A closed solar photobioreactor for cultivation of microalgae undersupra-high irradiance: basic design and performance

J. Masojídek1,2,*, Š. Papácek1,4, M. Sergejevová1, V. Jirka3, J. C{ervený1, J. Kunc1,Korecko3, O. Verbovikova1, J. Kopecký1,2, D. Štys1,2 and G. Torzillo5

1Institute of Physical Biology, University of South Bohemia, Zámek 136, Nové Hrady, CZ-37333, CzechRepublic; 2Department of Autotrophic Microorganisms, Institute of Microbiology, Academy of Science,Opatovický mlýn, Trebon, CZ-379 81, Czech Republic; 3ENKI public benefit corporation, Dukelská 145,Trebon, CZ-379 82, Czech Republic; 4Faculty of Mechatronics and Interdisciplinary Engineering Studies,Technical University of Liberec, Liberec, CZ-461 17, Czech Republic; 5Sezione di Firenze, Istituto per loStudio degli Ecosistemi del CNR, Sezigue di Firenze, Piazzale delle Cascine 28, Firenze, I-50144, Italy;*Author for correspondence (e-mail: masojidek@alga.cz; phone: +420-384-721831; fax: +420-384-721246)

Received 1 July 2002; accepted in revised form 17 February 2003

Key words: Chlorophyll fluorescence, Cyanobacterium, Spirulina, Heat exchange, High irradiance, Linear Fresnellenses, Non-photochemical quenching, Photoinhibition, Productivity

Abstract

An account is given of the setting up and use of a novel type of closed tubular photobioreactor at the Academicand University Centre in Nove Hrady, Czech Republic. This �penthouse-roof� photobioreactor was based on so-lar concentrators (linear Fresnel lenses) mounted in a climate-controlled greenhouse on top of the laboratorycomplex combining features of indoor and outdoor cultivation units. The dual-purpose system was designed foralgal biomass production in temperate climate zone under well-controlled cultivation conditions and with surplussolar energy being used for heating service water. The system was used to study the strategy of microalgal ac-climation to supra-high solar irradiance, with values as much as 3.5 times the ambient value, making the ap-proach unique. The cultivation system proved to be fully functional with sufficient mixing and cooling, efficientoxygen stripping and light tracking. Experimental results (measurement of the maximum photochemical yield ofPSII and non-photochemical quenching) showed that the cyanobacterium Spirulina (= Arthrospira) platensis cul-tivated under sufficient turbulence and biomass density was able to acclimate to irradiance values as high as 7mmol photon m−2 s−1. The optimal biomass concentration of Spirulina cultures in September ranged between 1.2to 2.2 g L−1, which resulted in a net productivity of about 0.5 g L−1 d−1 corresponding to a biomass yield of 32.5g m−2 d−1 (based on the minimum illuminated surface area of the photobioreactor).

Abbreviations: DCMU – 3-(3,4-dichlorophenyl)-1,1-dimethylurea, DW – dry weight, Fo, Fv, Fm – minimum –variable and maximum fluorescence yields of dark-adapted cultures, Fv/Fm – maximum quantum yield of PSII,i.d. – inner diameter, NP – non-photochemical quenching, PAR – photosynthetically active irradiance, PSII –Photosystem II

Introduction

Since early studies on microalgal biotechnology, themain concern of outdoor mass cultivation has beenaimed at obtaining effective utilisation of high lightintensities (Burlew 1953). It is characteristic of most

photosynthetic organisms exposed to increasing lightintensity that photosynthesis increases linearly in thefirst part of the curve (light-limited region), and thatafter a gradual transition, the saturation level isreached, when most of the light starts to be dissipatedas heat. If the irradiance further increases, photoinhi-

239Journal of Applied Phycology 15: 239–248, 2003.© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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bition may occur. In many cases the photosyntheticactivity of microalgae becomes saturated at about 200µmol photon m−2 s−1, that is, approximately 1/10 ofthe maximum solar irradiance in summer. The occur-rence of the �saturation effect� is one of the most se-rious limitations for the efficiency of solar energytransformation by microalgae (Burlew 1953).

