- Email: [email protected]
Enhancing neutral lipid content in Skeletonema marinoi through multiple phase growth in a bench photobioreactor
Algal Research 5 (2014) 32–36
Contents lists available at ScienceDirect
Algal Research journal homepage: www.elsevier.com/locate/algal
Enhancing neutral lipid content in Skeletonema marinoi through multiple phase growth in a bench photobioreactor Elena Bertozzini a,⁎, Luca Galluzzi a, Antonella Penna b, Mauro Magnani c a b c
Department of Biomolecular Sciences, Biotechnology Section, University of Urbino, Via Arco D'Augusto 2, 61032, Fano PU, Italy Department of Biomolecular Sciences, Environmental Biology Section, University of Urbino, Viale Trieste 296, 61121 Pesaro, PU, Italy Department of Biomolecular Sciences, University of Urbino, Via Saffi 2, 61029 Urbino, PU, Italy
a r t i c l e
i n f o
Article history: Received 31 July 2013 Received in revised form 14 March 2014 Accepted 9 May 2014 Available online xxxx Keywords: Biofuel Neutral lipids Nitrogen starvation Bench photobioreactor Growth strategy
a b s t r a c t The continuing increase in fuel demand, the dramatic situation of climate changes and global warming are bringing worldwide attention to identification of alternative energy source that can replace fossil fuel. Among all the feedstock considered for biodiesel production, microalgae are the most promising. In particular, it has been demonstrated that microalgae enhance their neutral lipids content in stress conditions, such as nutrient starvation. However, this increase in neutral lipids content is often coupled with a strong biomass decrease. In this study, in order to limit this side effect, a novel three-phase growth strategy was tested on the diatom Skeletonema marinoi. After a first phase directed to biomass production the culture, during a second phase of semi-continuous regimen characterized with reduction of nitrate concentration, achieved a neutral lipids content per cell of 10% of dry weight with slight impact on biomass. After these two phases the cells, gradually acclimatized to low nitrogen concentration, were still able to grow in a third maturation phase performed in batch. During this last phase, the culture further increased neutral lipid content up to the 30 % of dry weight. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The International Energy Agency (IEA) estimated the total primary energy supply in 2010 at 12,275 million tonnes of oil equivalent, with a further increase respect to the 2009 value. Fossil fuels, i.e. coal, oil and natural gas, constitute more than the 80% of this total energy supply [1]. These feedstocks are non-renewable natural resources, and they are one of the major contributors to the greenhouse effect and global warming. A great deal of attention across the globe has therefore been directed toward the identification of new, renewable energy sources that could be an alternative to fossil fuels. Biodiesel is one of those alternative sources [2]. The first crops used to produce biodiesel were edible crops such as rapeseed oil, sugarcane, sugar beet and maize [3], but the use of these feedstocks for biodiesel production would have a negative impact on global food markets and food security. Hence, a second generation of biodiesel feedstock was identified in inedible plants such as jatropha, mahua and tobacco seeds. However, to ensure high oil yield from these feedstocks, heavy fertilization, irrigation and good management practices are required [4], thus efforts to find a more sustainable biodiesel feedstock are now focused on microalgae [3].
⁎ Corresponding author. Tel.: +39 0722 304976; fax: +39 0722 304970. E-mail address: [email protected] (E. Bertozzini).
http://dx.doi.org/10.1016/j.algal.2014.05.004 2211-9264/© 2014 Elsevier B.V. All rights reserved.
Microalgae are considered one of the most promising biodiesel feedstocks due to their high growth rate and their ability to produce high amounts of neutral lipids and carbohydrates for biodiesel and bioethanol productions [5]. Moreover, microalgae can potentially produce more oil per acre than other feedstocks [6,7], they are not edible and can be grown on non-arable land [2], and they fix CO2 more efficiently than other plants and could potentially reduce the greenhouse effect through biofixation of waste carbon dioxide [6,8]. However, despite their great potential as a biodiesel feedstock, microalgae have yet to be exploited, in particular on the basis of Life Cycle Analysis (LCA). The LCA, defined from the ISO 14040.2 standard, quantifies the environmental impact of the processes that contribute to the supply of goods or services also considering how much fossil fuel energy is used for system maintenance [9]. In particular, the success of microalgae as a biodiesel feedstock strongly depends on the optimization of appropriate culture conditions that make it possible to produce high biomass and large amounts of neutral lipids [9,10]. Different approaches have been tested to achieve such conditions, and it has been clearly shown that nutrient limitation, in particular nitrogen limitation, enhances neutral lipid production however, it severely limits biomass production [11–16]. Several growth strategies have been proposed to limit this side effect. For example, it has been demonstrated that a gradual reduction of nitrate concentration in a semicontinuous regimen enhances the neutral lipid content of Skeletonema marinoi batch cultures with slight effects on biomass [17].
