Appl Biochem Biotechnol DOI 10.1007/s12010-013-0679-z
Cultivation of Scenedesmus obliquus in Photobioreactors: Effects of Light Intensities and Light–Dark Cycles on Growth, Productivity, and Biochemical Composition Barbara Gris & Tomas Morosinotto & Giorgio M. Giacometti & Alberto Bertucco & Eleonora Sforza
Received: 3 October 2013 / Accepted: 5 December 2013 # Springer Science+Business Media New York 2013
Abstract One of the main parameters influencing microalgae production is light, which provides energy to support metabolism but, if present in excess, can lead to oxidative stress and growth inhibition. In this work, the influence of illumination on Scenedesmus obliquus growth was assessed by cultivating cells at different light intensities in a flat plate photobioreactor. S. obliquus showed a maximum growth rate at 150 μmol photons m−2 s−1. Below this value, light was limiting for growth, while with more intense illumination photosaturation effects were observed, although cells still showed the ability to duplicate. Looking at the biochemical composition, light affected the pigment contents only while carbohydrate, lipid, and protein contents remained stable. By considering that in industrial photobioreactors microalgae cells are subjected to light–dark cycles due to mixing, algae were also grown under pulsed illumination (5, 10, and 15 Hz). Interestingly, the ability to exploit pulsed light with good efficiency required a pre-acclimation to the same conditions, suggesting the presence of a biological response to these conditions. Keywords Light intensity . Pulsed light . Biodiesel . Microalgae . Photosynthesis . Light use efficiency
Highlights • S.obliquus showed significant productivity at different light intensities • Saturation of photosynthesis was observed with irradiations over 150 μmol photons m−2 s−1 • Pulsed light effect was tested to simulate mixing in a photobioreactor • Cells grow efficiently with pulsed light only if acclimated to these conditions • Lipid accumulation is not affected by light regimes Electronic supplementary material The online version of this article (doi:10.1007/s12010-013-0679-z) contains supplementary material, which is available to authorized users.
B. Gris : A. Bertucco : E. Sforza (*) Department of Industrial Engineering, University of Padua, Via Marzolo 9, 35131 Padua, Italy e-mail: [email protected] T. Morosinotto : G. M. Giacometti Department of Biology, University of Padua, Via U. Bassi 58/B, 35121 Padua, Italy
Appl Biochem Biotechnol
Introduction Fossil fuel reserves are exhausting and their massive exploitation is also causing the accumulation of carbon dioxide in the atmosphere with possible consequences on the climate. There is, thus, the necessity to develop new clean sources of energy, alternative to fossil fuels. Biofuels, in particular biodiesel produced from algal biomass, have an interesting potential as a nontoxic, renewable, and biodegradable fuel [1]. Microalgae are among the most efficient photosynthetic organisms [2], and some species have much higher lipid yields than agricultural oleaginous crops, since their oil content may exceed 70 % (w/w), compared with the maximum 5 % of crops [3, 4]. Furthermore, microalgae do not require high-quality agricultural land, thus avoiding possible competition with food or feed production [5]. In addition, microalgae require only water, nutrients, CO2, coupled with sunlight, which are all available at low cost [2, 4]. Despite these advantages, several challenges remain to be addressed to make this technology competitive for commercial production of biodiesel [6–8]. Improved strains, in terms of volumetric productivity; appropriate lipid profile and metabolic convenience; improved photobioreactors, in terms of operation under high cell densities; gaseous mass transfer; transmittance of light; and less expensive downstream processing are all needed to make this technology feasible for large-scale production of biodiesel [4]. Among the several factors affecting microalgal productivity [9–11], light is one of the major parameters to be considered since it provides all the energy required to support metabolism, but, if present in excess, it can damage cells, leading to oxidative stress and photoinhibition and thus lower photosynthetic efficiency. To respond to excess light, photosynthetic organisms evolved two types of mechanisms. The first one consists in the repair of photosynthetic apparatus components damaged by excess illumination. This is known to involve in particular Photosystem II, through the continuous degradation and re-synthesis of its D1 subunit, which is preferentially damaged under illumination [12, 13]. The second mechanism involves the reduction of oxidative damage through thermal dissipation of energy in excess. Both mechanisms cause a reduction in light use efficiency and they must be minimized to reach an optimal productivity [14]. In photobioreactors, algal cultures reach high optical densities, which cause inhomogeneity in light distribution. As a consequence, cells on the surface, directly exposed to light, absorb most of the