Enhanced photosynthetic activity in Spinacia oleracea by spectral modification with a photoluminescent light converting material

The spectral conversion of incident sunlight by appropriate photoluminescent materials has been a widely studied issue for improving the efficiency of photovoltaic solar energy harvesting. By using phosphors with suitable excitation/emission properties, also the light conditions for plants can be adjusted to match the absorption spectra of chlorophyll dyes, in this way increasing the photosynthetic activity of the plant. Here, we report on the application of this principle to a high plant, Spinacia oleracea. We employ a calcium strontium sulfide phosphor doped with divalent europium (Ca0.4Sr0.6S:Eu, CSSE) on a backlight conversion foil in photosynthesis experiments. We show that this phosphor can be used to effectively convert green to red light, centering at a wavelength of ~650 nm which overlaps the absorption peaks of chlorophyll a/b pigments. A measurement system was developed to monitor the photosynthetic activity, expressed as the CO2 assimilation rate of spinach leaves under various controlled light conditions. Results show that under identical external light supply which is rich in green photons, the CO2 assimilation rate can be enhanced by more than 25% when the actinic light is modified by the CSSE conversion foil as compared to a purely reflecting reference foil. These results show that the phosphor could be potentially applied to modify the solar spectrum by converting the green photons into photosynthetically active red photons for improved photosynthetic activity. © 2013 Optical Society of America OCIS codes: (160.2540) Fluorescent and luminescent materials; (350.5130) Photochemistry; (350.6050) Solar energy; (170.1420) Biology References and links 1. L. Taiz and E. Zeiger, “Photosynthesis: the light reactions,” in Plant Physiology (Sinauer Associates, Inc., 2006), pp. 126–158. 2. N. R. Bulley, C. D. Nelson, and E. B. Tregunna, “Photosynthesis: action spectra for leaves in normal and low oxygen,” Plant Physiol. 44(5), 678–684 (1969). 3. J. B. Clark and G. R. Lister, “Photosynthetic action spectra of trees: I. Comparative photosynthetic action spectra of one deciduous and four coniferous tree species as related to photorespiration and pigment complements,” Plant Physiol. 55(2), 401–406 (1975). 4. K. J. McCree, “The action spectrum, absorptance and quantum yield of photosynthesis in crop plants,” Agric. Meteorol. 9, 191–216 (1972). 5. K. Inada, “Action spectra for photosynthesis in higher plants,” Plant Cell Physiol. 17, 355–365 (1976). 6. A. Andersen, “Comparison of fluorescent lamps as an energy source for production of tomato plants in a controlled environment,” Sci. Hortic. (Amsterdam) 28(1-2), 11–18 (1986). #188681 $15.00 USD Received 11 Apr 2013; revised 30 Jul 2013; accepted 31 Jul 2013; published 12 Sep 2013 (C) 2013 OSA 4 November 2013 | Vol. 21, No. S6 | DOI:10.1364/OE.21.00A909 | OPTICS EXPRESS A909 7. N. G. Bukhov, I. S. Drozdova, V. V. Bondar, and A. T. Mokronosov, “Blue, red and blue plus red light control of chlorophyll content and CO2 gas exchange in barley leaves: Quantitative description of the effects of light quality and fluence rate,” Physiol. Plant. 85(4), 632–638 (1992). 8. J. Ernstsen, I. E. Woodrow, and K. A. Mott, “Effects of growth-light quantity, growth-light quality and CO2 concentration on Rubisco deactivation during low PFD or darkness,” Photosynth. Res. 61(1), 65–75 (1999). 9. K. Humbeck, B. Hoffmann, and H. Senger, “Influence of energy flux and quality of light on the molecular organization of the photosynthetic apparatus in Scenedesmus,” Planta 173(2), 205–212 (1988). 10. N. G. Bukhov, I. S. Drozdova, and V. V. Bondar, “Light response curves of photosynthesis in leaves of sun-type and shade-type plants grown in blue or red light,” J. Photochem. Photobiol. B 30(1), 39–41 (1995). 11. H. Yu and B. Ong, “Effect of radiation quality on growth and photosynthesis of Acacia mangium seedlings,” Photosynthetica 41(3), 349–355 (2003). 12. G. D. Goins, N. C. Yorio, M. M. Sanwo, and C. S. Brown, “Photomorphogenesis, photosynthesis, and seed yield of wheat plants grown under red light-emitting diodes (LEDs) with and without supplemental blue lighting,” J. Exp. Bot. 48(7), 1407–1413 (1997). 13. G. Tamulaitis, P. Duchovskis, Z. Bliznikas, K. Breive, R. Ulinskaite, A. Brazaeityte, A. Novickovas, and A. Zukauskas, “High-power light-emitting diode based facility for plant cultivation,” J. Phys. D Appl. Phys. 38(17), 3182–3187 (2005). 14. J. W. Heo, K. S. Shin, S. K. Kim, and K. Y. Paek, “Light quality affects in vitro growth of grape 'Teleki 5BB',” J. Plant Biol. 49(4), 276–280 (2006). 15. S. Lian, C. Li, X. Mao, and H. Zhang, “H. “On application of converting green to red of CaS:Eu in agriculture,” Chin. Rare Earths. 23, 37–40 (2002). 16. L. Ma, D. Wang, Z. Mao, Q. Lu, and Z. Yuan, “Investigation of Eu–Mn energy transfer in A3MgSi2O8:Eu, Mn A=Ca,Sr,Ba for light-emitting diodes for plant cultivation,” Appl. Phys. Lett. 93(14), 144101 (2008). 17. G. Blasse and B. C. Grabmaier, Luminescent Materials (Springer, 1994). 18. G. Gao, S. Reibstein, M. Peng, and L. Wondraczek, “Tunable dual-mode photoluminescence from nanocrystalline Eu-doped Li2ZnSiO4 glass ceramic phosphors,” J. Mater. Chem. 21(9), 3156–3161 (2011). 19. G. Gao, N. Da, S. Reibstein, and L. Wondraczek, “Enhanced photoluminescence from mixed-valence Eu-doped nanocrystalline silicate glass ceramics,” Opt. Express 18(S4 Suppl 4), A575–A583 (2010). 20. P. F. Smet, I. Moreels, Z. Hens, and D. Poelman, “Luminescence in sulfides: a rich history and a bright future,” Mater. 3(4), 2834–2883 (2010). 21. Q. Xia, M. Batentschuk, A. Osvet, A. Winnacker, and J. Schneider, “Quantum yield of Eu2+ emission in (Ca1−xSrx)S:Eu light emitting diode converter at 20–420 K,” Radiat. Meas. 45(3-6), 350–352 (2009). 22. L. Wondraczek, M. Batentschuk, M. A. Schmidt, R. Borchardt, S. Scheiner, B. Seemann, P. Schweizer, and C. J. Brabec, “Solar spectral conversion for improving the photosynthetic activity in algae reactors,” Nat Commun 4, 2047 (2013), doi:10.1038/ncomms3047. 23. E. Danielson, A. Ellens, F. Jermann, W. Rossner, M. Devenney, D. Giaquinta, and M. Kobusch, “Light emitting device for generating specific colored light, including white light,” US Patent no. 6,850,002 B2 (2005). 24. S. Lian, “Ultramicro/nano solar dual conversion material, and its preparing method and use. Chin. Patent application. no. CN 1935937 A (2007). 25. Q. Xia, M. Batentschuk, A. Osvet, P. Richter, D.-P. Häder, J. Schneider, L. Wondraczek, A. Winnacker, and C. J. Brabec, “Red-emitting Ca(1-x)SrxS:Eu2+ phosphors as light converters for plant-growth applications. MRS Proc. 1342, mrss11-1342-v04-04 (2011). doi:10.1557/opl.2011.864. 26. H. A. Mooney, C. Field, C. V. Yanes, and C. Chu, “Environmental controls on stomatal conductance in a shrub of the humid tropics,” Proc. Natl. Acad. Sci. U.S.A. 80(5), 1295–1297 (1983). 27. G. E. Edwards and N. R. Baker, “Can CO2 assimilation in maize leaves be predicted accurately from chlorophyll fluorescence analysis?” Photosynth. Res. 37(2), 89–102 (1993).


