1 Introduction

In recent decades, energy crisis and global warming induced by CO₂ emissions are worldwide environmental problems (Kan et al., 2019). Biodiesel is a compatible alternative to fossil fuel, which is biodegradable, renewable, and clean-burning (Mwangi et al., 2015). Microalgae are among the most promising feedstock for biodiesel production (Chisti, 2007). They can assimilate and convert CO₂ from flue gas into biomass containing high amounts of lipids for subsequent biodiesel production (Parupudi et al., 2016); however, several challenges need to be overcome before microalgae can be successfully used for CO₂ biofixation and biodiesel production. A typical power plant flue contains 5%-15% CO₂ (Vuppaladadiyam et al., 2018), accompanied by toxic associated gases such as SO₂ (300 to 500 ppm), as well as high temperatures (more than 150℃) (Yoshihara et al., 1996; Kao et al., 2014). A continuous ventilation of flue gas will result in the decrease of pH in the liquid medium, which has an inhibitory effect on autotrophic algal growth (Ronda et al., 2014). These stresses are difficult to control when microalgae are cultivated in such a system. Therefore, the screening to find a suitable microalgae strain with high tolerance to the low pH, high temperature, and high CO₂ regime should be the premise for the CO₂ fixation from flue gas.

Microalgae of the genus Nannochloropsis have gradually become a research focus for those exploring their potential for CO₂ biofixation and biofuel production due to their relatively high photosynthetic efficiency, fast growth rates, and high lipid contents (Huang et al, 2013; Bartley et al., 2014). So far, five different Nannochloropsis species (N. gaditana, N. salina, N. granulata, N. oceanica, and N. oculata), recognized in saline habitats, are proposed to produce biodiesel (Chiu et al., 2009; Cai et al., 2013; Ma et al., 2014; Taleb et al., 2015; Cancela et al., 2019; Moraesa et al., 2019). However, the literature on freshwater Nannochloropsis species is limited. Moreover, the lipid accumulated in Nannochloropsis has high content of polyunsaturated fatty acids (PUFA) (Krienitz et al., 2006), which could be used in biotechnology and aquaculture.

In this context, the objective of this study was to evaluate the ability of Nannochloropsis sp. MASCC 11, a unicellular freshwater alga, to cope with different levels of CO₂, pH values, and temperatures, respectively, and algal growth and lipid accumulation were measured in this study. In order to determine the suitability of biomass formed under different CO₂ concentrations for biodiesel production, fatty acid composition of microalgae was also studied.

2 Methods 2.1 Microalgal Strain, Growth Medium, and Pre-Cultivation

Nannochloropsis sp. MASCC 11 was obtained from the Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China, and pre-cultured in 500 mL Erlenmeyer flasks at 25℃ with continuous illumination. Light intensity was set at 60 μmol photons m⁻² s⁻¹ and was measured by a portable light quantum meter (3415F type, pulse photoelectric sensor; Spectrum Technologies, Inc., USA). The flasks were manually shaken three times a day. The BG11 medium (Stanier et al., 1971) was used for the cultivation. Algal cells in the exponential growth phase were used for subsequent experiments.

2.2 Microalgal Tolerance Experiments 2.2.1 Low pH

Erlenmeyer flasks (500 mL) containing 300 mL BG11 medium were employed to study the effect of pH on growth of the microalga. The initial cell density was 1.5 × 10⁶ cells mL^–1. The pH values of the media were adjusted to 3.0, 3.5, 4.0, and 6.0 by adding 0.1 mol L^–1 H₂SO₄ after inoculation, then transferred they to the flasks. Then the algal cultures were incubated in an illuminated incubator with orbital shakers (PGX-250D-LED, Jiangsu Allison Instrument Manufacturing Co., Ltd., China) at 150 r min⁻¹. The other conditions used for culture were: continuousday (24-h light/0-h dark) illumination (120 μmol photons m⁻² s⁻¹) and a temperature of 25℃. During cultivation, the pH was controlled by a pH Stat System (Schott, Mainz, Germany) with an H₂SO₄ solution supplementary to the culture to maintain a constant pH. BG11 medium without H₂SO₄ addition served as the control (Initial pH, 9.0). All experiments were carried out in triplicate and ran for 10 d. At the end of cultivation, samples collected from each culture were used to determine the cell density, biomass (dry weight), and total lipid content. The specific growth rate and total lipid productivity were also calculated.