available radiation, but must also activate mechanisms of energy dissipation, to avoid oxidative damage. Instead, the cells in the dark zone of the photobioreactor (PBR) receive only a small part of the radiation, which is limiting for their growth [15]. The consequence of the light distribution is thus that algae in PBR use available energy with reduced efficiency. To avoid this issue, a reduction of light path could be beneficial, but thin reactors are unlikely to be economically sustainable on a large scale. In addition, in thin reactors, problems of photosaturation and inhibition are enhanced. Thus, finding other design solutions is crucial to enhance photosynthetic efficiency. A further source for complexity to be considered is that in photobioreactors, cells are actively mixed and move between the dark and light regions. As a consequence, they experience fast alternation of light and dark. Such dark–light cycles have been suggested to increase the photosynthetic efficiency in several cases [16–22]. Pulsating involves condensation of the whole energy into shorter periods, and in certain conditions, it causes no overall energy compromise. In particular, the kinetic of the photon absorption is in the range of 1–15 ms, a time needed to reset the system, prior to being ready to receive another photon [23]. However, the effect is strongly dependent on the frequency, and in some cases, these cycles can even be detrimental for productivity [14]. The culture system is also crucial to verify the positive effect on growth. For instance, Park and Lee [24] reported that cell concentration is a key parameter in
Appl Biochem Biotechnol
the exploitation of pulsed light, based on observation that at lower cell densities the instantaneous high light intensity caused photoinhibition anyway [24]. The technical feasibility of forcing the light regime in actual photobioreactors is currently under investigation [25, 26], with the aim of assuring mixing cycle up to 10 Hz which could yield increased microalgal productivity [27]. Scenedesmus obliquus is one of the most promising species as feedstock for biodiesel production [28, 29], since it presents several favorable features such as a fast growth, efficient CO2 fixation, and the ability to grow in wastewaters and accumulate lipids. In this paper, the influence of different light intensities, provided both continuously and at high-frequency pulsation, on S. obliquus growth and biochemical composition was assessed.
Materials and Methods Algae Strain and Cultivation S. obliquus 276.7, from SAG, was grown in sterile BG11 medium [30]. The medium and all materials were sterilized in an autoclave for 20 min at 121 °C in order to prevent any contamination. Cultures were maintained and propagated in the same medium with the addition of 10 g/L of plant agar, under continuous light. Pre-cultures for the inoculum were grown in flasks at 120 μmol photons m−2 s−1 and maintained in exponential phase. Cells with an optical density (OD) of 0.50 at 750 nm were inoculated in a 1.2-cm wide flat bed photobioreactor. The flat plate photobioreactors were built with transparent materials (polycarbonate) for maximum utilization of light energy. The working volume was 150 mL and the culture was mixed by an air-CO2 flow of 0.055 L/min of gas per liter of culture at 5 % of CO2, from a sparger placed in the bottom of the panel. The amount of CO2 in air was regulated by two flow meters. The gas flow supplied a non-limiting CO2 content to the culture, which was also responsible of cells mixing (see Sforza et al. [14] for the schematic of the reactor). Reactors were exposed to different constant light intensities ranging from 10 to 1,000 μmol m−2 s−1. For low light intensities (up to 150 μmol m−2 s−1), illumination was provided with a fluorescence lamp, while for higher intensities (from 200 to 1,000 μE m−2 s−1) and pulsed light, a LED lamp (Light Source SL 3500, Photon System Instruments) was employed. The latter was also employed for low light intensities to verify that the difference in light source did not affect growth kinetics. Light intensity was measured using a photo-radiometer (HD 2102.1, Delta Ohm). In experiments with pulsed light, the LED light source was programmed to generate square wave dark–light cycles with the desired intensity and frequency. Flashes were described from parameters as flash time (tf), dark time (td), flash light intensity (I0), and integrated light intensity (Ia), as reported in Table 2. In the experiments with pulsed light, three different frequencies were used (5, 10, and 15 Hz) and consistent with mixing cycle in the photobioreactor [31]. Temperature in growth chambers was kept at 23±1 °C. The medium was buffered with HEPES 10 mM, pH 8, to avoid acidification due to CO2 excess and to maintain the pH in the range of algal viability (between 7 and 8). Algal growth was measured by daily changes in OD at 750 nm (UV 500 UV-Visible Spectro, Spectronic Unicam), and cell number was monitored using Burker Counting Chamber (HBG, Germany). The specific growth rate was calculated from experimental measures in exponential phase, where nutrients were still not limiting, as the slope of the logarithmic phase for the number of cells or of the dry weight. The dry weight was measured by filtering a known volume of culture by cellulose acetate filters of 0.2 μm