E. Bertozzini et al. / Algal Research 5 (2014) 32–36
Another approach designed to reduce the negative effect of nutrient limitation strategies on biomass production, consists in a two-stage process. The first phase aims to achieve maximum biomass production, while in the second stage stress factors are introduced to increase neutral lipid content [8,18,19]. In this study, the diatom S. marinoi was used as a model in a bench photobioreactor to test a novel three-phase growth strategy that combines the two different strategies described above. In the first growth phase S. marinoi was grown in enriched f/2 medium, in order to obtain high biomass, in the second semicontinuous regimen-phase the nitrate concentration was gradually reduced to increase neutral lipid content [20], and in the final “maturation” stage, cultures were grown for an additional week without manipulation to further increase the neutral lipid content and biomass of the culture. In fact, recent literature has demonstrated that the association of culture aging with nitrogen depletion could further enhance neutral lipid productivity [21]. Biomass and neutral lipid content production were measured during the semicontinuous phase and the maturation phase. We found that this strategy successfully generated cultures with high neutral lipid content while at the same time ensuring high biomass production and limiting the use of photobioreactor space in the first two growth stages. 2. Materials and methods 2.1. Microalgal culture S. marinoi stock cultures were grown in f/2 medium [22,23] at 21 ± 1 °C, with a dark:light cycle of 12:12 h (Photosynthetic Active Radiation, PAR = 1.5 W/m2). 2.2. First culture phase: biomass production The three-stage growth strategy is illustrated in Fig. 1. A modified f/2 medium was used for the first phase of biomass production. The composition of this medium, which had been previously identified in our laboratory for optimal biomass and neutral lipid production, requires a double concentration of phosphate (10 mg/l) and a triple concentration of silicate and nitrate (900 mg/l and 225 mg/l respectively) compared to the standard f/2 composition [22,23]. Vitamins and trace metals were added according to the standard recipe. All components were added to 0.45 μm filtered natural seawater from the NW Adriatic. The final salinity of the culture media was adjusted by adding 10% distilled water. The inoculum was performed in the bench photobioreactor (PBR 2S — Stedim Sartorius) at a final concentration of 50,000 cells/ml in a total volume of 2.7 l of modified f/2 medium.
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The PBR 2S photobioreactor is a closed glass system in which microalgae are kept in suspension thanks to a peristaltic pump. The pH is maintained at 8.00 through active pH control (0.5 M NaOH and CO2 directly bubbled into the culture). At the moment of culture inoculum, 0.6 M NaHCO3 was added to the medium as an additional buffer. These growth conditions were maintained for 7 days at a 12–12 hour light–dark cycle with a light intensity of 5 W/m2 (Photosyntetic Active Radiation) without further manipulations. 2.3. Second culture phase: semicontinuous regimen with gradual reduction of nitrate concentration After 7 days of growth in the conditions described above, the second phase of the culture was carried out as follows: every day, for a week, half of the culture (1.350 l) was removed and replaced with the same volume of fresh f/2 medium without nitrate. 2.4. Third culture phase: sample maturation At each dilution step, a 600 ml aliquot from the subtracted culture volume was placed in a 1 l flask for the third experimental phase generating four “satellite” cultures called T1, T2, T3 and T4. T1, T2, T3 and T4 were grown for an additional 8 days at the same temperature and under the same light conditions (See 2.1 section) and tested at three different time points: day 1 (start), day 3 or 4 (intermediate), and day 8 (final) for biomass and neutral lipid content. 2.5. Nitrate analysis In order to confirm the effective dilution of nitrate in culture medium, culture supernatants were analyzed using a simple and rapid spectrophotometric method before and after the daily dilution in the second phase [24]. In this method, the absorbance of the samples at 220 nm (A220nm), is corrected for the absorbance at 275 nm (A275nm), which is related to organic nitrate absorption, using the following formula. A ¼ A220nm –2 A275nm : Spectrophotometric (analysis spectrophotometer UV-2401 PC, Shimadzu, Japan) was performed as follows: a 3 ml aliquot of each supernatant was filtered (0.22* μm) and acidified using 150 μl of 1 N HCl. The acidified sample was then transferred into a quartz cuvette for absorbance readings at the appropriate wavelengths. Serial dilution of NaNO3 was used for standard curve. 2.6. Fluorometric determination of neutral lipid content At each time point, three culture aliquots of 1.90 ml were collected and centrifuged at 3500 g for 20 min. The analysis of neutral lipid content was performed on the pellets using the fluorescent stain Nile red in association with the standard addition method as previously described [25]. The spectrofluorometric determinations were performed using a spectrofluorophotometer RF-5301PC (Shimadzu, Japan). Neutral lipid content expressed as % of dry weight was calculated dividing the amount of neutral lipids by the biomass expressed as mg/l (see Section 2.7). 2.7. Biomass determination
Fig. 1. Diagram of the three-stage growth strategy used for large scale cultivation of S. marinoi CBA4 at reduced nitrate concentrations.