2.2.2 Temperature

To study the effect of temperature, experiments were performed at 20, 25, 30, 35, and 40℃ in illuminated incubators. The pH values of algal culture were maintained at 9.0. The other conditions and growth/lipid parameters were determined as outlined in Section 2.2.1.

2.2.3 CO₂ concentration

Microalgae were inoculated to a bubble bioreactor comprising a 500 mL Erlenmeyer flask containing 300 mL BG11 medium (Initial pH, 9.0). The filtered air, supplemented with CO₂, was bubbled into the culture medium through a glass tube (2 mm i.d.) at a fixed aeration rate of 0.1 vvm (i.e., air volume/medium volume/min). CO₂ concentrations in the bubbled gas stream were regulated to 0.04% (air without excess CO₂), 5%, 10%, and 15% for different treatments. The temperature, inoculation density, duration and other conditions were as described in Section 2.2.1. Determination of sample pH was undertaken every day. At the end of cultivation, the microalgae were harvested and their biomasses, lipid contents, as well as total fatty acid concentrations and compositions were analyzed.

2.3 Analyses 2.3.1 Biomass

Dry weight was measured according to the method described by Ho et al. (2013). Microalgal culture (10 mL) was filtered through a 0.45 μm cellulose acetate membrane filter. Each pre-weighed loaded filter was then dried at 60℃ until a constant mass was reached. The microalgae biomass B (g L⁻¹) was calculated based on the following equation:

$ B=\frac{W_{2}-W_{1}}{V}, $

(1)

where W₁ is the mass of the blank filter (g), W₂ the mass of the filter loaded with microalgal cells (g), and V the volume of the culture (0.01 L).

2.3.2 Cell density and specific growth rate

Cell densities were measured by counting the number of cells using a hemocytometer (Marienfeld, Germany). The specific growth rate μ (d^–1) was calculated based on the following equation:

$ \mu=\frac{\ln N_{2}-\ln N_{1}}{t_{2}-t_{1}}, $

(2)

where N₂ and N₁ are the cell densities (cells mL⁻¹) at times t₂ and t₁ in the exponential growth phase, respectively.

2.3.3 Lipid content and lipid production of Nannochloropsis sp.

Cultures were centrifuged at 5000 r min⁻¹ for 10 min at 4℃. The resulting microalgal pellet was washed twice with distilled water to remove salts, and freeze dried at −50℃. Total lipid content was measured according to a modified method by Bligh and Dyer (1957). Briefly, 100 mg of freeze-dried microalgae were extracted with a 7.6 mL mixture of chloroform, methanol, and water (2.5/5/2, v/v) for 24 h at room temperature, followed by sonication (700 W, 5 s working, 5 s rest) for 2 min. After adding 1 mL of chloroform and 1 mL of water, the mixture was centrifuged at 5000 r min⁻¹ for 10 min to form three layers. The organic phase was collected into a pre-weighed glass tube and evaporated to dryness under nitrogen gas flow for weighing. The total lipid content ω (%) was calculated according to the following equation:

$ \omega=\frac{W_{1}}{W_{0}} \times 100 \%, $

(3)

where W₁ and W₀ are the lipid masses (g) and the dry cell masses (g), respectively.

The lipid production LP (g L⁻¹) of the microalgae was deduced from the following equation:

$ L P=B \times \omega, $

(4)

where B is the biomass (g L⁻¹), and ω is the total lipid content (%).