Appl Biochem Biotechnol
(Whatman). Filters were pre-dried for 10 min at 80 °C in order to remove any moisture. Biomass filtered was dried for 2 h at 80 °C and then weighed to calculate the dry weight in terms of grams per liter. All experiments were performed in at least two independent biological replicates and each measurement has at least three replicates. Analytical Procedures Microalgae composition, in terms of proteins, total sugar, and lipids, was measured for each light intensity at the end of batch culture, once the stationary phase was reached. Total sugar content was estimated using the Anthrone method [32, 33]: the reaction with sulfuric acid transforms all the sugars present in the sample in 5-hydroxymethylfurfural, which reacts with Anthrone to generate a blue-green complex absorbing light at 625 nm. The concentration of solubilized proteins extracted from about 35 mg of dry biomass was determined using bicinchoninic acid assay (BCA) [34]. Cells were ground with quartz powder in lysis buffer (1.5 mL, 50 mM Tris–HCl pH 8.0, 20 % guanidine hydrochloride, 10 mM EDTA, 10 mM DTT, 0.4 μM PMSF) and, after the addition of 0.05 % Triton X-100, centrifuged at 10,000 rpm for 10 min [35]. The supernatant was used for protein determination by the BCA assay; dilutions of bovine serum albumin were used to prepare a series of protein standards. Total protein was measured by Smith analysis with a commercially available kit (Sigma-Aldrich). Total lipids were extracted overnight from dried cells using chloroform/methanol (1:2 vol/vol) in accordance with Bligh&Dyer method, in a Soxhlet apparatus [36]. The lipid mass was measured gravimetrically after solvent removal using a rotary evaporator. Pigments were extracted from the cell sampled in the exponential phase, using 10 × 106 cells and DMSO as extraction solvent, after grinding with quartz powder (this protocol was specifically set up for the species) and incubation at 65 °C for 15 min to facilitate pigment extraction. Total pigment content was evaluated spectrophotometrically by absorbance in the spectrum from 350 to 750 nm. Absorbance at 480, 649, and 665 nm were used to calculate concentrations of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids [37]. The efficiency of Photosystem II was measured as Fv/Fm parameter, on the same day of pigment analysis, using Dual-Pam-100 (Measuring System), with previous acclimation of the sample in the dark for 20 min. Statistical Analysis Student’s t tests were applied to ascertain significant differences in specific growth rate, final cell concentration, final biomass concentration in terms of grams per liter, and cellular weight. The level of statistical significance was P
Cultivation of Scenedesmus obliquus in Photobioreactors: Effects of Light Intensities and Light–Dark Cycles on Growth, Productivity, and Biochemical Composition Barbara Gris & Tomas Morosinotto & Giorgio M. Giacometti & Alberto Bertucco & Eleonora Sforza
Received: 3 October 2013 / Accepted: 5 December 2013 # Springer Science+Business Media New York 2013
Abstract One of the main parameters influencing microalgae production is light, which provides energy to support metabolism but, if present in excess, can lead to oxidative stress and growth inhibition. In this work, the influence of illumination on Scenedesmus obliquus growth was assessed by cultivating cells at different light intensities in a flat plate photobioreactor. S. obliquus showed a maximum growth rate at 150 μmol photons m−2 s−1. Below this value, light was limiting for growth, while with more intense illumination photosaturation effects were observed, although cells still showed the ability to duplicate. Looking at the biochemical composition, light affected the pigment contents only while carbohydrate, lipid, and protein contents remained stable. By considering that in industrial photobioreactors microalgae cells are subjected to light–dark cycles due to mixing, algae were also grown under pulsed illumination (5, 10, and 15 Hz). Interestingly, the ability to exploit pulsed light with good efficiency required a pre-acclimation to the same conditions, suggesting the presence of a biological response to these conditions. Keywords Light intensity . Pulsed light . Biodiesel . Microalgae . Photosynthesis . Light use efficiency
Highlights • S.obliquus showed significant productivity at different light intensities • Saturation of photosynthesis was observed with irradiations over 150 μmol photons m−2 s−1 • Pulsed light effect was tested to simulate mixing in a photobioreactor • Cells grow efficiently with pulsed light only if acclimated to these conditions • Lipid accumulation is not affected by light regimes Electronic supplementary material The online version of this article (doi:10.1007/s12010-013-0679-z) contains supplementary material, which is available to authorized users.