Biomass values, expressed as cell concentrations, were determined on three replicates of 1 ml culture using spectrophotometric analysis (spectrophotometer UV-2401 PC, Shimadzu, Japan),[17]. Since the linear correlation between optical density (OD) and cell concentration is guaranteed for OD b0.25 [17], when needed, samples were appropriately diluted using artificial seawater.
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E. Bertozzini et al. / Algal Research 5 (2014) 32–36
In order to express biomass content as mg/l, cell concentrations were multiplied by 50 pg, which is the average dry weight of the S. marinoi cell [26]. 2.8. Statistics Each culture condition was analyzed on three technical replicates. Reported values are always the means of three values ± standard deviation. Statistical significance of the results was tested with Tukey's Multiple Comparative test (GraphPad-InStat 3.06 version). 3. Results 3.1. First phase: biomass production At the end of the first phase, the cell concentration was 2.06 × 109 ± 1.29 × 106 cells/l, with a neutral lipid content of 11.46 ± 0.25 mg/l corresponding to the 10.70 ± 0.23% of the dry weight. 3.2. Second phase: semicontinuous phase The nitrate analysis of culture supernatants before and after each dilution during the second phase of the experiment is shown in Table 1. During the semicontinuous regimen, S. marinoi continued growing until day 10 (Fig. 2 a), when the division rate was severely affected by the gradual decrease of nitrate concentration in the culture media (Table 2). The neutral lipid content of the culture in the photobioreactor showed the same trend observed for the biomass, and when the reduced nitrate concentration induced a decrease in biomass, neutral lipid content also decreased (Fig. 2 a). Nevertheless, in the last phase of the experiment, when biomass decreased, the percentage of neutral lipids respect to the dry weight increased significantly from 10 to 15% (Fig. 2 b). 3.3. Third phase — maturation During the semicontinuous regimen phase, each dilution generated a satellite culture that was grown for an additional 8 days without further manipulations. Each culture was tested for biomass and neutral lipid production at the start, after 3–4 days and at the end of this phase. Biomass trends and the values of T1, T2 and T3 cultures were very similar (Fig. 3): all cultures showed a peak at the intermediate time (day 3) and there were no significant differences among those values (p N 0.05), although the T3 culture biomass appeared slightly lower at the intermediate time. On the contrary, the T4 culture showed the typical trend of a culture in the stationary phase. Regarding neutral lipid content, the T1 and T2 cultures showed the lowest neutral lipid content as percentage of dry weight (Fig. 4 b). These cultures had the highest division rate (0.25 div/day from the start to the intermediate time). This is probably because the maturation
Table 1 Nitrate concentrations in culture supernatants before and after dilution, during the semicontinuous stage (see Section 2.3). Day
8 9 10 11
Nitrate concentration (μM) Before dilution
After dilution
1670.15 537.50 21.03 18.48
818.38 253.68 61.32 23.38
± ± ± ±
14.29 2.04 3.10(1) 5.03(1)
± ± ± ±
10.34 0.88 3.06(2) 1.53(1,2)
(1) In these samples A275nm N 10% of A220nm. At this condition the high level of organic nitrate could slightly affect the accuracy of the test [24]. (2) The increase in nitrate concentration is due to the addition of natural seawater that had a higher nitrate concentration than the culture media.
Fig. 2. Biomass and neutral lipids contents expressed as mg/l (2a) or as a percentage of dry weight (2b) in S. marinoi CBA4 culture at the second stage of the three-stage growth strategy (see Section 2.1). The values are means ± standard deviation of three technical replicates.
step of T1 and T2 cultures started from nitrate concentrations in culture media that were higher than those in the other cultures (Table 1). The T3 culture, which had a lower division rate (0.08 div/day from day 1 to day 3) and was withdrawn at a lower nitrate concentration, showed a significantly higher percentage of neutral lipid content than T1 and T2 at each time point. The T4 culture, which was in the stationary phase (Fig. 3), had the highest percentage of neutral lipid content of all the cultures and, although its biomass content was lower than that of the other cultures, at the end of the experiment it showed a neutral lipid content expressed as mg/l comparable to that of culture T3 (Fig. 4 a). 4. Discussion The success of microalgae as a feedstock for biodiesel production strongly depends on the ability to produce large amounts of biomass with high neutral lipid content. Growth strategies that involve nutrient depletion, in particular nitrogen depletion, are the most promising for increasing lipid productivity but, on the other hand, strongly affect biomass [27,28]. Several growth strategies have been investigated to reduce the negative effects of nutrient depletion, for example, two-stage growth
Table 2 Div/day in S. marinoi culture during the semicontinuous phase. Time range
Div/daya
From day 8 to day 9 From day 9 to day 10 From day 10 to day 11 From day 11 to day 12
0.8 0.5 −0.2 −0.8
a Div/day = (log2 Nt − log2 N0)/t. Nt = cells concentration at day “t”; N0 = initial cells concentration, t = days.