2.3.4 Fatty acid analysis

The fatty acid (FAs) composition of the lipids was analyzed using gas chromatography in tandem with mass spectrometry (GC–MS) (Kebelmann et al., 2013). Briefly, 20-50 mg freeze-dried microalgae samples were added to 1 mL saturated KOH-CH₃OH solution and allowed to stand for 1 h at 80℃ in a water bath. Then, 3 mL distilled water and 2 mL hexane were added. After being vortexed, the upper phase containing fatty acids were analysed using a gas chromatograph equipped with a flame ionisation detector (Perkin Elmer, Germany) and a DB-5MS chromatographic column (50 m × 250 μm × 0.25 μm). The injection temperature was 300℃, the initial column temperature was 150℃, and the temperature was increased to 300℃ using a temperature gradient of 10℃ min⁻¹, whereupon it was held for 5 min. All the analyses were carried out in duplicate and average values reported.

2.4 Calculation of Parameters Related to Biodiesel Fuel Properties

Four parameters related to biodiesel fuel properties were calculated based on fatty acid profiles in Nannochloropsis sp. MASCC 11 as described by the following equations (Hoekman et al., 2012):

$ {ADU = \Sigma \left({M \times {Y_i}} \right), } $

(5)

$ {kV = - 0.6316 \times ADU + 5.2065, } $

(6)

$ {SG = 0.0055 \times ADU + 0.8726, } $

(7)

$ {CN = - 6.6684 \times ADU + 62.876, } $

(8)

where ADU, kV, SG, and CN are average degree of unsaturation, kinematic viscosity, specific gravity, and cetane number, respectively; M is the number of C=C bonds; and Y_i is the mass fraction of each FA constituent.

2.5 Statistical Analysis

The results are expressed as mean ± standard deviation (SD) of three replicates except as specified above. Statistical analysis of data was performed by using SPSS 19.0 for Windows. One-way analysis of variance (ANOVA), followed by Tukey-Kramer multiple comparison, was used to analyze the differences among the treatments. A value of p < 0.05 was considered statistically significant.

3 Results 3.1 Effect of pH on Growth and Lipid Accumulation

During the cultivation period of 10 d, Nannochloropsis sp. MASCC 11 presented measurable specific growth rates at pH values of 4.0, 6.0, and 9.0 (control), but no growth was observed at pH 3.0 and 3.5 (Fig. 1). The maximum specific growth rate μ (0.27 d⁻¹) and biomass B (0.44 g L⁻¹) were observed at pH 9.0. Both μ and B at acidic pH levels were lower than those in control, accounting for 89% and 61% at pH 6.0, and 70% and 23% at pH 4.0, respectively. Unlike algal growth, the highest ω value (36.8%) was observed at pH 6.0, resulting in a maximum LP (108.2 mg L⁻¹) (Table 1). The lowest ω value obtained was 20.6% when algae were grown at pH 9.0.

3.2 Effect of Temperature on Growth and Lipid Accumulation

As shown in Fig. 2, the optimum temperature for proper growth of Nannochloropsis sp. MASCC 11 was 35℃, with a μ value of 0.30 d⁻¹ and a B value of 0.63 g L⁻¹, followed by 40℃ (μ = 0.28 d⁻¹ and B = 0.55 g L⁻¹) (Table 2). As the temperature decreased below 35℃, both μ and B also decreased. While no significant changes in ω were observed within the temperature range of 25 to 40℃ (P > 0.05), and the highest ω (28.34%) was found at 20℃, showing a significant difference from those at other temperatures (P < 0.05). Higher LP obtained at 35℃ (134.55 mg L⁻¹), 40℃ (123.26 mg L⁻¹), and 30℃ (118.42 mg L^–1) were 1.43, 1.31, and 1.26 times as high as that at 20℃, respectively (Table 2).

