J Appl Phycol (2008) 20:253–260 DOI 10.1007/s10811-007-9241-0 Growth and pigment composition in the red alga Halymenia floresii cultured under different light qualities José Luis Godínez-Ortega & Pauli Snoeijs & Daniel Robledo & Yolanda Freile-Pelegrín & Marianne Pedersén Received: 7 March 2007 / Revised and Accepted: 1 August 2007 / Published online: 9 September 2007 # Springer Science + Business Media B.V. 2007 Abstract Halymenia floresii is an edible species consumed in some Asian markets. In the Yucatan peninsula coast of Mexico, H. floresii dominates rocky substrata between 3 and 40 m where it grows up to 50 cm high. After analyzing the seasonal pattern of pigment content on H. floresii, we evaluate if and how the spectral composition of light affects growth and pigment dynamics under laboratory cultivation. Unialgal cultures were exposed to white, blue, red and green light in a 3-week experiment. Green light resulted in the highest algal growth rates. Synthesis of chlorophyll a, α-carotene and lutein, but not of β-carotene, was induced by white or green light. Phycocyanin synthesis was stimulated by blue light and phycoerythrin synthesis by blue or red light. Light quality treatments may be used to manipulate pigment composition in Halymenia floresii cultures. Keywords Halymenia floresii . Light quality . Pigments . Growth rate J. L. Godínez-Ortega : D. Robledo (*) : Y. Freile-Pelegrín Departmento de Recursos del Mar Cinvestav, Km 6 carretera antigua a Progreso, Cordemex 97310 AP 73, Mérida, Yucatán, Mexico e-mail: robledo@mda.cinvestav.mx P. Snoeijs Department of Ecology and Evolution, Evolutionary Biology Centre, Uppsala University, Villavägen 14, 75236 Uppsala, Sweden M. Pedersén Department of Botany, Stockholm University, 10691 Stockholm, Sweden Abbreviations chl a chlorophyll a fw algal fresh weight PE phycoerythrin PC phycocyanin Introduction Red algae have evolved a number of long and short term acclimation strategies to survive under constant changing light fields, involving changes in the thallus morphology (Monroe and Poore 2005), cell wall composition at the individual level (Carmona et al. 1998), differences in chloroplast morphology and thylakoid organization at the cellular level (long-term adaptation), and alterations in pigmentation, photosynthetic membrane composition and functionality at the molecular level (short-term acclimation) (Talarico and Maranzana 2000). In particular, light quality and the modulation of the ratios between spectral components seem to play the role of photomorphogenic ‘signals’ regulating algal metabolism and growth (Lüning 1992). Many experiments have demonstrated that light sources of different wave lengths, besides affecting metabolism and growth, produce finely modulated responses in the relative pigment composition and structure. By using white and monochromatic lights, alone or combined in different proportions at low and high irradiances, both short-term and long-term experiments on various species of red algae indicated that both irradiance and spectral composition greatly influence pigment composition, metabolism and growth (Corallina elongata, Algarra et al. 1991; Porphyra umbilicalis, P. leucosticta, P. laciniata, Chondrus crispus, Plocamium cartilagineum, Palmaria palmata, López- DO09241; No of Pages 254 Figueroa and Niell 1990; Rüdiger and López-Figueroa 1992; and Audouinella saviana, Talarico and Cortese 1993). More recently, Franklin et al. (2002) found an increase in phycoerythrin, phycocyanin and chlorophyll a concentrations in Chondrus crispus grown under blue, red and green light, respectively, when compared to white-light controls. The manipulation of pigment composition by the use of different light qualities besides explaining physiological acclimation or adaptation processes in algae may be of use during cultivation of edible species either to improve appearance and attractiveness or to improve nutritional composition, particularly for potentially economically important species (Robledo and Freile-Pelegrín 1997). Nowadays, Asian markets for edible seaweeds are looking for species with textures and colors that may be attractive for traditional consumers. New species have been added to the list of species for human consumption such as Callophyllis variegata ‘carola’ (McHugh 2003) and Gayralia spp. (Pellizzari et al. 2007), while others have been treated or manipulated commercially to obtained different colors and/ or textures (Chondrus crispus ‘hana nori’; McHugh 2003). Halymenia floresii is the type species for the genus Halymenia, with several species including the former being consumed in Asian countries where they are appreciated (H. discoidea, H. durvillaei, H. venusta, H. dilatata, H. formosa) (Kawaguchi 2004). Some recent studies on Hawaiian species of Halymenia, known