B. Gris : A. Bertucco : E. Sforza (*) Department of Industrial Engineering, University of Padua, Via Marzolo 9, 35131 Padua, Italy e-mail: [email protected] T. Morosinotto : G. M. Giacometti Department of Biology, University of Padua, Via U. Bassi 58/B, 35121 Padua, Italy
Appl Biochem Biotechnol
Introduction Fossil fuel reserves are exhausting and their massive exploitation is also causing the accumulation of carbon dioxide in the atmosphere with possible consequences on the climate. There is, thus, the necessity to develop new clean sources of energy, alternative to fossil fuels. Biofuels, in particular biodiesel produced from algal biomass, have an interesting potential as a nontoxic, renewable, and biodegradable fuel [1]. Microalgae are among the most efficient photosynthetic organisms [2], and some species have much higher lipid yields than agricultural oleaginous crops, since their oil content may exceed 70 % (w/w), compared with the maximum 5 % of crops [3, 4]. Furthermore, microalgae do not require high-quality agricultural land, thus avoiding possible competition with food or feed production [5]. In addition, microalgae require only water, nutrients, CO2, coupled with sunlight, which are all available at low cost [2, 4]. Despite these advantages, several challenges remain to be addressed to make this technology competitive for commercial production of biodiesel [6–8]. Improved strains, in terms of volumetric productivity; appropriate lipid profile and metabolic convenience; improved photobioreactors, in terms of operation under high cell densities; gaseous mass transfer; transmittance of light; and less expensive downstream processing are all needed to make this technology feasible for large-scale production of biodiesel [4]. Among the several factors affecting microalgal productivity [9–11], light is one of the major parameters to be considered since it provides all the energy required to support metabolism, but, if present in excess, it can damage cells, leading to oxidative stress and photoinhibition and thus lower photosynthetic efficiency. To respond to excess light, photosynthetic organisms evolved two types of mechanisms. The first one consists in the repair of photosynthetic apparatus components damaged by excess illumination. This is known to involve in particular Photosystem II, through the continuous degradation and re-synthesis of its D1 subunit, which is preferentially damaged under illumination [12, 13]. The second mechanism involves the reduction of oxidative damage through thermal dissipation of energy in excess. Both mechanisms cause a reduction in light use efficiency and they must be minimized to reach an optimal productivity [14]. In photobioreactors, algal cultures reach high optical densities, which cause inhomogeneity in light distribution. As a consequence, cells on the surface, directly exposed to light, absorb most of the available radiation, but must also activate mechanisms of energy dissipation, to avoid oxidative damage. Instead, the cells in the dark zone of the photobioreactor (PBR) receive only a small part of the radiation, which is limiting for their growth [15]. The consequence of the light distribution is thus that algae in PBR use available energy with reduced efficiency. To avoid this issue, a reduction of light path could be beneficial, but thin reactors are unlikely to be economically sustainable on a large scale. In addition, in thin reactors, problems of photosaturation and inhibition are enhanced. Thus, finding other design solutions is crucial to enhance photosynthetic efficiency. A further source for complexity to be considered is that in photobioreactors, cells are actively mixed and move between the dark and light regions. As a consequence, they experience fast alternation of light and dark. Such dark–light cycles have been suggested to increase the photosynthetic efficiency in several cases [16–22]. Pulsating involves condensation of the whole energy into shorter periods, and in certain conditions, it causes no overall energy compromise. In particular, the kinetic of the photon absorption is in the range of 1–15 ms, a time needed to reset the system, prior to being ready to receive another photon [23]. However, the effect is strongly dependent on the frequency, and in some cases, these cycles can even be detrimental for productivity [14]. The culture system is also crucial to verify the positive effect on growth. For instance, Park and Lee [24] reported that cell concentration is a key parameter in
Appl Biochem Biotechnol
the exploitation of pulsed light, based on observation that at lower cell densities the instantaneous high light intensity caused photoinhibition anyway [24]. The technical feasibility of forcing the light regime in actual photobioreactors is currently under investigation [25, 26], with the aim of assuring mixing cycle up to 10 Hz which could yield increased microalgal productivity [27]. Scenedesmus obliquus is one of the most promising species as feedstock for biodiesel production [28, 29], since it presents several favorable features such as a fast growth, efficient CO2 fixation, and the ability to grow in wastewaters and accumulate lipids. In this paper, the influence of different light intensities, provided both continuously and at high-frequency pulsation, on S. obliquus growth and biochemical composition was assessed.