3.3 Effect of CO₂ Concentration on the Growth, Lipid Accumulation, and Fatty Acid Composition

Fig. 3 and Table 3 show the effects of CO₂ concentration on the growth and lipid accumulation in Nannochloropsis sp. MASCC 11 cells. The highest values of μ and B were obtained at 5% CO₂ (0.42 d⁻¹ and 2.27 g L⁻¹), followed by those at 10% CO₂ (0.35 d⁻¹ and 1.14 g L⁻¹). When aerated with 15% CO₂, the microalgae presented lower values of μ and B (0.29 d⁻¹ and 0.54 g L⁻¹), but without obvious inhibition of growth compared to those obtained in the control group (i.e., aeration with air) (P > 0.05). Similarly, higher lipid contents (38.60% and 34.47 %) were also observed at 10% and 5% CO₂, respectively, compared to those at 15% CO₂ (20.90%) and in the control group (24.19%). LP reached its highest value of 782.7 mg L⁻¹ at 5% CO₂, followed by that at 10% CO₂ (437.4 mg L⁻¹), with much lower LP at 0.04% and 15% CO₂.

During the cultivation, the pH changes greatly with the increase of CO₂ concentration. When aerated with ambient air (the control), the pH of the culture dropped to 8.33 at the first day after cultivation, followed by a slow increase during the exponential growth phase. The final pH remained constant with minor variations between 9.50 and 9.75. However, when cultures were aerated with high concentrations of CO₂ (5%-15%), the pH values fell sharply below 7.0 or even 6.0 in the initial stage of cultivation and then increased rapidly due to the exponential cell growth. Finally, they stabilized about 8.43, 8.11 and 7.51 respectively (Fig. 4).

During the cultivation, the pH changes greatly with the increase of CO₂ concentration. When aerated with ambient air (the control), the pH of the culture dropped to 8.33 at the first day after cultivation, followed by a slow increase during the exponential growth phase. The final pH remained constant with minor variations between 9.50 and 9.75. However, when cultures were aerated with high concentrations of CO₂ (5%-15%), the pH values fell sharply below 7.0 or even 6.0 in the initial stage of cultivation and then increased rapidly due to the exponential cell growth. Finally, they stabilized about 8.43, 8.11 and 7.51 respectively (Fig. 4).

At each concentration of CO₂, the majority of the FAs were palmitic acid (C16:0), hexadecatrienoic acid (C16:3), linoleic acid (C18:2), and linolenic acid (C18:3) (Table 4). The sum of them accounted for 89.1% (air), 86.7% (5% CO₂), 85.3% (10% CO₂), and 88.2% (15% CO₂) of the total fatty acid. C16 and C18 fatty acids comprised 97.6 % to 98.6% of the total lipids. Increasing CO₂ concentration led to a general downtrend in the levels of saturated fatty acids (SFAs, e.g., C16:0 and C18:0), and a concomitant increase in the levels of unsaturated fatty acids (UFAs), mainly due to the increase of polyunsaturated fatty acid (PUFA) contents. In general, a high CO₂ concentration enhanced the synthesis of C16:3 and C16:2 FAs, while C18:2 and C18:3 FAs showed the opposite tendency.

4 Discussion 4.1 Tolerance of Nannochloropsis sp. MASCC 11 to Low pH

It is estimated that about 220 ppm of SO_X is produced in a typical power plant flue gas (Kumar et al., 2011). Most microalgae are unable to grow at all when exposed to SO₂ at a concentration exceeding 100 ppm (Hauck et al., 1996). And the major toxicity of SO₂ to microalgae seems to result from the enhanced acidic conditions (Matsumoto et al., 1997). Matsumoto et al. (1997) reported that pH decreased to less than 4.0 after 20 h of aeration with 400 ppm of SO₂, however, when the pH was maintained at 8.0 by adding NaOH solution, no significant reduction in algal growth rate was observed. A typical optimum pH for most microalgal species is approximately 8.2 to 8.7 (Havlik et al., 2013). Under low pH conditions, the increasing chemical gradient between cytoplasm and medium leads to a higher H⁺ influx to the cells, requiring active transport of H⁺ to maintain an internal pH suitable for normal metabolic processes (Santikul, 2000). In the present study, Nannochloropsis sp. MASCC 11 grew best at pH 9.0 (Table 1), an optimum value similar to N. salina (8–9, Bartley et al., 2014), Nannochloropsis sp. (8.5, Khatoon et al., 2014), Spirulina (Arthospira) platensis (8.2–8.7, Kim et al., 2007), and Pavlova lutheri (8–9, Shah et al., 2014). It is noteworthy that Nannochloropsis sp. MASCC 11 tolerated pH as low as 4.0, which strongly inhibited growth in some Nannochloropsis species such as N. salina (Bartley et al., 2014).