E. Bertozzini et al. / Algal Research 5 (2014) 32–36
Fig. 3. Biomass values of the four S. marinoi CBA4 cultures at the third stage of the threestage growth strategy (see Section 2.1). Values are means ± standard deviation of three technical replicates. ***: highly significantly different from the other conditions (p b 0.001).
strategies, with a first phase for biomass production and a second phase for neutral lipid accumulation [18], or the gradual reduction of nitrate in the culture media through a semicontinuous regimen [17]. Our new approach consists of a three-stage growth strategy. In the first phase, microalgae are grown to achieve high levels of biomass using an enriched f/2 medium. In the second phase, cells continue to grow but the nitrate concentration is gradually reduced through daily dilution of the cultures in a semicontinuous regimen. In the third phase, when low nitrogen levels have been achieved, microalgae are grown for an additional period to further increase biomass and to induce neutral lipid accumulation related to culture aging [21]. The first and second phases were performed in a closed system using a bench photobioreactor, while for the last phase the culture was transferred in batch. In the second growth phase, both culture and nitrate concentration in culture media were half-diluted, and biomass, neutral lipid content and nitrate concentration were measured at each dilution time (Fig. 2 a–b). In some cases, the dilution factors of biomass and nitrate concentration did not match perfectly. This could be due to several factors: concerning biomass, it is possible that, despite the circulation of the culture in the photobioreactor, a non homogeneous withdrawal could have been performed. Regarding nitrate concentration, the f/2 medium, used for each dilution and prepared using natural and not artificial seawater,
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was not absolutely free of nitrate. In addition, it is noteworthy that, especially in the final stages of the semicontinuous phase, the values of nitrate concentration detected with the spectrophotometric method could be less accurate due to the high level of organic nitrate in the culture. In any case, during the semicontinuous phase, low satisfactory levels of nitrate concentration were achieved. Indeed, the percentage of neutral lipids in the cells gradually increased from day 9 to the end of this phase. During the second phase, the removed fraction of the culture was transferred in batches for the last maturation phase, at each dilution time, and the four derived cultures showed different behaviors. T1 and T2 cultures did not appear to be influenced by nitrate depletion based on the parameters that we monitored during the experiment. Indeed, they did not show any stress. During the eight days of maturation their biomass showed an increase followed by a natural decline related to culture aging, and the neutral lipid content in these cultures did not increase (Figs. 3 and 4). This may stem from the fact that these cultures were derived from the first two dilutions of the culture in a photobioreactor, and nitrate concentration in T1 and T2 culture supernatant was still 1.67 mM, and 537 μM, respectively (Table 1). On the contrary, T3 and T4 cultures started their third phase at very low levels of nitrate concentration (21 and 18 μM respectively) and showed interesting behaviors. The T4 culture was in the stationary phase, with slight variations in biomass values during the seven days of maturation (Fig. 3). The percentage of neutral lipids in this culture was consistently significantly higher than percentages measured in the other cultures, and although biomass values were always significantly lower than those measured in the other cultures, the neutral lipid content of this culture was consistently higher. Nevertheless, the best results were achieved with the T3 culture. In this culture the biomass trend was never significantly different from that of T1 and T2 (Fig. 3), and the neutral lipid content, expressed as percentage of dry weight, was significantly higher (p b 0.001) than that found in the other cultures (Fig. 4 a and b). The neutral lipid contents of the T3 and T4 cultures were very similar at the end of the third phase, but the T3 culture showed the best results in terms of biomass. This new approach, which combines the use of a photobioreactor and batch culturing, can be scaled up and is therefore suitable for large scale cultivation. In this case, the aliquots subtracted for the culture dilution in the firsts days of the semicontinuous regimen, not ready to start the maturation phases (T1 and T2), might be employed in different ways. As an example, they could be transferred in parallel photobioreactors to carry on the same culture strategy or, as an alternative, it should be possible to perform, instead of semicontinuous regimen a semicontinuous harvesting regimen as described in our previous work [17]. In this way no part of the culture would be lost or underemployed. In conclusion, it has been demonstrated that biomass gradually acclimatized, even for 3–4 days, to low nitrogen concentration in a semicontinuous regimen, can be grown in batch culture conditions to further increase biomass and neutral lipid content, reaching, in our case, a tryglyceride content which ranged from 20% to 30% of the dry weight. In addition, since microalgal growth in photobioreactors is productive but very expensive because of the energy required [29], this growth strategy, which limits the use of the photobioreactor to the first phases of growth, may also be more cost effective than other strategies. Acknowledgments This work was funded by the Department of Biomolecular Science of the University of Urbino “Carlo Bo”.
Fig. 4. Neutral lipids content expressed as mg/l (4a) or as a percentage of dry weight (4b) in the four S. marinoi CBA4 cultures at the third stage of the three-stage growth strategy (see Section 2.1). Values are means ± standard deviation of three technical replicates. **: significantly different from the other conditions (p b 0.01) ***: highly significantly different compared to the other conditions (p b 0.001).