Introduction
Chlorophylls -along with other accessory pigments of high plants -capture photosynthetically active radiation (PAR) from solar energy to carry out photosynthetic reactions.The absorption spectra of chlorophyll a and b have two major peaks in the blue (400 -500nm) and red (600 -700nm) regions [1].The action spectra based on various plant species show similar profiles with major peaks in the two mentioned spectral regions [2][3][4][5].Green photons (500 -600 nm), however, are less active for photosynthetic actions such as CO 2 assimilation and biomass production, although they account for as much as 35% of the whole solar PAR energy.
Great efforts have been made to investigate the dependence of photosynthetic activity on light quality and intensity, using with various artificial light sources such as incandescent, fluorescent, and halogen lamps [6][7][8], lamps combined with optical filters [9][10][11], and more recently light emitting diodes (LEDs) [12][13][14].For about a decade, alongside the rapid development of solid state lighting (SSL) for general illumination applications, increasing attention has been paid to the use of luminescent phosphors as a new solution for artificial plant cultivation light, owing to the broad spectral versatility which can be provided in this way [15,16].For this purpose, phosphors must have suitable photoluminescent properties in terms of excitation and emission spectra.For example, divalent europium doped materials show a broad-band red emission band of which the position can be tuned widely by changing the host lattice composition [17][18][19].In particular, sulfides of the type Ca x Sr 1-x-y Eu y S have been of great interest in this context [20].The excitation spectrum of these phosphors exhibits a broad band in the spectral range of 450 -580 nm [21,22].Using this material, several applications have been reported for plant cultivation, such as luminescent conversion LEDs (LUCOLEDs) based on blue LED chips [23], agriculture membranes with solar-spectral conversion functionality [24] and solar spectral conversion for improving the efficiency of algae reactors [22].However, very little information is available which directly supports the assumption of higher photosynthetic activity when green photons of the solar spectrum are converted by functional red emitting-phosphors also for higher plants.
In the present report, we discuss the application of a CSSE conversion to convert incident light which is rich in green photons and its effect on the photosynthetic activity of Spinacia oleracea (S.o.), green living spinach leaves.We show that in this way, about 30% higher CO 2 assimilation rates can be achieved experimentally as compared to equivalent reference conditions without spectral converter, and we directly attribute this improvement to the light conversion process.