Although pH 9.0 favored biomass formation in Nannochloropsis sp. MASCC 11, it did not result in the highest lipid content. Relatively low pH, e.g., 6.0, was ideal with regard to maximizing both lipid content and lipid production (Table 1). Low lipid production at pH 4.0 was due to the poor growth, not the low lipid content, which is equal to the control (pH 9.0). Shah et al. (2014) also observed a decreasing trend in algal lipid accumulation with decreased pH in Pavlova lutheri. Acetyl-CoA carboxylase, a key enzyme in fatty acid synthesis, may be inhibited in more acidic media (Sasaki et al., 1997). In contrast, Bartley et al. (2014) found no significant effect of pH change (over 7 to 9) on lipid accumulation in N. salina. Therefore, the relationship between lipid accumulation and pH value is species-specific.

4.2 Tolerance of Nannochloropsis sp. MASCC 11 to High Temperature

Temperature is one of the key factors affecting microalgal growth, because photosynthesis in algal cells consists of a series of temperature-dependent reactions (Zhao and Su, 2014). Low temperatures reduce the activity of enzymes related to photosynthesis (and other metabolic reactions). Moderate high temperatures could promote the rate of respiratory action, but extremely high temperatures will restrain metabolic activity and respiration in microalgae (Breuer et al., 2013). Generally, temperatures greater than 35℃ are too harsh for many microalgal species (Zhao and Su, 2014). If microalgae are used to eliminate CO₂ from flue gas, they must exhibit a certain tolerance to high temperatures to reduce the cost of cooling production systems (Wang et al., 2008). Nannochloropsis sp. MASCC 11 was high-temperature tolerant, with rapid growth (μ = 0.28 to 0.30 d⁻¹, B = 0.51 to 0.63 g L⁻¹) within the range 30 to 40℃. This thermotolerance conferred potential for CO₂ removal from power station flue gases. Another freshwater Nannochloropsis strain, N. limnetica Krienitz (SAG 18.99), showed the highest dry-mass value at 22℃ (1.03 ± 0.04 g L⁻¹) when cultured in OHM culture medium adjusted to an N concentration of 10 mmol L⁻¹ KNO₃ (Freire et al., 2016). N. oculata has an optimal temperature of 20℃, with a μ value of 0.13 d⁻¹, more than double the value at 15 and 25℃ (Converti et al., 2009). N. salina grows between 17 to 32℃, with an optimum of 28℃ (Boussiba et al., 1987). 30℃ is very close to the limit of survivability of N. gaditana strain B-3, and the maximum biomass productivity (0.429 g L⁻¹ d⁻¹) was achieved at 25℃, with an average irradiance of 170 μmol s⁻¹ m⁻¹ and a dilution rate of 0.3 d⁻¹ (Camacho-Rodríguez et al., 2015). Clearly, compared with other Nannochloropsis strains, Nannochloropsis sp. MASCC 11 possess higher thermotolerance, thus conferring the potential for use in CO₂ removal from power station flue gases.

