Journal of Biotechnology 192 (2014) 114–122
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Light modulation of biomass and macromolecular composition of the diatom Skeletonema marinoi Raghu Chandrasekaran a,1 , Lucia Barra a,b,1 , Sara Carillo c , Tonino Caruso d , Maria Michela Corsaro c , Fabrizio Dal Piaz d , Giulia Graziani e , Federico Corato a , Debora Pepe a , Alessandro Manfredonia a , Ida Orefice a , Alexander V. Ruban f , Christophe Brunet a,∗ a
Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Napoli, Italy CNR-IBBR, Via Università 133, 80055 Portici (NA), Italy c University of Naples “Federico II”, Dipartimento di Scienze Chimiche, Complesso Universitario M.S. Angelo, Via Cintia 4, 80126 Napoli, Italy d University of Salerno, Dipartimento di Chimica e Biologia, Via Giovanni Paolo II 132, 84084 Fisciano (SA), Italy e University of Naples “Federico II”, Department of Agricultural and Food Science, Parco Gussone, 80055 Portici (NA), Italy f School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, UK b
a r t i c l e
i n f o
Article history: Received 25 June 2014 Received in revised form 8 October 2014 Accepted 13 October 2014 Available online 27 October 2014 Keywords: Biotechnology Blue light Carotenoids Microalgae Lipids
a b s t r a c t The biochemical profile and growth of the coastal diatom Skeletonema marinoi was investigated under four different daily blue light doses (sinusoidal light peaking at 88, 130, 250 and 450 mol photons m−2 s−1 , respectively). Ability of cells to regulate the light energy input caused alterations in growth and different biosynthetic pathways. The light saturation index for photosynthesis (Ek ), which governs the photoacclimative processes, ranged between 250 and 300 mol photons m−2 s−1 . Cells that were adapted to low light ( 0.05, Fig. 2). This agrees with a potential increase in photorespiration rate induced by high light (Brunet et al., 2011; Schnitzler Parker et al., 2004), being this process a way for cells to dissipate the excess biochemical energy, and to the fact that silicate metabolism is more linked to respiration and cell cycle than photosynthesis (Norici et al., 2011). The RNA content per cell did not show significant variation among the light conditions (p > 0.05, n = 15), ranging from 0.30 to 0.70 pg cell−1 (Table 1). The RNA content per cell displayed circadian oscillation in the cells (data not shown), that might be related to variations in the cellular content of proteins, lipids, carbohydrates or pigments (Fábregas et al., 2002). In all conditions, the RNA content per cell increased at midday compared to the morning and afternoon samples, while some studies (e.g. Berdalet et al., 1992) showed enhanced RNA content at the beginning of the light period. This discrepancy is due to the light distribution, provided in a sinusoidal way in our study and in a quadratic way for the other studies, as recently demonstrated (Orefice, personal communication).
3.2. Light modulation of carotenoids and chlorophylls The main photosynthetic pigments, chlorophyll a (Chl a) and fucoxanthin (Fuco), were significantly correlated (p < 0.001, n = 36) presenting a significant decrease under the high light conditions (p < 0.05, n = 9; Fig. 3a and b). Under low light (E < Ek , 88 and 130 mol photon m−2 s−1 ), the total carotenoid content and Chl a per cell was similar (Fig. 3a, Table 3), despite the difference in daily light dose (2.2 and 3.2 mol m−2 day−1 ). Fuco/Chl a was similar among the different light conditions (p > 0.05, n = 9), except for 450 mol photon m−2 s−1 where it significantly decreased (p < 0.01, n = 9). Thus, S. marinoi tends to modify the antenna structure by decreasing Fuco compared to the Chl a molecules in the reaction center under the latter condition (Dimier et al., 2009), while under low light condition, S. marinoi tends to modify all the reaction center and antenna structure to maintain similar the pigment ratio. On the opposite of Fuco and Chl a, chlorophyll c (Chl c) content per cell exponentially decreased from the lowest to the highest light dose (Fig. 3a), in agreement with the results obtained on Pseudonitzschia multistriata (Brunet et al., 2014). The highest Chl c content under 88 mol photon m−2 s−1 (two times more compared to the other light conditions) enhances the ability of cells to cope with extreme low light, since this pigment is known to efficiently transfer energy to Chl a (Di Valentin et al., 2013). The -carotene (-car) content did not show any significant trend over the light gradient (Fig. 3c), while a significantly higher
Table 3 Biochemical properties of S. marinoi under the four blue fluence rates.
