide uptake was detected.

The pO2 signal was useful to follow the metabolic activityin all the culture modes. Consumption of glucose is relatedwith a diminishing of oxygen dissolved in the medium. Afterglucose extinction, the level of pO2 returned around thebasal value. In addition, for the autotrophic growth, a faintproduction of oxygen could be detected with a continuousincrease in the oxygen dissolved in the medium.

Figure 6. Determination 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.



Figure 7. A comparison of the heat-flux from metabolic activity, qbio (continuous

line), with the changes in glucose concentration (squares) and dissolved oxygen

(dashed line), during mixotrophic growth conditions.

The heat-flux from metabolic activity qbio was also arepresentative signal of the algal growth, as showed inFigure 7 where the on-line measurement for qbio productionis in relation to the glucose consumption and thecorresponding pO2 signal.

During the lag time, before a significant glucose uptake, aslight drop of qbio was frequently observed. After the totalglucose consumption, in heterotrophic conditions, the totalheat production remained constant. However, when lightwas on, now in autotrophic conditions, an immediateconsumption of heat could always be observed. Especiallywith the fluorescent lamp, the consumption of qbio wasremarkable from lag time to total glucose uptake. A contrastbetween heterotrophic and mixotrophic conditions isshowed in Figure 8 for the integrated valued of qbio, thetotal biological heat exchange Qbio. With these results, the

Figure 8. Differences of the total biological heat exchange, Qbio, 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 Qbio ¼�70 kJ � L�1.


heat yield per biomass formed can be calculated at differentstages of the cellular metabolism for every experiment.

As a compilation of the different experiments, Table Ishows a variety of the conditions and results for each of thebatch and fed-batch experiments. Mean values werecomputed in order to remark the differences among thethree growth modes and even between the two lamps. Byinstance, the growth specific rate, m, is the smallest for theautotrophic mode, but the maximum for the mixotrophicmode. It is possible to observe that the biomass yield is notfavored when the fluorescent lamp was used. A discussionabout the differences in the heat yields is widely presentedlater on.

Some test experiments were additionally performed inorder to check the utility of performing on-line measure-ments with dielectric and infrared spectroscopies. Fordielectric measurements, the differences of the conductanceat two frequencies were correlated with the off-line biomassdetermination. Figure 9 shows one of the best examples forthis experimental correlation. As it can be seen, a linearcorrelation was found only at biomass concentrations below1 g � L�1. For higher biomass concentrations, the con-ductance in the medium seems to remain constant.

From infrared experiments, the profiles of the mostimportant metabolites in the medium broth (glucose,nitrate, and phosphate) were obtained during heterotrophicgrowth of algae (Fig. 10). A good agreement of these profileswas found with the values measured off-line by HPLC. Itshould be noted, however, that for each experimental batcha previous calibration with the matrix of standard solutionsis fundamental for the success of this analytical method.

Discussion

The total chemical process for the Chlorella vulgaris growthcould be regarded in a general simplified set of equations asfollows:

2:45 CH2Oþ 0:09 NO3� þ 1:07 O2

¼ CH1:76O0:35N0:09 þ 1:45CO2 þ 1:52H2O

þ 0:09 OH� (5)

2 H2Oþ 2 NADPþ ¼ O2 þ 2 NADPHþ 2 Hþ (6)

CO2 þ 2 NADPH þ 2 Hþ

¼ CH2Oþ 2 NADPþ þH2O (7)

Equation (5) represents normal metabolic growth asgenerally described for heterotrophic organisms. An externalsource of carbohydrates (CH2O) is used as the source ofchemical energy for biosynthetic reactions that lead to theformation of new biomass. The process is not totallyefficient, and some of the energy is lost as heat and

DOI 10.1002/bit

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 —

x8 h 85� 13 0.36� 0.06 12.0� 0.6 �495� 17

x a 6� 1 — — 381� 81

x m 111� 23 0.63� 0.15 — �215� 26

x m* �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*, mixotrophicwith fluorescent lamp; (2) glucose substrate; (3) biomass product as dry weight; (4) growth specific rate; (5) molar yield of biomass from glucose; (6) molaryield of biomass from nitrate; (7) heat yield per molar biomass; (8) mean values with uncertainties being the standard deviation of the mean.

production of carbon dioxide. In the case of Chlorella, thechemical composition of biomass was taken from previousreports for microalgae (Duboc et al., 1999). It should beremarked that this consideration is approximate, since thechemical composition of the microalgal cells may vary formixotrophic, heterotrophic, and autotrophic growth,although the differences cannot be very large. Unfortunately,the elemental analysis of the biomass produced in everyexperiment was not determined. The stoichiometriccoefficient for carbohydrates was computed from theaverage value for the batch experiments with the hetero-