Phosphor synthesis and converter fabrication
The CSSE phosphor of composition Ca 40 Sr 59 S:Eu 1 (mol%) was synthesized via a solid state reaction from a stoichiometric mixture of CaS, SrS (99.5%, Sigma Aldrich) and Eu 2 O 3 (99.99%,Alfa Aesar) to which 3 wt.% of NH 4 Br (99.99 + %, Sigma Aldrich) and sulfur (99.99%,Carl Roth) were add as flux agents.After thoroughly mixing in a ball-mill, the batch was sintered under an atmosphere of 95 vol.%N 2 and 5 vol.%H 2 at 1250 °C for 3 h.Further details of the synthesis procedure and characterization of the phosphor are discussed in a previous publication [21].
CSSE particles with an average size of 20 µm were obtained after a post-treatment which consisted of a milling and a sedimentation step.The particles were then embedded in resin and coated on a highly reflective aluminum surface (~325 cm 2 ) by doctor blading method.The active layer was finally encapsulated with a matt polymer foil to protect it from ambient moisture and oxidation.In the following, this multi-layer system will be referred to as the converter foil (C-foil).Correspondingly, a reference foil (R-foil) was prepared in the same way, but using neutral MgO particles (99.99%,Alfa Aesar) as filler particles instead of CSSE.Absorption, remission and photoluminescence excitation and emission spectra of both foils were recorded with a UV-VIS spectrophotometer (Perkin Elmer Lambda 900) and a highresolution spectrofluorometer (Horiba Jobin-Yvon Fluorolog 3-22), respectively.