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Contents lists available at ScienceDirect
Algal Research journal homepage: www.elsevier.com/locate/algal
Enhancing neutral lipid content in Skeletonema marinoi through multiple phase growth in a bench photobioreactor Elena Bertozzini a,⁎, Luca Galluzzi a, Antonella Penna b, Mauro Magnani c a b c
Department of Biomolecular Sciences, Biotechnology Section, University of Urbino, Via Arco D'Augusto 2, 61032, Fano PU, Italy Department of Biomolecular Sciences, Environmental Biology Section, University of Urbino, Viale Trieste 296, 61121 Pesaro, PU, Italy Department of Biomolecular Sciences, University of Urbino, Via Saffi 2, 61029 Urbino, PU, Italy
a r t i c l e
i n f o
Article history: Received 31 July 2013 Received in revised form 14 March 2014 Accepted 9 May 2014 Available online xxxx Keywords: Biofuel Neutral lipids Nitrogen starvation Bench photobioreactor Growth strategy
a b s t r a c t The continuing increase in fuel demand, the dramatic situation of climate changes and global warming are bringing worldwide attention to identification of alternative energy source that can replace fossil fuel. Among all the feedstock considered for biodiesel production, microalgae are the most promising. In particular, it has been demonstrated that microalgae enhance their neutral lipids content in stress conditions, such as nutrient starvation. However, this increase in neutral lipids content is often coupled with a strong biomass decrease. In this study, in order to limit this side effect, a novel three-phase growth strategy was tested on the diatom Skeletonema marinoi. After a first phase directed to biomass production the culture, during a second phase of semi-continuous regimen characterized with reduction of nitrate concentration, achieved a neutral lipids content per cell of 10% of dry weight with slight impact on biomass. After these two phases the cells, gradually acclimatized to low nitrogen concentration, were still able to grow in a third maturation phase performed in batch. During this last phase, the culture further increased neutral lipid content up to the 30 % of dry weight. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The International Energy Agency (IEA) estimated the total primary energy supply in 2010 at 12,275 million tonnes of oil equivalent, with a further increase respect to the 2009 value. Fossil fuels, i.e. coal, oil and natural gas, constitute more than the 80% of this total energy supply [1]. These feedstocks are non-renewable natural resources, and they are one of the major contributors to the greenhouse effect and global warming. A great deal of attention across the globe has therefore been directed toward the identification of new, renewable energy sources that could be an alternative to fossil fuels. Biodiesel is one of those alternative sources [2]. The first crops used to produce biodiesel were edible crops such as rapeseed oil, sugarcane, sugar beet and maize [3], but the use of these feedstocks for biodiesel production would have a negative impact on global food markets and food security. Hence, a second generation of biodiesel feedstock was identified in inedible plants such as jatropha, mahua and tobacco seeds. However, to ensure high oil yield from these feedstocks, heavy fertilization, irrigation and good management practices are required [4], thus efforts to find a more sustainable biodiesel feedstock are now focused on microalgae [3].
⁎ Corresponding author. Tel.: +39 0722 304976; fax: +39 0722 304970. E-mail address: [email protected] (E. Bertozzini).
http://dx.doi.org/10.1016/j.algal.2014.05.004 2211-9264/© 2014 Elsevier B.V. All rights reserved.
Microalgae are considered one of the most promising biodiesel feedstocks due to their high growth rate and their ability to produce high amounts of neutral lipids and carbohydrates for biodiesel and bioethanol productions [5]. Moreover, microalgae can potentially produce more oil per acre than other feedstocks [6,7], they are not edible and can be grown on non-arable land [2], and they fix CO2 more efficiently than other plants and could potentially reduce the greenhouse effect through biofixation of waste carbon dioxide [6,8]. However, despite their great potential as a biodiesel feedstock, microalgae have yet to be exploited, in particular on the basis of Life Cycle Analysis (LCA). The LCA, defined from the ISO 14040.2 standard, quantifies the environmental impact of the processes that contribute to the supply of goods or services also considering how much fossil fuel energy is used for system maintenance [9]. In particular, the success of microalgae as a biodiesel feedstock strongly depends on the optimization of appropriate culture conditions that make it possible to produce high biomass and large amounts of neutral lipids [9,10]. Different approaches have been tested to achieve such conditions, and it has been clearly shown that nutrient limitation, in particular nitrogen limitation, enhances neutral lipid production however, it severely limits biomass production [11–16]. Several growth strategies have been proposed to limit this side effect. For example, it has been demonstrated that a gradual reduction of nitrate concentration in a semicontinuous regimen enhances the neutral lipid content of Skeletonema marinoi batch cultures with slight effects on biomass [17].