Materials and Methods Algae Strain and Cultivation S. obliquus 276.7, from SAG, was grown in sterile BG11 medium [30]. The medium and all materials were sterilized in an autoclave for 20 min at 121 °C in order to prevent any contamination. Cultures were maintained and propagated in the same medium with the addition of 10 g/L of plant agar, under continuous light. Pre-cultures for the inoculum were grown in flasks at 120 μmol photons m−2 s−1 and maintained in exponential phase. Cells with an optical density (OD) of 0.50 at 750 nm were inoculated in a 1.2-cm wide flat bed photobioreactor. The flat plate photobioreactors were built with transparent materials (polycarbonate) for maximum utilization of light energy. The working volume was 150 mL and the culture was mixed by an air-CO2 flow of 0.055 L/min of gas per liter of culture at 5 % of CO2, from a sparger placed in the bottom of the panel. The amount of CO2 in air was regulated by two flow meters. The gas flow supplied a non-limiting CO2 content to the culture, which was also responsible of cells mixing (see Sforza et al. [14] for the schematic of the reactor). Reactors were exposed to different constant light intensities ranging from 10 to 1,000 μmol m−2 s−1. For low light intensities (up to 150 μmol m−2 s−1), illumination was provided with a fluorescence lamp, while for higher intensities (from 200 to 1,000 μE m−2 s−1) and pulsed light, a LED lamp (Light Source SL 3500, Photon System Instruments) was employed. The latter was also employed for low light intensities to verify that the difference in light source did not affect growth kinetics. Light intensity was measured using a photo-radiometer (HD 2102.1, Delta Ohm). In experiments with pulsed light, the LED light source was programmed to generate square wave dark–light cycles with the desired intensity and frequency. Flashes were described from parameters as flash time (tf), dark time (td), flash light intensity (I0), and integrated light intensity (Ia), as reported in Table 2. In the experiments with pulsed light, three different frequencies were used (5, 10, and 15 Hz) and consistent with mixing cycle in the photobioreactor [31]. Temperature in growth chambers was kept at 23±1 °C. The medium was buffered with HEPES 10 mM, pH 8, to avoid acidification due to CO2 excess and to maintain the pH in the range of algal viability (between 7 and 8). Algal growth was measured by daily changes in OD at 750 nm (UV 500 UV-Visible Spectro, Spectronic Unicam), and cell number was monitored using Burker Counting Chamber (HBG, Germany). The specific growth rate was calculated from experimental measures in exponential phase, where nutrients were still not limiting, as the slope of the logarithmic phase for the number of cells or of the dry weight. The dry weight was measured by filtering a known volume of culture by cellulose acetate filters of 0.2 μm
Appl Biochem Biotechnol
(Whatman). Filters were pre-dried for 10 min at 80 °C in order to remove any moisture. Biomass filtered was dried for 2 h at 80 °C and then weighed to calculate the dry weight in terms of grams per liter. All experiments were performed in at least two independent biological replicates and each measurement has at least three replicates. Analytical Procedures Microalgae composition, in terms of proteins, total sugar, and lipids, was measured for each light intensity at the end of batch culture, once the stationary phase was reached. Total sugar content was estimated using the Anthrone method [32, 33]: the reaction with sulfuric acid transforms all the sugars present in the sample in 5-hydroxymethylfurfural, which reacts with Anthrone to generate a blue-green complex absorbing light at 625 nm. The concentration of solubilized proteins extracted from about 35 mg of dry biomass was determined using bicinchoninic acid assay (BCA) [34]. Cells were ground with quartz powder in lysis buffer (1.5 mL, 50 mM Tris–HCl pH 8.0, 20 % guanidine hydrochloride, 10 mM EDTA, 10 mM DTT, 0.4 μM PMSF) and, after the addition of 0.05 % Triton X-100, centrifuged at 10,000 rpm for 10 min [35]. The supernatant was used for protein determination by the BCA assay; dilutions of bovine serum albumin were used to prepare a series of protein standards. Total protein was measured by Smith analysis with a commercially available kit (Sigma-Aldrich). Total lipids were extracted overnight from dried cells using chloroform/methanol (1:2 vol/vol) in accordance with Bligh&Dyer method, in a Soxhlet apparatus [36]. The lipid mass was measured gravimetrically after solvent removal using a rotary evaporator. Pigments were extracted from the cell sampled in the exponential phase, using 10 × 106 cells and DMSO as extraction solvent, after grinding with quartz powder (this protocol was specifically set up for the species) and incubation at 65 °C for 15 min to facilitate pigment extraction. Total pigment content was evaluated spectrophotometrically by absorbance in the spectrum from 350 to 750 nm. Absorbance at 480, 649, and 665 nm were used to calculate concentrations of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids [37]. The efficiency of Photosystem II was measured as Fv/Fm parameter, on the same day of pigment analysis, using Dual-Pam-100 (Measuring System), with previous acclimation of the sample in the dark for 20 min. Statistical Analysis Student’s t tests were applied to ascertain significant differences in specific growth rate, final cell concentration, final biomass concentration in terms of grams per liter, and cellular weight. The level of statistical significance was P