as Limu lepe `ula`ula, Limu Lepe-o-hina, have shown its nutritional and dietary fiber composition (McDermid and Stuercke 2003; McDermid et al. 2005). In the Yucatan peninsula coast of Mexico, H. floresii dominates rocky substrates between 3 and 40 m where it grows up to 50 cm high. The objective of our experiment was to evaluate if and how the spectral composition of light affects growth and pigment dynamics in this tropical red alga, in order to manipulate pigment composition under controlled cultivation conditions. Determining pigment content under different light qualities may help in understanding the physiological status of the alga providing data for utilization of natural populations as well as for the culture of this species on the Yucatan coast. J Appl Phycol (2008) 20:253–260 on basal, medial and apical sections of H. floresii fronds. Pigments were analyzed according to the methods described in the following sections. Healthy specimens collected in September 2003 were selected and cultivated in the laboratory. Light quality experiments Short-term (48 h) light-intensity experiment and a long-term (21 day) light-quality experiment were carried out under controlled laboratory conditions. Stock cultures were grown in 100 L plexiglass cylinders in filtered (0.2 μm) natural seawater (33 psu) at 24±2°C supplied with continuous air bubbling and water filtration. The cultures were irradiated with white fluorescent tubes (60 μmol photons m−2 s−1) with a 12:12 h light:dark cycle. The seawater was exchanged once every second week, and at the same time the algae were placed in pulse feeding for 24 h with full ESP medium (Enriched Seawater Provasoli medium). For each light treatment (W=white, B=blue, G=green and R=red light), 12 apical branches of H. floresii of about 0.1 g fresh weight (fw) were placed in 12 separate flasks with 1 L of filtered seawater and continuous air-bubbling. The cultures were irradiated at 200 μmol photons m−2 s−1 using W (400–700 nm, Sylvania Octron 4100 K F 032/841 32 W), B (400–500 nm, Philips TLD 36 W/18), G (540– 560 nm, Philips TLD 36 W/17) and R (620–690 nm, Philips TLD 36W/15) light fluorescent lamps (Color Rendering Index, CRI=85). The experiment was carried out in a thermostated room (24°C) and an extra refrigerated recirculator was used to maintain the temperature of the experiment at 24±2°C. Algal growth was measured weekly over the course of 3 weeks, by weighing the algae after gently drying them with tissue paper to remove excess water. Daily growth rate was calculated using the formula: r=[ln(Nt/No)/t] 100%, where r stands for daily growth rate in percent, No is the initial biomass and Nt is the biomass at day t. Each week, four branches were randomly sampled and immediately frozen at −80°C until pigment analyses took place. For statistical comparisons, all data over the 3week (21 days) growth period were analyzed as follows: first week (days 0–7), second week (days 7–14) and third week (days 14–21). Materials and methods Pigment analysis Halymenia floresii floresii sporophytes were collected from the sublittoral zone at Punta Holchit (21°37′N, 88° 06′W), north of the Yucatan Peninsula, Mexico. In order to evaluate seasonal variation on pigment composition, plants were collected in June (dry season), September (rainy season) and December (cold season) of 2003. To evaluate seasonal pigment acclimation, chlorophyll a, phycobiliproteins and total carotenoids were analyzed Phycobiliproteins From each algal sample, a fragment of ca. 0.1 g fw was weighed, submerged into 5 mL 0.1 M phosphate buffer (pH 6.8) at 4°C and sonicated with a Vibracell sonicator (pulse 2, amplitude 100) for 6 min in a test tube inserted in water cooled with ice. The extracts with the algae were left J Appl Phycol (2008) 20:253–260 overnight in darkness at 4°C. Immediately before measurement they were centrifuged at 36,000 g in a MPW-350RS laboratory centrifuge for 20 min. The supernatant was used for the measurement of light absorption in a Helios Alpha Unicam DW double-beam scanning spectrophotometer scanning from 400 to 700 nm at bandwidth 2 nm and data interval 0.2 nm. Concentrations of phycoerythrin (PE) and phycocyanin (PC) in μg (g fw)−1 were calculated by the formulae proposed by Beer and Eshel (1985). The completeness of extraction was verified by re-homogenizing pellets and measuring PE and PC concentrations. Chlorophyll a and carotenoids From each algal sample, a fragment of ca. 0.02 g fw was weighed, submerged in 2 mL methanol at 4°C and sonicated with a Vibracell sonicator (pulse 2, amplitude 100) for 2 min in a test tube inserted in water cooled with ice. After addition of 40 μl ammonium acetate buffer, the extracts with the algae were left overnight in darkness at 4°C. The completeness of 255 extraction was verified by