Total carotenoids Total carbohydrates Total lipids Neutral lipids Phospholipids Glycolipids DU Total proteins Total amino acids IDF (wt%, insoluble fibers) SDF (wt%, soluble fibers) POC PON
88 mol photons m−2 s−1
130 mol photons m−2 s−1
250 mol photons m−2 s−1
450 mol photons m−2 s−1
0.015 ± 0.006 0.62 ± 0.12 1.25 ± 0.16 0.54 ± 0.13 0.03 ± 0.02 0.68 ± 0.04 28 26.73 ± 3.45 0.00199 11.85 2.15 14.16 ± 1.87 2.62 ± 0.89
0.019 ± 0.003 0.79 ± 0.24 2.60 ± 0.25 0.90 ± 0.10 0.96 ± 0.16 0.74 ± 0.01 92 45.03 ± 7.88 0.00356 12.45 1.89 10.99 ± 0.13 3.86 ± 0.60
0.009 ± 0.003 2.60 ± 1.52 0.66 ± 0.06 0.21 ± 0.08 0.29 ± 0.07 0.16 ± 0.04 5 23.87 ± 0.73 0.00231 16.80 nd 19.37 ± 2.78 3.78 ± 1.31
0.005 ± 0.001 2.52 ± 0.06 0.68 ± 0.14 0.42 ± 0.08 0.18 ± 0.15 0.08 ± 0.04 34 15.89 ± 0.79 0.00038 12.28 nd 15.18 ± 0.83 2.11 ± 0.63
DU, degree of unsaturation [(MUFA, wt%) + 2 × (PUFA, wt%)]; POC, particulate organic carbon (pg cell−1 ); PON, particulate organic nitrogen (pg cell−1 ). Data represent mean and standard deviation (for POC, PON, n = 15; carotenoids and proteins, n = 9; and for lipids, fibers, and carbohydrates, n = 3).
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showed an opposite trend to Dd, being significantly higher under the highest intensity compared to the other conditions (p < 0.05, n = 9; Fig. 3c). Dt concentration was very low under all the light conditions, reinforcing that cells are not able to develop photoprotective process under blue light (Brunet et al., 2014). Indeed, the absence of red spectrum strongly limits Dt synthesis and therefore the development of non-photochemical quenching (NPQ, excessenergy dissipative process, Table 2). 3.3. Light modulation of macromolecular composition
Fig. 3. Variations of photosynthetic and photoprotective pigments content. (a) Chlorophyll a and Chlorophyll c content per cell (Chl a and Chl c; pg cell−1 ); (b) Fucoxanthin content per cell (Fuco; pg cell−1 ); (c) -carotene, diadinoxanthin and diatoxanthin content per cell (-car, Dd and Dt; pg cell−1 ); maximum deepoxidation state [DESmax = Dt/(Dd + Dt)] (data represent mean ± SD; n = 9 except for DESmax , n = 3).
-car/Chl a was found under the two high light conditions (p < 0.05, n = 9) compared to the low light conditions (data not shown). This feature is related to the Chl a decrease and to the involvement of -car in the biosynthesis of the xanthophyll cycle (XC) pigments (Dambek et al., 2012). Diadinoxanthin over chlorophyll a ratio (Dd/Chl a) followed the same trend as -car/Chl a and was significantly higher under high light than low light (p < 0.05, n = 9). As Fuco, Dd concentration decreased from the 130 mol photon m−2 s−1 to the highest light intensity (Fig. 3c). The 88 mol photon m−2 s−1 condition was out of this trend, revealing a peculiar physiological response of the cells grown under this extremely light limiting condition. Diatoxanthin (Dt; photoprotective pigment)
Although the protein and lipid content per cell were significantly correlated to the growth rate (p < 0.05, n = 4), the biochemical property depends on the light experienced by cells. Similar to carotenoid content, cells increased lipid and protein content under low light compared to high light (Table 3, p < 0.01). The highest concentration was found under 130 mol photon m−2 s−1 . The high lipid content under the low light conditions may serve as an effective energy and carbon storage (Fábregas et al., 2002) providing a larger sink of the available energy. It also decreases the membrane permeability by forming thick membrane layers and thus reducing the energy loss by passive diffusion of organic matter (Cherrier et al., 2014). The classes of glycolipids and neutral lipids followed the same trend as total lipids, being enhanced under low light, with a maximal concentration at 130 mol photon m−2 s−1 (Table 3). The third class, the phospholipids, did not follow any trend over the light gradient (Table 3), while its concentration was strongly enhanced under 130 mol photon m−2 s−1 (Table 3). The low phospholipid content under high light suggests that the membrane permeability was higher under this condition compared to the other light conditions. This feature could allow cells to remove excess energy by exudation of organic matter when the photosynthesis outpaces cell growth (Cherrier et al., 2014). Furthermore, the increase in the neutral lipid contribution under the highest light condition (≈62 wt% vs