Usually, when subjected to stressful conditions such as high irradiance and temperature, microalgae can accumulate lipids (Wijffels and Barbosa, 2010), mainly in the form of triacylglycerols (TAGs), whereas under optimal growth conditions, they produce only small amounts of lipids. Our results showed that a lower temperature, i.e., 20℃, seems to be most favorable to lipid accumulation in Nannochloropsis sp. MASCC 11, on the contrary, lower lipid contents were found in the groups exposed to higher temperatures. This was not consistent with the findings of previous studies. Converti et al. (2009) found that increasing the cultivation temperature from 20 to 25℃ doubled the lipid content in N. oculata (from 7.90% to 14.9%), and the largest biomass and lipid content in N. oculata were both obtained at 25℃. This can be explained by the fact that the Nannochloropsis sp. MASCC 11 used in the present study may be a mesophilic species which prefer moderate high temperatures, and lower temperatures, rather than higher temperatures, constitute a stress condition. Based on the data in Table 2, and the cultivation period, the calculated biomass productivity and lipid productivity of Nannochloropsis sp. MASCC 11 (63 mg L⁻¹ d⁻¹ and 13.5 mg L⁻¹ d⁻¹ at 35℃; 55 mg L⁻¹ d⁻¹ and 12.3 mg L⁻¹ d⁻¹ at 40℃) could be comparable with those of Monoraphidium sp. SB2 at 35℃ (93 mg L⁻¹ d⁻¹ and 29 mg L⁻¹ d⁻¹), whereas the growth of the latter at 40 ℃ was too weak to produce a detectable lipid content (Wu et al., 2013). Biomass productivity and lipid productivity of Nannochloropsis have not been reported at the similar temperatures, perhaps due to the poor tolerance to high temperatures.

4.3 Tolerance of Nannochloropsis sp. MASCC 11 to High CO₂ Concentrations

CO₂ is an essential carbon source for algal autotrophic growth. Supplementing a microalgae culture with an appropriate level of CO₂ can promote photosynthetic efficiency by enhancing the CO₂ concentration around RuBisCO, and then increasing the ratio of the rates of carboxylation to oxygenation and the efficiency of CO₂ fixation (Long et al., 2006). However, when the CO₂ supply is much higher than that with which the microalgal metabolism can cope, the culture pH will decrease, possibly to less than 5.0 at 15% or 30% CO₂ (Meier et al., 2015), and the acidic pH would produce an adverse effect upon microalgal growth by inhibiting the activity of extracellular enzymes such as carbonic anhydrase (Tang et al., 2011). Another explanation for the low biomass productivity at higher CO₂ concentrations is that most of the CO₂ was consumed for metabolic activity to combat higher CO₂ stress, and less CO₂ was used in biomass synthesis accordingly (Chiu et al., 2009). Therefore, highly CO₂-tolerant species would be necessary for efficient carbon capture from flue gases. According to this study, the use of high CO₂ concentrations (from 5% to 15%) led to a short decline of pH in the medium (mainly in the lag phase), but there was a rapid increase in pH levels due to the exponential cell growth (Fig. 4). In the groups of 5% and 10% CO₂, it only took two days to rise to be higher or close to pH 7.0 which was favorable to the elimination of growth inhibition from acidification, while pH rebounding needed a longer time in the 15% CO₂ group. Nevertheless, Nannochloropsis sp. MASCC 11 could grow well within a CO₂ range of 5% to 15% without any inhibition compared to the control group. The highest biomass productivity of 0.23 g L⁻¹d⁻¹ was obtained at a 5% CO₂ concentration. It has been reported that N. oculata grew best at 2% CO₂ and was completely inhibited at CO₂ concentrations of 5%, 10%, and 15% (Chiu et al., 2009). In another study with N. oculta (NAO), the biomass growth was completely inhibited by bubbling the medium with 10% CO₂, accompanied by a pH decrease to approximately 5.0 (Hsueh et al., 2009). Growth inhibition was observed at ≥ 6.5% CO₂ in N. salina (Arudchelvam and Nirmalakhandan, 2012), and ≥ 9% CO₂ in N. gaditana (Meier et al., 2015). The effects of high CO₂ concentrations on the growth of the genus Nannochloropsis are strain-dependent: the Nannochloropsis sp. MASCC 11, used in this study, was more tolerant to high CO₂ concentrations than other strains. Miyachi et al. (2003) proposed a tolerance classification scheme in which microalgae that can tolerate 2 to 5%, 5 to 20%, and 20 to 100% CO₂ concentrations are classified as CO₂ tolerant, highly-CO₂ tolerant, and extremely highly-CO₂ tolerant, respectively. From this point of view, Nannochloropsis sp. MASCC 11 should be classed as highly-CO₂ tolerant.