re-homogenizing pellets and measuring pigment concentrations. Before HPLC determination, the extracts were centrifuged at 36,000 g for 4 min and filtered through a 0.45 μm PTFE/PP filter. The pigments were separated by high performance liquid chromatography (HPLC) using the method of Wright and Jeffrey (1997) with a slightly modified solvent system program (Pinto et al. 2002). A sample volume of 150 μL was injected into the HPLC system using an auto injector (Holland Spark). A reversed-phase C-18 column, Spherisorb 5ODS, 250×4.60 mm, 5-μm particle size, Phenomenex was used. An Optiguard 436 nm with variable wavelength (1×46 mm, RP-18) was used with a UV detector (Milton Roy, Spectro Monitor 3100) coupled to a multiple solvent delivery system (Milton Roy, CM 4000). The software Chromcard (version 1.2 for Windows) was used for chromatogram analysis. Pigment standards (21 algal pigments; see Andersson et al. 1988) were run routinely to ensure the validity of pigment retention times. Pigment concentrations in μg (g fw)−1 were calculated Fig. 1 Seasonal pigment composition of apical (a), middle (b) and basal (c) zones of Halymenia floresii thalli. PE Phycoerythrin, Chl a chlorophyll a, TC total carotenoid content. Standard deviation indicated (n=27) 256 Fig. 2 Absorption spectra of Halymenia floresii extracts in phosphate buffer (pH 6.8) from green and red light treatments (a) and HPLC chromatogram of H. floresii extracts in methanol with 2% ammonium acetate buffer from white light treatment (b). Carotene consisted of four successive peaks for α and β-carotene using a five-point calibration curve for pigment standards of β,β-carotene (β-carotene), β,ɛ-carotene (α-carotene), chlorophyll a (chl a) and lutein obtained from Sigma™. Statistical analysis Pigment composition in field collected Halymenia floresii was analyzed by one-way ANOVA and Tukey test to evaluate significant differences between season and thallus zones. For light quality treatments correlations were calculated using Pearson’s product moment correlation coefficient (rP) and ANOVA and t-test were used to test for differences between means with the programme MINITAB™ Version 14. To summarize the variation in pigment composition among the samples, principal component analysis (PCA), implemented with the program CANOCO Version 4.51 (ter Braak and Šmilauer 2002), was applied on log-transformed pigment concentrations. J Appl Phycol (2008) 20:253–260 Six major pigments, chl a, PE, PC, lutein, α-carotene and β-carotene, were detected in all 82 samples analyzed. Absorption spectra of crude algal extracts of H. floresii are presented in Fig. 2a. PE absorption spectra showed three peaks: two at about 495 and 545 nm, and one main peak at 564 nm. PC showed one absorption peak at 618 nm. Figure 2b shows HPLC retention times for lutein, chlorophyll a and carotene. The major pigments were PE [up to ca. 2,500 μg (g fw)−1] and Chl a [up to ca. 150 μg (g fw)−1] and the major carotenoid was lutein [up to ca. 50 μg (g fw)−1]. Light quality experiment The growth rate of H. floresii varied between 2 and 6% day−1 (Fig. 3). Two-way ANOVA showed significant differences in growth rate between treatments and cultivation time (P< 0.05). Growth rates were 50% higher in green light compared to the other light-quality treatments. Over the course of 21 days, the average growth rate in white light was 2.3±1.0, 3.3±0.7 and 5.0±1.0% day−1 for days 0–7, 7–14 and 14–21, respectively. In the green light treatment, growth rates increased significantly from the first (4.4±1.6% day−1) to the third week (5.9±1.8% day−1) (t-test, P<0.05). On the contrary, in the blue light (3.0±0.5 to 3.9±2.4% day−1) and red light (3.1±0.5 to 2.9±0.7% day−1) no significant differences in growth rate were observed between the first and third week (t-test, P>0.05). The pigment concentrations in the H. floresii thalli exhibited different response patterns in relation to the Results Thallus pigment composition Pigment content on apical, middle and basal zones of Halymenia floresii are shown in Fig. 1. Significant differences in PE content were found between season and thallus zone (F0.05 (1),4,18 =15.3). Chl a changed significantly between thallus zone (F0.05(1),2,18 =7.4), whereas, total carotenoids showed significant differences only by season (F0.05(1),2,17 =9.6). Significant differences in PE and chl content were found between apical, middle and basal zones during the dry season (P<0.05). The PE content was lower in the apical zone [70 μg (g fw)−1] when compared to basal zone [200 μg (g fw)−1]. The contrary was observed during cold season when PE apical content was higher. Fig. 3 Growth rates of Halymenia floresii (% day−1) subjected to four different light quality treatments (white, green, blue and red) during 3 weeks. Standard deviation