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Light modulation of biomass and macromolecular composition of the diatom Skeletonema marinoi Raghu Chandrasekaran a,1 , Lucia Barra a,b,1 , Sara Carillo c , Tonino Caruso d , Maria Michela Corsaro c , Fabrizio Dal Piaz d , Giulia Graziani e , Federico Corato a , Debora Pepe a , Alessandro Manfredonia a , Ida Orefice a , Alexander V. Ruban f , Christophe Brunet a,∗ a
Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Napoli, Italy CNR-IBBR, Via Università 133, 80055 Portici (NA), Italy c University of Naples “Federico II”, Dipartimento di Scienze Chimiche, Complesso Universitario M.S. Angelo, Via Cintia 4, 80126 Napoli, Italy d University of Salerno, Dipartimento di Chimica e Biologia, Via Giovanni Paolo II 132, 84084 Fisciano (SA), Italy e University of Naples “Federico II”, Department of Agricultural and Food Science, Parco Gussone, 80055 Portici (NA), Italy f School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, UK b
a r t i c l e
i n f o
Article history: Received 25 June 2014 Received in revised form 8 October 2014 Accepted 13 October 2014 Available online 27 October 2014 Keywords: Biotechnology Blue light Carotenoids Microalgae Lipids
a b s t r a c t The biochemical profile and growth of the coastal diatom Skeletonema marinoi was investigated under four different daily blue light doses (sinusoidal light peaking at 88, 130, 250 and 450 mol photons m−2 s−1 , respectively). Ability of cells to regulate the light energy input caused alterations in growth and different biosynthetic pathways. The light saturation index for photosynthesis (Ek ), which governs the photoacclimative processes, ranged between 250 and 300 mol photons m−2 s−1 . Cells that were adapted to low light ( 0.05, Fig. 2). This agrees with a potential increase in photorespiration rate induced by high light (Brunet et al., 2011; Schnitzler Parker et al., 2004), being this process a way for cells to dissipate the excess biochemical energy, and to the fact that silicate metabolism is more linked to respiration and cell cycle than photosynthesis (Norici et al., 2011). The RNA content per cell did not show significant variation among the light conditions (p > 0.05, n = 15), ranging from 0.30 to 0.70 pg cell−1 (Table 1). The RNA content per cell displayed circadian oscillation in the cells (data not shown), that might be related to variations in the cellular content of proteins, lipids, carbohydrates or pigments (Fábregas et al., 2002). In all conditions, the RNA content per cell increased at midday compared to the morning and afternoon samples, while some studies (e.g. Berdalet et al., 1992) showed enhanced RNA content at the beginning of the light period. This discrepancy is due to the light distribution, provided in a sinusoidal way in our study and in a quadratic way for the other studies, as recently demonstrated (Orefice, personal communication).
3.2. Light modulation of carotenoids and chlorophylls The main photosynthetic pigments, chlorophyll a (Chl a) and fucoxanthin (Fuco), were significantly correlated (p < 0.001, n = 36) presenting a significant decrease under the high light conditions (p < 0.05, n = 9; Fig. 3a and b). Under low light (E < Ek , 88 and 130 mol photon m−2 s−1 ), the total carotenoid content and Chl a per cell was similar (Fig. 3a, Table 3), despite the difference in daily light dose (2.2 and 3.2 mol m−2 day−1 ). Fuco/Chl a was similar among the different light conditions (p > 0.05, n = 9), except for 450 mol photon m−2 s−1 where it significantly decreased (p < 0.01, n = 9). Thus, S. marinoi tends to modify the antenna structure by decreasing Fuco compared to the Chl a molecules in the reaction center under the latter condition (Dimier et al., 2009), while under low light condition, S. marinoi tends to modify all the reaction center and antenna structure to maintain similar the pigment ratio. On the opposite of Fuco and Chl a, chlorophyll c (Chl c) content per cell exponentially decreased from the lowest to the highest light dose (Fig. 3a), in agreement with the results obtained on Pseudonitzschia multistriata (Brunet et al., 2014). The highest Chl c content under 88 mol photon m−2 s−1 (two times more compared to the other light conditions) enhances the ability of cells to cope with extreme low light, since this pigment is known to efficiently transfer energy to Chl a (Di Valentin et al., 2013). The -carotene (-car) content did not show any significant trend over the light gradient (Fig. 3c), while a significantly higher
Table 3 Biochemical properties of S. marinoi under the four blue fluence rates.