E. Bertozzini et al. / Algal Research 5 (2014) 32–36
Another approach designed to reduce the negative effect of nutrient limitation strategies on biomass production, consists in a two-stage process. The first phase aims to achieve maximum biomass production, while in the second stage stress factors are introduced to increase neutral lipid content [8,18,19]. In this study, the diatom S. marinoi was used as a model in a bench photobioreactor to test a novel three-phase growth strategy that combines the two different strategies described above. In the first growth phase S. marinoi was grown in enriched f/2 medium, in order to obtain high biomass, in the second semicontinuous regimen-phase the nitrate concentration was gradually reduced to increase neutral lipid content [20], and in the final “maturation” stage, cultures were grown for an additional week without manipulation to further increase the neutral lipid content and biomass of the culture. In fact, recent literature has demonstrated that the association of culture aging with nitrogen depletion could further enhance neutral lipid productivity [21]. Biomass and neutral lipid content production were measured during the semicontinuous phase and the maturation phase. We found that this strategy successfully generated cultures with high neutral lipid content while at the same time ensuring high biomass production and limiting the use of photobioreactor space in the first two growth stages. 2. Materials and methods 2.1. Microalgal culture S. marinoi stock cultures were grown in f/2 medium [22,23] at 21 ± 1 °C, with a dark:light cycle of 12:12 h (Photosynthetic Active Radiation, PAR = 1.5 W/m2). 2.2. First culture phase: biomass production The three-stage growth strategy is illustrated in Fig. 1. A modified f/2 medium was used for the first phase of biomass production. The composition of this medium, which had been previously identified in our laboratory for optimal biomass and neutral lipid production, requires a double concentration of phosphate (10 mg/l) and a triple concentration of silicate and nitrate (900 mg/l and 225 mg/l respectively) compared to the standard f/2 composition [22,23]. Vitamins and trace metals were added according to the standard recipe. All components were added to 0.45 μm filtered natural seawater from the NW Adriatic. The final salinity of the culture media was adjusted by adding 10% distilled water. The inoculum was performed in the bench photobioreactor (PBR 2S — Stedim Sartorius) at a final concentration of 50,000 cells/ml in a total volume of 2.7 l of modified f/2 medium.
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The PBR 2S photobioreactor is a closed glass system in which microalgae are kept in suspension thanks to a peristaltic pump. The pH is maintained at 8.00 through active pH control (0.5 M NaOH and CO2 directly bubbled into the culture). At the moment of culture inoculum, 0.6 M NaHCO3 was added to the medium as an additional buffer. These growth conditions were maintained for 7 days at a 12–12 hour light–dark cycle with a light intensity of 5 W/m2 (Photosyntetic Active Radiation) without further manipulations. 2.3. Second culture phase: semicontinuous regimen with gradual reduction of nitrate concentration After 7 days of growth in the conditions described above, the second phase of the culture was carried out as follows: every day, for a week, half of the culture (1.350 l) was removed and replaced with the same volume of fresh f/2 medium without nitrate. 2.4. Third culture phase: sample maturation At each dilution step, a 600 ml aliquot from the subtracted culture volume was placed in a 1 l flask for the third experimental phase generating four “satellite” cultures called T1, T2, T3 and T4. T1, T2, T3 and T4 were grown for an additional 8 days at the same temperature and under the same light conditions (See 2.1 section) and tested at three different time points: day 1 (start), day 3 or 4 (intermediate), and day 8 (final) for biomass and neutral lipid content. 2.5. Nitrate analysis In order to confirm the effective dilution of nitrate in culture medium, culture supernatants were analyzed using a simple and rapid spectrophotometric method before and after the daily dilution in the second phase [24]. In this method, the absorbance of the samples at 220 nm (A220nm), is corrected for the absorbance at 275 nm (A275nm), which is related to organic nitrate absorption, using the following formula. A ¼ A220nm –2 A275nm : Spectrophotometric (analysis spectrophotometer UV-2401 PC, Shimadzu, Japan) was performed as follows: a 3 ml aliquot of each supernatant was filtered (0.22* μm) and acidified using 150 μl of 1 N HCl. The acidified sample was then transferred into a quartz cuvette for absorbance readings at the appropriate wavelengths. Serial dilution of NaNO3 was used for standard curve. 2.6. Fluorometric determination of neutral lipid content At each time point, three culture aliquots of 1.90 ml were collected and centrifuged at 3500 g for 20 min. The analysis of neutral lipid content was performed on the pellets using the fluorescent stain Nile red in association with the standard addition method as previously described [25]. The spectrofluorometric determinations were performed using a spectrofluorophotometer RF-5301PC (Shimadzu, Japan). Neutral lipid content expressed as % of dry weight was calculated dividing the amount of neutral lipids by the biomass expressed as mg/l (see Section 2.7). 2.7. Biomass determination
Fig. 1. Diagram of the three-stage growth strategy used for large scale cultivation of S. marinoi CBA4 at reduced nitrate concentrations.