Lipid contents in Nannochloropsis sp. MASCC 11 aerated with 5% and 10% CO₂ were 1.42 times and 1.60 times as high as that in the control group, respectively (Table 3). This enhancement is consistent with the chlorophyte, Ettlia sp., which had high growth rate and lipid content at 5% to 10% CO₂ (Yoo et al., 2013). Chlorella vulgaris NIOCCV, cultivated in seafood processing industry wastewater, showed an increase trend in total lipid content at CO₂ concentration from 5% to 10% (Jain et al., 2019). Lipid contents in Monoraphidium sp. QLZ-3 aerated with 12% CO₂ were 1.18-fold higher than those of the control (Dong et al., 2019). Therefore, it is feasible to increase the lipid content in these species by using a high concentration of CO₂. However, the lipid contents in other microalgae showed different response patterns to high CO₂ concentrations. Lv et al. (2010) propose that the lipid content in C. vulgaris decreases when the CO₂ concentration exceeds 1%. In research by our team, lipid contents in C. vulgaris underwent a slight change (from 17.8% to 23.1%) when exposed to CO₂ at concentrations of 0.04% to 10%, with a maximum lipid content found at 2.5% CO₂ (Wang et al., 2015). Similarly, lipid contents (between 18.1% and 18.7%) in C. vulgaris grown in a column-type photobioreactor did not change significantly with the increase in CO₂ concentration (from 0.03% to 5 %) (Lam and Lee, 2013). Also, a mere 1% to 6% increase in the lipid contents in Scenedesmus obliquus and Chlorella pyrenoidosa under high CO₂ (10% to 15%) was observed (Ho et al., 2010; Tang et al., 2011). In the present study, a CO₂ concentration of 5% produced the best lipid productivity (0.078 g L^–1 d^–1) in Nannochloropsis sp. MASCC 11. Compared to other studies on the genus Nannochloropsis in batch culture, this lipid productivity is superior to those of N. oculta (NAO) at 5% and 8% CO₂ (Hsueh et al., 2009), N. salina at 6.5% and 9.5% CO₂ (Arudchelvam and Nirmalakhandan, 2012), and four strains of Nannochloropsis sp. at 5% CO₂ (Rodolfi et al., 2009) (Table 5). Even our second-highest value of lipid productivity (0.044 g L⁻¹ d⁻¹) obtained at 10% CO₂ was near to or higher than those of many Nannochloropsis species. The greater tolerance to high CO₂ concentrations and the higher lipid productivity made Nannochloropsis sp. MASCC 11 a suitable microalga for CO₂ mitigation from flue gases containing 5% to 10% CO₂ while offering efficient lipid production.

4.4 Fatty Acid Profiles at High CO₂ Concentrations and Their Suitability for Biodiesel Applications

In addition to lipid productivity, an appropriate FA composition in the microalgal lipids needs to be considered when evaluating the suitability of microalgae for biodiesel production. C16–C18-enriched fatty acids, including C16:0, C18:0, C18:1, C18:2, and C18:3, are the most suitable feedstocks for biodiesel production (Knothe, 2009). The high proportion of C16–C18 FAs (greater than 97%, Table 4) in Nannochloropsis sp. MASCC 11 can meet this requirement. Different CO₂ supply could influence intracellular fatty acid composition of microalgae (Wang et al., 2014; Mudimu et al., 2015; Dong et al., 2019). The PUFA content in Nannochloropsis sp. MASCC 11 was greater than 80% in the control group and showed an increasing trend when the microalgae were subjected to increased CO₂ concentrations, a trend mainly contributed to the increase in the amounts of C16:3 and C16:2. Tang et al. (2011) also found that high levels of CO₂ enhanced the accumulation of PUFAs in S. obliquus SJTU-3 and C. pyrenoidosa SJTU-2. The increase in PUFA content probably arises because feeding the culture medium with a high concentration of CO₂ leads to a relative decrease in the O₂ concentration, thus affecting enzymatic desaturation (Vargas, 1998).