Total carotenoids Total carbohydrates Total lipids Neutral lipids Phospholipids Glycolipids DU Total proteins Total amino acids IDF (wt%, insoluble fibers) SDF (wt%, soluble fibers) POC PON
88 mol photons m−2 s−1
130 mol photons m−2 s−1
250 mol photons m−2 s−1
450 mol photons m−2 s−1
0.015 ± 0.006 0.62 ± 0.12 1.25 ± 0.16 0.54 ± 0.13 0.03 ± 0.02 0.68 ± 0.04 28 26.73 ± 3.45 0.00199 11.85 2.15 14.16 ± 1.87 2.62 ± 0.89
0.019 ± 0.003 0.79 ± 0.24 2.60 ± 0.25 0.90 ± 0.10 0.96 ± 0.16 0.74 ± 0.01 92 45.03 ± 7.88 0.00356 12.45 1.89 10.99 ± 0.13 3.86 ± 0.60
0.009 ± 0.003 2.60 ± 1.52 0.66 ± 0.06 0.21 ± 0.08 0.29 ± 0.07 0.16 ± 0.04 5 23.87 ± 0.73 0.00231 16.80 nd 19.37 ± 2.78 3.78 ± 1.31
0.005 ± 0.001 2.52 ± 0.06 0.68 ± 0.14 0.42 ± 0.08 0.18 ± 0.15 0.08 ± 0.04 34 15.89 ± 0.79 0.00038 12.28 nd 15.18 ± 0.83 2.11 ± 0.63
DU, degree of unsaturation [(MUFA, wt%) + 2 × (PUFA, wt%)]; POC, particulate organic carbon (pg cell−1 ); PON, particulate organic nitrogen (pg cell−1 ). Data represent mean and standard deviation (for POC, PON, n = 15; carotenoids and proteins, n = 9; and for lipids, fibers, and carbohydrates, n = 3).
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showed an opposite trend to Dd, being significantly higher under the highest intensity compared to the other conditions (p < 0.05, n = 9; Fig. 3c). Dt concentration was very low under all the light conditions, reinforcing that cells are not able to develop photoprotective process under blue light (Brunet et al., 2014). Indeed, the absence of red spectrum strongly limits Dt synthesis and therefore the development of non-photochemical quenching (NPQ, excessenergy dissipative process, Table 2). 3.3. Light modulation of macromolecular composition
Fig. 3. Variations of photosynthetic and photoprotective pigments content. (a) Chlorophyll a and Chlorophyll c content per cell (Chl a and Chl c; pg cell−1 ); (b) Fucoxanthin content per cell (Fuco; pg cell−1 ); (c) -carotene, diadinoxanthin and diatoxanthin content per cell (-car, Dd and Dt; pg cell−1 ); maximum deepoxidation state [DESmax = Dt/(Dd + Dt)] (data represent mean ± SD; n = 9 except for DESmax , n = 3).
-car/Chl a was found under the two high light conditions (p < 0.05, n = 9) compared to the low light conditions (data not shown). This feature is related to the Chl a decrease and to the involvement of -car in the biosynthesis of the xanthophyll cycle (XC) pigments (Dambek et al., 2012). Diadinoxanthin over chlorophyll a ratio (Dd/Chl a) followed the same trend as -car/Chl a and was significantly higher under high light than low light (p < 0.05, n = 9). As Fuco, Dd concentration decreased from the 130 mol photon m−2 s−1 to the highest light intensity (Fig. 3c). The 88 mol photon m−2 s−1 condition was out of this trend, revealing a peculiar physiological response of the cells grown under this extremely light limiting condition. Diatoxanthin (Dt; photoprotective pigment)
Although the protein and lipid content per cell were significantly correlated to the growth rate (p < 0.05, n = 4), the biochemical property depends on the light experienced by cells. Similar to carotenoid content, cells increased lipid and protein content under low light compared to high light (Table 3, p < 0.01). The highest concentration was found under 130 mol photon m−2 s−1 . The high lipid content under the low light conditions may serve as an effective energy and carbon storage (Fábregas et al., 2002) providing a larger sink of the available energy. It also decreases the membrane permeability by forming thick membrane layers and thus reducing the energy loss by passive diffusion of organic matter (Cherrier et al., 2014). The classes of glycolipids and neutral lipids followed the same trend as total lipids, being enhanced under low light, with a maximal concentration at 130 mol photon m−2 s−1 (Table 3). The third class, the phospholipids, did not follow any trend over the light gradient (Table 3), while its concentration was strongly enhanced under 130 mol photon m−2 s−1 (Table 3). The low phospholipid content under high light suggests that the membrane permeability was higher under this condition compared to the other light conditions. This feature could allow cells to remove excess energy by exudation of organic matter when the photosynthesis outpaces cell growth (Cherrier et al., 2014). Furthermore, the increase in the neutral lipid contribution under the highest light condition (≈62 wt% vs