In dense cultures of microalgae the light intensityreaching the cells decreases with the thickness of cul-ture layer and, at a certain point, the light intensityjust equals the saturation intensity. All the light thatpenetrates deeper is absorbed/utilised with the maxi-mum efficiency, falling in the light-limited region ofthe growth curve. It is evident that the irradiance sat-urating the growth represents the most important fac-tor in determining the light utilisation efficiency foroutdoor cultures.

The limitations of growth imposed by the light-saturation and light-limitation effect has prompted al-gal biotechnologists to look for solutions to attenuatethis effect in outdoor cultures. Two practical strategieshave been proposed: (i) to increase population den-sity and the radial mixing of the cultures, and (ii) touse special designs of photobioreactors in order toimprove light utilisation efficiency by the culture.

The first approach has been pursued by Richmondand co-workers since the introduction of outdoor al-gal biotechnology in Israel (e.g., Richmond and Von-shak (1978)), particularly with the use of ultra-highcell densities of Spirulina and other microalgae in flatphotobioreactors (Hu et al. 1996). The second ap-proach to avoiding the saturation effect has focussedon the design of sophisticated photobioreactors,which promote the so-called �light dilution� (Burlew1953). Such photobioreactors have been designed inthe recent years (Torzillo et al. 1993; Tredici andChini Zittelli 1998; Pulz 2001). One possibility toimprove the performance of the photobioreactors is tooptimize the light/dark cycle of cells inside the tubelumen. This goal has been only partially reached, ei-ther by modifying the photobioreactor design or byintroducing static mixers inside the tubes (Carlozziand Torzillo 1996; Ugwu et al. 2002).

Due to the light saturation effect, information onthe behaviour of microalgal cultures exposed to veryhigh irradiance is relatively sparse and most of it hasbeen obtained in laboratory experiments. However,high irradiance is essential for inducing efficient syn-thesis of some metabolites in stressed cultures, suchas astaxanthin in Haematococcus pluvialis.

High mixing rates coupled with high biomass con-centrations have been indicated as the way to circum-vent the onset of the saturation effect in outdoor cul-tures. In such a way cells may experience lightintermittently, which has been considered essential to�dilute� strong irradiance for effective use by the cul-ture (Richmond 2000).

The main aim of the study reported here was toinvestigate acclimation and growth of cultures undervery high solar irradiance, as much as 2 to 4 timesambient values, while at the same time making use ofsurplus solar energy to warm service water for labo-ratory use and maintain cultures at night.

Materials and methods

Photobioreactor design

A photobioreactor was set-up at the Academic andUniversity Centre in Nové Hrady, Czech Republic(48.5° N, l14.5° E). This �penthouse-roof� photo-bioreactor was based on solar concentrators (linearFresnel lenses) and designed to combine features ofindoor and outdoor cultivation units to fulfil the fol-lowing features:

• Mounted in a closed, temperature-controlled en-vironment (e.g., rooftop greenhouse, see Fig-ure 1), which make it possible to extend culti-vation season in temperate climate zone formost of the year;

• Use of collectors concentrating solar irradiance(linear Fresnel lenses) into the cultivation sys-tem;

• Fully controlled cultivation parameters (e.g.,flow rate, irradiance, temperature, pH, dissolvedoxygen concentration) with sufficient mixingand effective heat and gas exchange;

• On-line monitoring of chlorophyll fluorescenceto evaluate the physiological status of the cul-ture.

Description of photobioreactor

Two units of the photobioreactor were mounted in aclimate-controlled greenhouse construction built ontop of the laboratory complex, with a southern expo-sure and a roof-angle of 40° (Figure 1). This construc-tion was covered by insulating double-glazing withlinear Fresnel lenses (6 panels of 9 m2 correspondingto one unit: Figure 1). The illumination system (Lin-

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ear Fresnel lenses) concentrates the direct componentof incident sunlight directly on glass cultivation tubes.The tubular loop system is placed on a movableframe, which enable automatic focusing of direct so-lar irradiance during the day. Each of the two photo-bioreactor units comprises of seven subsystems: (i)loop reactor, (ii) degasser, (iii) light supply, (iv)pump, (v) CO2 supply, (vi) measurement and control,and (vii) heat exchange. The main part of the cultiva-tion unit represents a 24-m loop system of 6 parallelhorizontal glass tubes (i. d. 48 mm), connected byglass U-bends with watertight flanges. The workingvolume of the photobioreactor is 65 L, of which 50 Lflow in the glass tubes and 15 L in the darkened partof the reactor (i.e., degasser, circulating pump anddark PVC connections). The minimum illuminatedarea of the reactor is about 1.2 m2. The loop is fixedto a metallic movable frame (Figure 2). A peristalticcirculation pump is connected to the inlet tube of theloop taking algal suspension from the degasser (aera-tion or retention tank, see Figure 2). It is coupled to afrequency inverter to vary the flow speed of suspen-sion.