indicated (n=144) J Appl Phycol (2008) 20:253–260 257 Fig. 4 Pigment concentrations of Halymenia floresii μg (g fw)−1 subjected to four different light quality treatments (white, green, blue and red) during 3 weeks. (a) Chlorophyll-a; (b) Phycoerythrin; (c) Phycocyanin; (d) Lutein; (e) α carotene; (f) β carotene. Standard deviation indicated (n=54) light-quality treatments (Fig. 4). Two-way ANOVA analyses with spectral quality and sampling week as predictors and pigment concentrations as response variables showed significant variations for PE, PC, lutein and α-carotene with spectral quality, but not for chlorophyll a and β-carotene. After 3 weeks of cultivation, PE concentrations were significantly higher (t-tests, P<0.05) in the blue- and redlight treatments than in the white- and green-light treat- ments (Fig. 4b). PC concentrations were significantly higher only in blue light compared with the other three treatments (Fig. 4c). Over time, PC:PE ratios decreased in red light and increased in green light. On the other hand, the blue-light adaptation was already fully induced after 1 week for both PE and PC while the red-light adaptation of PE increased with time. Simultaneously, chl a, lutein and α-carotene showed the opposite pattern with concentrations 258 Fig. 5 Principal Component Analysis (PCA) ordination plot showing pigment dynamics in Halymenia floresii in relation to the four light quality treatments (white, green, blue and red) and growth rate remaining at initial levels in blue or red light and increasing in the white- and green-light treatments. In green light, the concentrations of α-carotene were always higher than in white, blue and red light. The pigment dynamics of H. floresii in response to the spectral light qualities are summarized in Fig. 5. This analysis is based on the concentrations of the six major pigments in all 48 samples. Light-quality treatment (as dummy variables), sampling week and growth rate were tested on the results of the ordination by multiple regression analysis (i.e., they were not used as constraints; the ordination which is solely based on pigment composition). The variation in treatments and growth rate were significantly related to the ordination results, but sampling week was not. Axes 1 and 2 of the ordination together explained 91% of the variation in the data and the eigenvalues of the first four axes were 0.56, 0.29, 0.10 and 0.03, respectively. The centroids for the light quality treatments showed a clear pattern in which green and white light, in the lower part of the ordination, are associated with the scores of lutein and α-carotene and growth rate. The centroids for blue and red light were situated in the upper part of the ordination, and were associated with the scores of PE and PC. The centroid for red light was situated in an opposite direction to growth rate, lutein and α-carotene, indicating that these variables were negatively associated with red light. Discussion The experimental responses obtained in this study under light quality treatments can be related to the environment J Appl Phycol (2008) 20:253–260 where Halymenia floresii is found. Changes in tropical red algal pigmentation appear to take place within individual thalli as results of photoacclimation (Beach and Smith 1996). This was evident in H. floresii pigment content over the year. The PE concentration augmented in the basal zone of the H. floresii thalli. This is a common observation in algae found in the sublittoral zone where light is naturally limited (deep and turbidity) and the algae itself contributes to self shading. For most algae, the total pigment concentration is higher in low-exposed samples than in samples exposed to high light intensity (Falkowski and Raven 1991). Similar trends have been reported in previous works on tropical red algae. On this regard Hydrolithon onkodes showed higher content of PE in thallus exposed to lower light intensity (Payri et al. 2001). The variations in the pigment proportions are likely to reflect the adaptation of the thalli to the light level. The observed pattern for PE content in H. floresii diminishing from the basal to apical zones in rainy season may reflect the increase in the light availability. Furthermore, the highest content of total carotenoids in the apical zone in Halymenia was observed during the rainy season, when higher light intensities occurred (5.81–8.34 mol photons−2 day−1). Thalli exposed to a high rate of solar radiation increased their carotenoid content, including photoprotectant substances, whereas their phycobiliprotein pigments decreased. A higher content of total carotenoids has been also reported by Payri et al. (2001) for Hydrolithon onkodes at high intensities (6.48 μg cm−2). This may also suggest that Halymenia floresii needs protection to increased