When using microalgal oil as diesel, some issues should be taken into consideration, i.e., oxidation stability and cold flow, as well as kV, CN, and SG. For Nannochloropsis sp. MASCC 11 microalgal oil, the presence of large amounts of PUFAs resulted in a higher average degree of unsaturation (ADU) ranging from 2.23 at 0.04 % CO₂ to 2.40 at 15% CO₂ (Table 6) than those of other Nannochloropsis strains (0.72 to 1.43) as reported elsewhere (Ma et al., 2014). There are two different effects generated by high levels of unsaturation: lowering of the melting point (MP) to make the biodiesel appear less viscous in cold weather (Isioma et al., 2013), and increasing the susceptibility of biodiesel to oxidation (Sarin et al., 2009). In fact, the oxidation stability can be improved by adding synthetic antioxidants such as pyrogallol, propyl gallate, and so on, which have been found to be very effective in biodiesels (Agarwal et al., 2015). The kV value is a measure of the flow resistance of a biodiesel. A high kV may lead to poor atomisation performance (Alptekin and Canakci, 2008), while a lower viscosity is insufficient to ensure the proper lubrication of the injector pump (Mohammad-Ghasemnejadmaleki et al., 2014). CN is an indicator of fuel ignition quality and a higher CN value is conducive to decreasing ignition delay and avoiding diesel knock (Mohammad-Ghasemnejadmaleki et al., 2014). Both parameters should be in accordance with two worldwide standards, i.e., European standard (EN 14214) and American standard (ASTM D6751), however, the latter one is considered to be more adapted to biodiesel from microalgae (ASTM 2015). In this study, the estimated values of kV and CN for Nannochloropsis sp. MASCC 11 biodiesel are well within the specifications of ASTM standards (ASTM 2015), except for that of CN at 15% CO₂ (Table 6). SG is a measure of the density of liquid fuels, which has direct effects on the amount of injected fuel, the injection timing, and injection spray pattern (Lee et al., 2002). Furthermore, high-density fuel always leads to an increase in particulate matter (PM) and NOx emission in diesel engines (Szybist et al., 2007). SG limits are not specified in ASTM D6751, but the estimated SG value for Nannochloropsis sp. MASCC 11 biodiesel is within the range of EN 14214 specifications (0.86 to 0.90 kg L⁻¹) for biodiesel (B100) and biodiesel blends (Mohammad-Ghasemnejadmaleki et al., 2014).

5 Conclusions

Compared to other Nannochloropsis species, Nannochloropsis sp. MASCC 11, a freshwater species, was highly tolerant to acidic pH (4.0), high temperatures (40 ℃), and high CO₂ concentrations (15%). Changes in these conditions significantly affected the algal biomass production, with the highest values occurring at a pH of 9.0 (0.44 g L⁻¹), a temperature of 35℃ (0.63 g L⁻¹), and a CO₂ concentration of 5% (2.27 g L⁻¹). The maximum lipid yields were obtained at a pH of 6.0 (108.21 mg L⁻¹), a temperature of 35℃ (134.55 mg L⁻¹), and a CO₂ concentration of 5% (782.7 mg L⁻¹). The fatty acid profiles of Nannochloropsis sp. MASCC 11 grown at CO₂ concentrations of between 5% and 10% featured a high degree of unsaturation, mainly due to the presence of large amounts of PUFAs such as C16:3, C18:2, and C18:3, leading to an algae-based biodiesel with a lower oxidation stability and better cold flow properties, and the values of kV, CN, and SG met ASTM, or EN 14214, biodiesel specifications.

Acknowledgements

This study was supported by the National Key Technology R & D Programme (No. 2011BAD14B04). We gratefully acknowledge all members of the key laboratory who participated in the research. We also thank Dr. Yongfu Li for his valuable assistance with the experiments.