The outlet from the loop in its highest position isconnected to the counter-flow heat exchanger. Then,the suspension flows back to the degasser, which isplaced in the lowest position of the cultivation sys-tem and can store the total volume of photobioreac-tor. The efficient gas exchange is supported by a tan-gential flow of algal suspension on the conic surfaceof the upper part of the degasser.

The heat exchange circuit represents a special partof the photobioreactor and guarantees efficient tem-

perature control (cooling/heating). It consists of accu-mulation reservoirs for service water that are con-nected to the secondary circuits of 2 heat exchangers(a double-jacket in the lower cylindrical part of thedegasser and the counter-flow cooler). The controlsystem of this circuit operates circulation pumps andelectromagnetic valves according to temperature sen-sors placed in various positions of the primary (cul-tivation) and secondary (temperature regulation) cir-cuits.

Linear Fresnel double-lenses, the basic element ofthe unique Solar Collectors SG-1 (SOLARGLASS™system, Glaverbel Czech Ltd., Jirka et al. (1999))transmit diffuse light and concentrate the direct com-ponent of solar radiation from the whole surface ofthe lens (0.75 × 2 m) into two linear bands of a cer-tain width (focal stripe) located about 500 mm underthe longitudinal axis of the lens (Figure 3). As the sunchanges its position, an automatic tracking system(light sensors and a servo-drive) keeps glass tubes inthe focus of the collector lenses by movement of theframe (Figure 3). According to the measurements inthis paper, the glass tubes with algal suspension(placed in the focal stripe) can receive irradiance be-tween 2 to 7 mmol photon (PAR) m−2 s−1 for most ofthe diel cycle.

Pure carbon dioxide is supplied into the suspen-sion before the inlet to the circulation pump accord-ing to the pH value. Alternatively, sodium bicarbonatewas also added to growth medium.

The sensors allow measurement of solar irradi-ance, temperature (in the cultivation system and in thesecondary circuit of the heat exchangers), pH value,

Figure 1. Greenhouse construction of a solar photobioreactor built on top of the laboratory complex, with a southern exposure and a roof-angle of 40°. The construction is covered by insulating double-glazing with linear Fresnel lenses of 18 m2 for two separate units (9 squaremeters of area correspond to one unit, i.e., 6 panels of linear Fresnel lenses with dimensions 0.75 m × 2 m, see crossed area in this figure).

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dissolved O2 concentration and variable chlorophyllfluorescence.

More technical and operation details of the dual-purpose photobioreactor were shown at the World Re-newable Energy Congress in Cologne, Germany(Jirka et al. 2002). The costs of one unit (about 9square metres) of this experimental photobioreactor,which includes 9 m2 of Fresnel lenses, 24 m of glasstubes, a metallic frame holding tubes, plastic hoses,light tracking system, degasser, heat exchanger, peri-staltic pump, and sensors (pH, light and temperature)with a control box were about 8,700 Euro.

Organisms and culture conditions

Spirulina platensis strain M2 (culture collection ofIstituto per lo Studio degli Ecosistemi, CNR, Firenze,Italy) was used as a test organism. The strain appearsto be relatively resistant to high irradiance. The cul-tures were grown at 35 ± 1 °C and circulated at 0.2m s−1, which corresponded to a Reynolds number of5000. Biomass concentration ranged between 0.5 and2.5 g L−1. Dry weight was determined in duplicate25-mL samples using GF/C glass microfibre filters(Whatman International Ltd, England). The cultureswere operated in a semicontinuous regime. Eachmorning, the addition of fresh Zarrouk’s medium di-luted the culture. The pH of the culture was kept at9.4 by automatic addition of pure CO2. Average dis-

Figure 2. Inside view of two units of a penthouse-roof photobioreactor based on solar concentrators (linear Fresnel lenses). The main partsare indicated: movable frame, heat exchanger, cultivation tubes, degasser, pump, sensors.