Biomass values, expressed as cell concentrations, were determined on three replicates of 1 ml culture using spectrophotometric analysis (spectrophotometer UV-2401 PC, Shimadzu, Japan),[17]. Since the linear correlation between optical density (OD) and cell concentration is guaranteed for OD b0.25 [17], when needed, samples were appropriately diluted using artificial seawater.
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In order to express biomass content as mg/l, cell concentrations were multiplied by 50 pg, which is the average dry weight of the S. marinoi cell [26]. 2.8. Statistics Each culture condition was analyzed on three technical replicates. Reported values are always the means of three values ± standard deviation. Statistical significance of the results was tested with Tukey's Multiple Comparative test (GraphPad-InStat 3.06 version). 3. Results 3.1. First phase: biomass production At the end of the first phase, the cell concentration was 2.06 × 109 ± 1.29 × 106 cells/l, with a neutral lipid content of 11.46 ± 0.25 mg/l corresponding to the 10.70 ± 0.23% of the dry weight. 3.2. Second phase: semicontinuous phase The nitrate analysis of culture supernatants before and after each dilution during the second phase of the experiment is shown in Table 1. During the semicontinuous regimen, S. marinoi continued growing until day 10 (Fig. 2 a), when the division rate was severely affected by the gradual decrease of nitrate concentration in the culture media (Table 2). The neutral lipid content of the culture in the photobioreactor showed the same trend observed for the biomass, and when the reduced nitrate concentration induced a decrease in biomass, neutral lipid content also decreased (Fig. 2 a). Nevertheless, in the last phase of the experiment, when biomass decreased, the percentage of neutral lipids respect to the dry weight increased significantly from 10 to 15% (Fig. 2 b). 3.3. Third phase — maturation During the semicontinuous regimen phase, each dilution generated a satellite culture that was grown for an additional 8 days without further manipulations. Each culture was tested for biomass and neutral lipid production at the start, after 3–4 days and at the end of this phase. Biomass trends and the values of T1, T2 and T3 cultures were very similar (Fig. 3): all cultures showed a peak at the intermediate time (day 3) and there were no significant differences among those values (p N 0.05), although the T3 culture biomass appeared slightly lower at the intermediate time. On the contrary, the T4 culture showed the typical trend of a culture in the stationary phase. Regarding neutral lipid content, the T1 and T2 cultures showed the lowest neutral lipid content as percentage of dry weight (Fig. 4 b). These cultures had the highest division rate (0.25 div/day from the start to the intermediate time). This is probably because the maturation
Table 1 Nitrate concentrations in culture supernatants before and after dilution, during the semicontinuous stage (see Section 2.3). Day
8 9 10 11
Nitrate concentration (μM) Before dilution
After dilution
1670.15 537.50 21.03 18.48
818.38 253.68 61.32 23.38
± ± ± ±
14.29 2.04 3.10(1) 5.03(1)
± ± ± ±
10.34 0.88 3.06(2) 1.53(1,2)
(1) In these samples A275nm N 10% of A220nm. At this condition the high level of organic nitrate could slightly affect the accuracy of the test [24]. (2) The increase in nitrate concentration is due to the addition of natural seawater that had a higher nitrate concentration than the culture media.
Fig. 2. Biomass and neutral lipids contents expressed as mg/l (2a) or as a percentage of dry weight (2b) in S. marinoi CBA4 culture at the second stage of the three-stage growth strategy (see Section 2.1). The values are means ± standard deviation of three technical replicates.
step of T1 and T2 cultures started from nitrate concentrations in culture media that were higher than those in the other cultures (Table 1). The T3 culture, which had a lower division rate (0.08 div/day from day 1 to day 3) and was withdrawn at a lower nitrate concentration, showed a significantly higher percentage of neutral lipid content than T1 and T2 at each time point. The T4 culture, which was in the stationary phase (Fig. 3), had the highest percentage of neutral lipid content of all the cultures and, although its biomass content was lower than that of the other cultures, at the end of the experiment it showed a neutral lipid content expressed as mg/l comparable to that of culture T3 (Fig. 4 a). 4. Discussion The success of microalgae as a feedstock for biodiesel production strongly depends on the ability to produce large amounts of biomass with high neutral lipid content. Growth strategies that involve nutrient depletion, in particular nitrogen depletion, are the most promising for increasing lipid productivity but, on the other hand, strongly affect biomass [27,28]. Several growth strategies have been investigated to reduce the negative effects of nutrient depletion, for example, two-stage growth
Table 2 Div/day in S. marinoi culture during the semicontinuous phase. Time range
Div/daya
From day 8 to day 9 From day 9 to day 10 From day 10 to day 11 From day 11 to day 12
0.8 0.5 −0.2 −0.8
a Div/day = (log2 Nt − log2 N0)/t. Nt = cells concentration at day “t”; N0 = initial cells concentration, t = days.