Analyses of photosynthetic activity
The photosynthetic activity of S.o. was assessed indirectly via monitoring the CO 2 assimilation rate as a function of time with and without the converter.For that, a reactor was constructed as shown in Fig. 1.The primary incident light was generated by four metal halide lamps (Philips type 13117, 17 V, 150 W) combined with dichroic filters (green, Edmund Optics).The photosynthetic photon flux density (PPFD) could be adjusted from 200.5 to 4059.8 µmol/(m 2 s) by decreasing the distance between the lamps and the light entrance into the reaction cell.In order to prevent undesired heating of the reactor, the infrared spectral part of the incoming irradiation was filtered-out with a 7 cm-thick water shield which was placed between the reaction cell and the light source.As the reaction unit, a gas-tight glass cell with an inner volume of 5 l was used.This cell was blackened on all sides except for a top light entrance with an area of 150 cm 2 .Intact S. o. leaves were cut into six squares of 6 x 6 cm 2 each and their surface was sterilized with an ethanol spray and rinsed three times in tap water.They were then fixed on a sterilized sponge as humidity reservoir, shaded from the top and exposed to solely either converted or reflected light from the C-/R-foils as depicted in Fig. 1.The incoming spectra for both light conditions were recorded with a UV-VIS spectrometric probe (Carry 500, Varian).The CO 2 concentration, relative humidity and temperature inside the reaction cell were measured with a CO 2 sensor (Voltcraft, CO-10) and a humidity/temperature sensor (Extech, 445815).Data from all sensors and the spectrometer were logged by a computer.The internal gas distribution was homogenized with a ventilation fan.For each measurement the starting CO 2 concentration was fixed at 443 ± 8 ppm by flushing compressed air through water bottle, and CO 2 concentrations (ppm) were logged every minute for 20 min at starting temperatures of about 22 °C.The relative humidity was maintained above 90% with the sponge as wet medium.The absorbance the S. o. chloroplasts was also measured ex situ.For that, several S. o. leaves were milled in methanol, the obtained suspension was filtered and then filled into a cuvette which was placed into the UV-VIS spectrometer.
During each measurements, the reaction cell remained sealed so that a continuous decline of the CO 2 concentration within the cell was observed over the measurement time.In order to test the reproducibility, every measurement was replicated three times.For that, the cell was opened at the gas outlet after each measurement and flushed with compressed air until the starting CO 2 concentration and temperature of ~443 ppm and 22 °C, respectively, were achieved.Only after this point, the next measurement was started.In this way, comparative measurements were carried-out with C-and R-foil, respectively.Over a complete experimental series, the primary incident photon flux density was decreased step by step from 4059.8 to 200.5 µmol/(m 2 s) and eventually to 0 µmol/(m 2 s) (dark respiration scan).

Spectral modification
In Fig. 2, the spectral light conditions in the presence of the R-foil and the C-foil are compared for equivalent primary incident light.The primary incident light is particularly rich in green photons with wavelengths from 486 to 586 nm.It is reflected without any spectral modification by the MgO surface of the R-foil.When the C-foil is applied, the photon flux in the red spectral region dramatically increases due to luminescent emission from the CSSE phosphor.The Eu 2+ -related broad-band emission peaks at a wavelength of ~650 nm and exhibits a bandwidth at half maximum of 68 nm.The remaining green radiation in the presence of the C-foil results from the remitted green photons which are not participating in the photoluminescence process.Corresponding PPFD values and percentages of photon flux in the relevant spectral ranges are listed in Table 1, obtained by integration of the spectra in Fig. 2. In the unmodified conditions, green photons account for 95.02% of the total photons, while the photosynthetically more active blue and red photons are almost negligible.In comparison, in the modified condition, the percentage of red photons is dramatically increased to more than 50% of the total photon flux due green-to-red conversion by the CSSE phosphor.Also the total PPFD increases from 5.48 to 6.38 µmol/m 2 s, which may provide an additional contribution to the photosynthetic activity.Fig. 2. PPFD spectra in the presence of the R-foil and the C-foil, respectively, in comparison to that of the primary incident light.For clarity, the intensity of the latter was divided by factor of 20.As shown in Fig. 3, the spectral distribution of the effectively absorbed PPFD can be estimated by multiplying the PPFD spectra of Fig. 2 with the relative absorbance spectrum of the chloroplasts of S. o. (inset Fig. 3).Absorbance of S. o. is substantially low at wavelengths of 500-600 nm, but exhibits strong bands in the blue and red spectral ranges.This provides a good match to the photoluminescence spectrum of the CSSE phosphor (Fig. 2), which leadswhen employed -to an improvement of photon absorbance.By integrating the spectra in Fig. 3, one can quantitatively analyze the spectral distribution of the absorbed photons (Table 2).We find that in the presence of the C-foil, in principle, the number of absorbed photons can be increased by a factor of 2.8.  Figure 4 shows the total PPFD in the reaction cell and the fraction which is absorbed on the S.o.surface in the presence of the C-and R-foil, respectively, as a function of the primary incident photon flux.The values are obtained by integrating the corresponding spectra over the wavelength range of 400 to 700 nm.It is clear that in both cases, the value of the PPFD increases linearly with the number of incident primary photons, and that the presence of the C-foil results in a notable increase of overall PPFD and absorbed PPFD as compared to using the R-foil.