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solved oxygen concentration was between 10 and 15mg L−1. Most measurements were carried out be-tween 08:00 and 18:00 h (GMT + 1).

Chlorophyll fluorescence measurements

The steady-state, fluorescence level was recorded insitu using solar irradiance as actinic light, placing thefibre-optic light guide of a pulse-amplitude-modula-tion fluorimeter (PAM 101–103, H. Walz, Germany)directly in a holder on glass tubes of the photobiore-actor (Figure 4). Then, a single, high-intensity flash(> 15 mmol photon m−2 s−1, 0.8 s in duration) wasapplied to raise fluorescence yield to the Fm� value(the maximum fluorescence yield in the light-adaptedstate). The effective photochemical quantum yield of

PSII, �F/Fm� = (Fm� − F)/Fm�, was measured (Gentyet al. 1989). Non-photochemical quenching coeffi-cient (NPQ) was calculated as the Stern-Volmerquenching coefficient NPQ = Fm/Fm� − 1 (Bilger andBjörkman 1990).

The maximum quantum yield of PSII, Fv/Fm wasdetermined in dark-adapted (10 min) culture samplestaken from photobioreactors. The fibre-optic lightguide was placed close to a stirred cuvette (the ED-101US unit). Minimum fluorescence Fo was mea-sured by modulated light (< 0.3 µmol photon m−2 s−1)from a light-emitting diode (peak wavelength at 655nm). In case of cyanobacteria, DCMU has to be usedunder continuous illumination (0.2 mmol photon m−2

s−1) to achieve Fm. The nomenclature of chlorophyll

Figure 3. View of Fresnel lenses focusing solar irradiance on cultivation tubes. An insert figure shows the design and dimensions of the lenspanel (SOLARGLASS™ system, Jirka et al. (1999)).

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fluorescence parameters follows Van Kooten and Snel(1990).

All measurements were repeated during severaldays; then one typical experiment was taken for pre-sentation in figures.

Light and oxygen concentration measurement

Photosynthetically active radiation (PAR) was mea-sured with the LI-190SA quantum sensor (cosine cor-rected up to 80° angle of incidence) coupled to theLI-250 Light Meter (Li-Cor, USA).

A pH/Oxi340i meter and electrodes (WTW, Ger-many) were used to control pH and oxygen concen-tration.

Results

Several series of experiments using Spirulina platen-sis were carried out in the tubular photobioreactor inMarch and September 2002, mostly on sunny days,when the maximum ambient irradiance peaked be-tween 1.5 and 1.8 mmol photon m−2 s−1. The irradi-ance condensed to the cultivation tubes by linearFresnel lenses was higher than 2 mmol photon m−2

s−1 between 1000 and 1700 h and at its maximum (at12:00 h) reached almost 7 mmol photon m−2 s−1. Theshape of the irradiance curves created by Fresnellenses is given by their construction (Figure 5A, 6)with the maximum at noon and several minor maximain the afternoon. The construction of this illuminationsystem allows reaching irradiance intensities, which,as far as we know, has never been tested outdoors.

In the first series of experiments, acclimation oflaboratory cultures to high irradiance was tested forthree successive days (Figure 5). The biomass con-centration during the cultivation ranged between 1and 3 g L−1. Changes of photochemical activities andgrowth were followed as the Fv/Fm ratio and biomassconcentration. In �healthy� cultures, the Fv/Fm ratioreached about 0.6 in the morning. If this value waslower, the culture was not in a good physiologicalcondition. The decrease of this parameter during thediel cycle reflects when the culture experiences highirradiance stress.

During Day 1 of exposure to high light, the Fv/Fm

ratio decreased to 0.47 at mid-afternoon and the re-covery towards the evening was rather slow. On Day2, the decline of the Fv/Fm ratio was much lower thanthat observed on Day 1. The culture recovered com-pletely overnight as Fv/Fm in the morning of Day 3

Figure 4. In situ measurement of chlorophyll fluorescence characteristics using a fibreoptic guide of a pulse-amplitude-modulation fluorom-eter (PAM 101-103, H. Walz, Germany).