E. Bertozzini et al. / Algal Research 5 (2014) 32–36
Fig. 3. Biomass values of the four S. marinoi CBA4 cultures at the third stage of the threestage growth strategy (see Section 2.1). Values are means ± standard deviation of three technical replicates. ***: highly significantly different from the other conditions (p b 0.001).
strategies, with a first phase for biomass production and a second phase for neutral lipid accumulation [18], or the gradual reduction of nitrate in the culture media through a semicontinuous regimen [17]. Our new approach consists of a three-stage growth strategy. In the first phase, microalgae are grown to achieve high levels of biomass using an enriched f/2 medium. In the second phase, cells continue to grow but the nitrate concentration is gradually reduced through daily dilution of the cultures in a semicontinuous regimen. In the third phase, when low nitrogen levels have been achieved, microalgae are grown for an additional period to further increase biomass and to induce neutral lipid accumulation related to culture aging [21]. The first and second phases were performed in a closed system using a bench photobioreactor, while for the last phase the culture was transferred in batch. In the second growth phase, both culture and nitrate concentration in culture media were half-diluted, and biomass, neutral lipid content and nitrate concentration were measured at each dilution time (Fig. 2 a–b). In some cases, the dilution factors of biomass and nitrate concentration did not match perfectly. This could be due to several factors: concerning biomass, it is possible that, despite the circulation of the culture in the photobioreactor, a non homogeneous withdrawal could have been performed. Regarding nitrate concentration, the f/2 medium, used for each dilution and prepared using natural and not artificial seawater,
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was not absolutely free of nitrate. In addition, it is noteworthy that, especially in the final stages of the semicontinuous phase, the values of nitrate concentration detected with the spectrophotometric method could be less accurate due to the high level of organic nitrate in the culture. In any case, during the semicontinuous phase, low satisfactory levels of nitrate concentration were achieved. Indeed, the percentage of neutral lipids in the cells gradually increased from day 9 to the end of this phase. During the second phase, the removed fraction of the culture was transferred in batches for the last maturation phase, at each dilution time, and the four derived cultures showed different behaviors. T1 and T2 cultures did not appear to be influenced by nitrate depletion based on the parameters that we monitored during the experiment. Indeed, they did not show any stress. During the eight days of maturation their biomass showed an increase followed by a natural decline related to culture aging, and the neutral lipid content in these cultures did not increase (Figs. 3 and 4). This may stem from the fact that these cultures were derived from the first two dilutions of the culture in a photobioreactor, and nitrate concentration in T1 and T2 culture supernatant was still 1.67 mM, and 537 μM, respectively (Table 1). On the contrary, T3 and T4 cultures started their third phase at very low levels of nitrate concentration (21 and 18 μM respectively) and showed interesting behaviors. The T4 culture was in the stationary phase, with slight variations in biomass values during the seven days of maturation (Fig. 3). The percentage of neutral lipids in this culture was consistently significantly higher than percentages measured in the other cultures, and although biomass values were always significantly lower than those measured in the other cultures, the neutral lipid content of this culture was consistently higher. Nevertheless, the best results were achieved with the T3 culture. In this culture the biomass trend was never significantly different from that of T1 and T2 (Fig. 3), and the neutral lipid content, expressed as percentage of dry weight, was significantly higher (p b 0.001) than that found in the other cultures (Fig. 4 a and b). The neutral lipid contents of the T3 and T4 cultures were very similar at the end of the third phase, but the T3 culture showed the best results in terms of biomass. This new approach, which combines the use of a photobioreactor and batch culturing, can be scaled up and is therefore suitable for large scale cultivation. In this case, the aliquots subtracted for the culture dilution in the firsts days of the semicontinuous regimen, not ready to start the maturation phases (T1 and T2), might be employed in different ways. As an example, they could be transferred in parallel photobioreactors to carry on the same culture strategy or, as an alternative, it should be possible to perform, instead of semicontinuous regimen a semicontinuous harvesting regimen as described in our previous work [17]. In this way no part of the culture would be lost or underemployed. In conclusion, it has been demonstrated that biomass gradually acclimatized, even for 3–4 days, to low nitrogen concentration in a semicontinuous regimen, can be grown in batch culture conditions to further increase biomass and neutral lipid content, reaching, in our case, a tryglyceride content which ranged from 20% to 30% of the dry weight. In addition, since microalgal growth in photobioreactors is productive but very expensive because of the energy required [29], this growth strategy, which limits the use of the photobioreactor to the first phases of growth, may also be more cost effective than other strategies. Acknowledgments This work was funded by the Department of Biomolecular Science of the University of Urbino “Carlo Bo”.
Fig. 4. Neutral lipids content expressed as mg/l (4a) or as a percentage of dry weight (4b) in the four S. marinoi CBA4 cultures at the third stage of the three-stage growth strategy (see Section 2.1). Values are means ± standard deviation of three technical replicates. **: significantly different from the other conditions (p b 0.01) ***: highly significantly different compared to the other conditions (p b 0.001).
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