Figure 9. The differences of capacitance DC at two measurement frequencies

(0.21 and 5.75 MHz) correlated with the off-line measurements of biomass.

trophic growth from the glucose. The nitrogen balance waseasily computed from the experiments where the pH wasfixed by addition of diluted nitric acid, and correspondsperfectly to the value for the elemental composition ofmicroalgae. The other coefficients were added to completethe total balance. As it was said before, the exchange ofoxygen and carbon dioxide during the growth was not veryclear and it was useless to include them in the balances.Equation (5) also represents the experimental observationfor the consumption of nitrates and the rising of the pH inthe medium along the microbial growth. It should be noted

Figure 10. On-line concentrations of glucose, nitrate, and phosphate as com-

puted from infrared spectra in the broth for heterotrophic growth.



that potassium, phosphorous, and magnesium are alsoimportant in the composition of biomass, but theseelements could be ignored in this work since they have asmall contribution compared with the others elements in theformula (Mandalam and Palsson, 1998).

A simplified description of photosynthesis can be seen inEquations (6) and (7). Equation (6) corresponds to aprocess known as the light phase, in which photons fromlight are absorbed to keep the corresponding energy as ATPmolecules, needed for the second process called dark phase,and represented by Equation (7). In order to get the balancesof charge and hydrogen in both equations, the nicotineadenine dinucleotide phosphate (NADPH) and the corre-sponding oxidized molecule are presented. However, it iseasy to find that the addition of these two reactions gives asimplified equation in which carbon dioxide and waterparticipate to be transformed in oxygen and carbohydrates.It should be remarked that the production of ATP wasomitted in the first equation, as well as the consumption ofATP in the second reaction. This is because the balance ofATP is kept in the total photosynthetic process. Actually,energy from light photons is not kept by ATP molecules, buttransformed to the so-called chemical energy stored ascarbohydrate molecules.

Nevertheless, as showed before, the glucose is not storedbut consumed during the Chlorella growth. The hetero-trophic and autotrophic growth modes were always relatedto external glucose transformation through exothermicglobal processes, as expected from results of previous reportsfor non-autotrophic microorganisms (von Stockar and Liu,1999; von Stockar et al., 2006). A clear difference is observedbetween these two modes when released heat is compared:the heterotrophic growth has more energetic heat yields thanthe mixotrophic growth. This can be reasonably understoodwhen the only two autotrophic growth experiments areobserved to be markedly endothermic. In this sense, theheterotrophic growth, as represented by Equation (5),releases heat, while the autotrophic growth, as representedby Equations (5), (6), and 7), absorbs it. It is concluded thatEquations (6) and (7) together are clearly endothermic asexpected from previous thermodynamic results (Johanssonand Wadso, 1997). Moreover, the mixotrophic growth hasintermediate values of energy between the heterotrophic andthe autotrophic growths, with a final value depending onwhich of both modes is favored. By instance, when thefluorescent lamp was used, the increment in light intensity(compared with the Xe lamp illumination) allowed anendothermic mixotrophic growth because photosynthesiswas significantly favored.

Even finer details can be obtained with the heat yields:between the batch and fed-batch mixotrophic growth, thesecond process releases more energy than the former. Thiscould be explained if an additional exothermic process isconsidered in addition to the heterotrophic growth and thephotosynthesis. This process may be related to death of cells,and it could be crudely represented as the combustion ofbiomass 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 connectingall the processes in a unique cellular process, but thesimplified model used here helps to understand a linkbetween the biological process and the correspondingmeasurements during the microalgae growth in thebioreactor.

Although numerous works have been reported in relationto the growth of Chlorella vulgaris and other microalgae,very little is found about the metabolic differences betweenthe autotrophic, heterotrophic, and mixotrophic growthmodes. A wide variety of techniques were tested here inorder to follow and understand the algal growth, beingsignificantly useful. Traditional techniques were combinedwith some others presented for the first time in a study ofmicroalgae, being meaningful to distinguish three differentmetabolic modes: (i) the calorimetry, to detect whenphotosynthesis is favored; (ii) the nitric acid pH regulation,to follow the kinetics of growth; and (iii) the on-line infraredspectroscopy, to follow the changes of nutrients in the broth.

Rodrigo Patino thanks Conacyt-Mexico and EPFL-Switzerland for the

stipendium as postdoctoral researcher.

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