light intensities utilizing the xanthophylls (i.e. lutein). Light quality experiment H. floresii pigment composition was influenced by the light quality treatment. After 3 weeks, PE and PC contents were influenced positively by blue light, while green light induced chl a synthesis when compared with white light treatments. This is in accordance with the results obtained for the red alga Chondrus crispus by Franklin et al. (2002). Considering the acclimation process of algae to red- and blue-light quality treatments the phycobiliproteins are capable of extending the absorption range toward the shortest wavelengths blue and green light (Talarico and Maranzana 2000). These has been also shown in Porphyra leucosticta were the presence of a great density of phycobilisomes was related to exposition to blue light (800 μm−2) in comparison to red and green light treatments (250 μm−2 and 180 μm−2, respectively) (Tsekos et al. 2002). In the course of 21 days, H. floresii increased its growth in green light treatments in concert with chl a concentration. Leukart and Lüning (1994) demonstrated that green light, even at very low intensity (0.5 μmol photons m−2 s−1), was J Appl Phycol (2008) 20:253–260 more effective than red or blue light for germling growth in several red algae cultivated for at least 15 weeks. In the present study, the higher growth rate of H. floresii in green light compared to red and blue light can be explained by the higher chl a concentrations and thereby a higher efficiency of light absorption. PC and, especially PE, were highly concentrated within the stroma, for PE from 1,400 to 1,500 μg (g fw)−1 under W and G to 2,400 μg (g fw)−1 under B, versus a maxima around 50–60 μg (g fw)−1 for PC under B. For the red alga Porphyra umbilicalis LópezFigueroa et al. (1995) showed that growth rate (in terms of weight and area, carbon content and intercellular matrix dimensions) was higher in red light. The same authors also found that concentrations of soluble protein and photosynthetic pigments (Chl a, PC and PE), package effect and cell volume were higher in blue light. Thus, red light seemed to stimulate thallus expansion and increased cell size, consequently high cell density per thallus area were produced, whereas, blue light mainly stimulated the accumulation of nitrogen compounds in the form of soluble protein and phycobiliproteins. In H. floresii we found no signs of growth stimulation or induction of chl a or PC synthesis by red light, but the accumulation of phycobiliproteins stimulated by blue light seems to be a general feature in red algae. No carotenoids other than β-carotene and α-carotene, and the xanthophyll lutein were detected in H. floresii. Carotenoids may have a key role in the photosynthesis by transferring excitation energy to chlorophyll during light harvesting (Anderson et al. 2006), however H. floresii did not seem to mobilize its carotenoid pigments for photoprotection at the experimental light-intensity level of 200 μmol photons m−2 s−1, since β-carotene levels remained stable throughout the experiment. In green light, the concentrations of α-carotene were always higher than in white, blue and red light, suggesting that the synthesis of this carotenoid was enhanced by green light. Moreover, the increases in lutein and it’s precursor α-carotene constitute a strong indication that a new xanthophylls cycle is activated by green light to optimize photosynthesis by increasing the light-harvesting function of lutein (Anderson et al. 2006). A strong increase of lutein was observed under W rather in the short term (days 0–7) stabilizing in the long term (days 14– 21). These results lead to hypothesize, an acclimation (at short and medium terms) to higher irradiance (excess light) with photoprotection against photoinhibition, because α-carotene- not just β-carotene-derived xanthophylls, also contribute the nonphotochemical quenching of excess light energy. Lutein, in particular, has been proven both to stabilize chl-protein complexes (CPs) within PS and to prevent their photo-oxidation possibly caused by ROS formation during photosynthetic activity (Baroli et al. 2004). 259 In conclusion, light quality treatments influenced the growth and pigment composition of Halymenia floresii in culture. Under green light H. floresii optimizes its growth and induces chlorophyll-a and lutein synthesis, blue and red light induce phycobiliprotein synthesis. The use of light quality treatments in cultivation of red algae can be use to manipulate pigment composition. The lutein content of H. floresii may be of particular interest for the market of edible seaweeds. Further research is required to determine how the spectral composition of light controls pigments biosynthesis pathways. 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