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reached 0.59. On Day 3, the decline of the Fv/Fm ra-tio was less than 10% of the morning value, indicat-

ing that the culture had fully acclimated to high lightconditions. The time course of irradiance measuredon the tube surface (condensed) and the ambientvalue on a horizontal roof surface during a sunny dayin September is shown in Figure 6. It demonstratedthat Fresnel lenses allowed about a 3.5-fold increaseof irradiance intensity on the tube surface in the mid-dle of day.

The second series of experiments was carried outto assess the optimal biomass concentration for Spir-ulina in the photobioreactor. The biomass concentra-tion tested ranged from 0.7 to 2.3 g L−1 dry weight.Optimal biomass concentration was achieved whenthe morning dry weight was set at about 1.2 g L−1,while a further increase in biomass concentration didnot improve productivity significantly (Figure 7). Inorder to get a better evaluation of growth during thewhole daylight cycle, both the productivity obtainedduring the daylight period (gross productivity) andthe one referred to a 24-h period (net productivity)were measured. Gross productivity was almost con-stant over a wide range of concentrations (0.7 to 2.3g L−1). However, when the cultures were grown atlower biomass concentration (0.7 g L−1), up to 46%biomass synthesised during the day was lost at night(Figure 7). At 1.2 and 2.3 g L−1 the loss decreased to5.8% and 1.4%, respectively (results not shown).

In order to obtain better understanding of the pho-toadaptation behaviour of Spirulina under supra-opti-

Figure 5. Daily course of irradiance reaching the cultivation tubesof the solar photobioreactor between 08:00 and 18:00 h in March(panel A). Diel changes of the Fv/Fm ratios were recorded during 3subsequent days in the same time period (panel B).

Figure 6. Daily course of the ambient irradiance measured on thehorizontal roof (dashed curve) and the irradiance focused on thecultivation tubes of the solar photobioreactor between 09:00 and17:00 h in September.

Figure 7. Gross (daylight) and net (24 h) productivities of Spir-ulina platensis cultures grown in the solar photobioreactor with thebiomass concentration of 0.7, 1.2 and 2.3 g L−1 DW.

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mal light intensities, measurements of photochemicalactivities were performed using chlorophyll fluores-cence. The daily time course of the Fv/Fm ratio wasmeasured at time intervals during the day in Spirulinacultures having different biomass concentrations (Fig-ure 8A). In all the cultures, a decline of the Fv/Fm ra-tio was observed in the middle of the day, correspond-ing with the highest light intensities. The Fv/Fm

decline increased when the cultures were grown atlower biomass concentrations (by about 38% in theculture at 0.7 g L−1), while it was much lower (by16%) at the highest concentration tested. It was con-cluded that cultures grown at a biomass lower than1.2 g L−1 can experience significant photoinhibitionof PSII evaluated as the Fv/Fm decline. Analysis ofnon-photochemical quenching clearly confirmedthese observations since the cultures grown at lowerbiomass concentrations developed a very high levelof non-photochemical quenching as compared withthe dense cultures. It indicated that the low-biomasscultures were subjected to high irradiance stress (Fig-ure 8B).

Since the cultures were exposed to very high irra-diance, tests were carried out on the effect of in-creased turbulence. Cultures (2 g L−1 biomass con-centration) were compared with circulation atvelocities of 0.2 and 0.5 m s−1, which correspond toReynolds numbers of 5000 and 12,500. There was nodifference in gross or net productivity (results notshown) nor apparently was there any increased frag-mentation of the filaments.

Discussion

For the first time, a multifunctional photobioreactorhas been assembled within the laboratory complex ofa research campus. The intention was to evaluate thefunction and usability of this integrated collector-pho-tobioreactor system, which employed solar concentra-tors under the climatic conditions of central Europe.Besides the production of algal biomass and the heat-ing of service water, this solar photobioreactor can beused as an experimental tool for basic photosynthesisresearch.

Our experiments with Spirulina have shown that itis possible to operate cultures under supra-high irra-diance, provided the biomass concentration is high.The cyanobacterium showed a very high ability toadapt to high light conditions through the develop-ment of high non-photochemical quenching capabil-

ity. Even the low-biomass cultures were able to copewith high irradiance by developing a high level ofnon-photochemical quenching, which can be consid-ered a good parameter to assess the ability of an or-ganism to adapt to stress. The nature of this quench-ing capability should be further investigated.