Photosynthesis and CO2 assimilation
In Fig. 5(a), the CO 2 concentration inside the cell is plotted as a function of time for exemplary experiments (primary PPF of ~1014.9µmol/m 2 s) in the presence of the C-and Rfoil, respectively [25].As the photosynthetic activity is sensitive to temperature and relative humidity , also the environmental parameters were recorded (not shown): During each individual experiment, the temperature inside the reaction cell increased slightly from a fixed starting temperature of about 22.1 °C to 23.8 °C; the relative humidity remained constant at a value above 90%.No deviations could be observed between the cells equipped with C-or Rfoil.From the CO 2 concentration curves, the decline rates were evaluated to be 11.0 and 13.9 ppm/min in the presence of the R-foil and the C-foil, respectively.The small magnitudes of error bars reflect the good reproducibility of the measurements.Taking into account the experimental parameters of total leaf surface area of 216 cm 2 and the cell volume of 5 l, one can calculate the specific CO 2 assimilation rate for both conditions as 1.89 µmol CO 2 /(m 2 s) for the R-foil and 2.39 µmol CO 2 /(m 2 s) for the C-foil.This means that as a result of spectral conversion, ambient CO 2 is assimilated by S.o.more than 25% faster.In the presence of the C-foil, the CO 2 assimilation curves were obtained for various values of primary PFD as shown in Fig. 6.When S.o. were held in the dark, a mitochondrial respiration rate of about 7.27 ppm/min (1.3 µmol/(m 2 s)) was found.With increasing primary PFD, CO 2 assimilation increased significantly due to increasing photosynthetic activity.The specific CO 2 assimilation rates in the presence of C-and R-foil, respectively, are compared in Fig. 5(b).Both data sets show a typical Levenberg-Marquardt profile which is characterized by an initial linear increase with increasing primary PFD followed by a light saturation region which starts at about 649 µmol/(m 2 s): where P x is the specific CO 2 assimilation rate, P m is the maximum value of P x , R x is the specific respiration rate and I o is the incident PPFD.The fitting parameters P m and K are listed are inset in Fig. 6.In agreement with the data shown in Fig. 4, in the presence of the C-foil, the CO 2 assimilation rates are about 28.1 ± 7.5% higher as compared to the R-foil.From Fig.
5(b) it can be deduced that the light compensation points -the PFD where CO 2 assimilation rate equals the respiration rate -is about 77 µmol/(m 2 s) lower in the presence of the C-foil as compared to the R-foil.This is taken as a direct result of the higher photosynthesis activity which is achieved by spectral conversion.

Conclusions
In summary, we investigated the influence of spectral photoluminescent conversion of the incident light on the photosynthetic activity of a higher green plant, S.o..For that, the CO 2 assimilation rate of S. o. leaves was monitored under controlled illumination.A calcium strontium sulfide phosphor doped with divalent europium was used on a backlight conversion foil to effectively adjust the incoming light spectrum to the region of optimal absorption of the plants chloroplasts.We show that this phosphor can be used to convert green to red light, centering at a wavelength of ~650 nm which overlaps the absorption peaks of chlorophyll a/b.Under identical external light supply which is rich in green photons, the CO 2 assimilation rate can be enhanced by more than 25% when the actinic light is modified by conversion foil as compared to a purely reflecting reference foil.These results show that the phosphor could be potentially applied to modify the solar spectrum by converting the green photons into photosynthetically active red photons for improved photosynthetic activity.

Fig. 3 .
Fig. 3. Calculated spectra of the effective incident light, derived from multiplication of the PPFD spectra of the C-and R-foil, respectively, with the absorption spectrum of the S.o.chloroplasts.The inset shows the absorption spectrum of the S. o. chloroplasts.

Fig. 4 .
Fig. 4. Incident PPFD in the reaction cell (left axis) and absorbed fraction of PPFD (right axis) in the presence of C-and R-foil, respectively, as a function of primary incident photon flux density PFD.

Fig. 5 .
Fig. 5. (a) CO 2 concentration inside the reaction cell recorded over time in the presence of Cand R-foil, respectively.Data in (a) was adopted from Ref [25].(b) Specific CO 2 assimilation rate in the presence of C. and R-foil, respectively, under various primary photon flux densities.The dashed lines represent fits of the data to a Levenberg-Marquardt function, Eq. (1).

Fig. 6 .
Fig. 6.CO 2 concentration in the presence of the C-foil under various primary photon flux densities as a function of time.