The Fv/Fm ratio represents another parameter indi-cating the status and behaviour of microalgal culture,which is easy to record. The experience gathered witha number of experiments have demonstrated that adecrease of Fv/Fm between 20 and 25% during theday can be still considered physiological, since it oc-curred even in dense Spirulina cultures (Torzillo et al.1996; Richmond 2000).

The biomass yields obtained during Septemberwith the photobioreactor described in this paper wereby about 30% higher than the ones described earlier(Torzillo et al. 1991) for a tubular photobioreactorhaving similar basic characteristics (e.g., tube diam-eter, volume and illuminated surface), but without thepossibility to increase the intensity of solar irradiance

Figure 8. Diurnal changes in the Fv/Fm ratio (panel A) and thenon-photochemical quenching coefficient NPQ (panel B) in Spir-ulina cultures grown at different biomass concentrations (0.7, 1.2and 2.3 g DW L−1) in the solar photobioreactor.

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reaching the surface of tubes. In the photobioreactordescribed here, the maximum productivity reached0.5 g L−1 d−1 corresponding to an area yield (basedon the minimum surface area) of about 32.5 g m−2 d−1

which is a relatively high value if we consider that itwas obtained in the month of September. Hence, al-though the photobioreactor has been constructed toinduce the synthesis of valuable compounds understress conditions, the possibility to use it for normalbiomass production at high cell densities is not pre-cluded.

Optimal biomass concentration of the cultureranged between 1 and 2 g L−1 demonstrating that withSpirulina the range of optimal biomass concentrationis not very restricted. Yet, the possibility to use densercultures is desirable since it allows a reduction ofharvesting costs.

In this photobioreactor, the increase of culture flowrate did not have any effect on the productivity. Thesefindings confirmed previous observations with Spir-ulina in which further increase in the turbulent regionof the flow do not produce further increase in produc-tivity (Carlozzi and Torzillo 1996). However, our re-sults contrast with those obtained by Hu et al. (1996)in a flat-panel photobioreactor. Most likely the muchlonger light path used in our case could prevent thebetter exploitation of high light. As a matter of fact,short light path represents an important condition toachieve intermittent light regime.

The results on the night biomass loss are in agree-ment with those of Torzillo et al. (1991) who reportedthat the biomass concentration strongly influence thisparameter. It was concluded that the night biomassloss depended on the temperature and irradiance ex-perienced by the cells in the preceding day, due tosignificant effect of both temperature and irradianceon the cell composition. Spirulina cells exposed to ahigh irradiance direct biomass synthesis towards car-bohydrates, which can be used as a sink to store anexcess of reducing power. However, during night car-bohydrates are lost due to dark respiration, thus in-creasing the biomass loss during night (Torzillo et al.1991).

One weak point of the photobioreactor is the factthat a considerable part of the culture is kept in thedark and this �dilutes� the increase in biomass con-centration obtained in the illuminated section. Thisproblem, common to most of tubular photobioreactorprototypes, can be avoided by increasing the unit sizeand a partial change of the set-up in the dark sectionof the photobioreactor (in progress now).

At present it is difficult to make any reasonablecalculation of biomass production costs, since weplan to improve further the photobioreactor perform-ance. However, it is clear that this photobioreactordesign represents a valuable instrument for basic andapplied research, especially for investigating the be-haviour of microalgal cultures under high irradiance.Furthermore, the light concentrating system makes itpossible to extend considerably the cultivation sea-son, and thus is promising for cultivation of microal-gae in the northern parts of Europe. At the momentvertical Fresnel lenses are also being developed(project No.103/99/0562 �Development of the Ener-getic Facade using Glass Optical Rasters�, GrantAgency of the Czech Republic), which can bemounted in a building wall.

Acknowledgements

This research was supported by projects LN00A141�Mechanism, Ecophysiology and Biotechnology ofPhotosynthesis�, and No. 020001 �Solar Energetic ofNatural and Technological Systems� of the Ministryof Education of the Czech Republic. A partial fund-ing of travel expenses was provided in the frameworkof the Bilateral Scientific Agreement between the Na-tional Research Council of Italy and the Academy ofSciences of the Czech Republic.

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