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Enhancing total fatty acids and arachidonic acid production by the red microalgae Porphyridium purpureum
Bioresources and Bioprocessing volume 3, Article number: 33 (2016)
This study investigated the effect of aeration rate and light intensity on biomass production and total fatty acids (TFA) accumulation by Porphyridium purpureum. The red microalgae is also known to accumulate considerable amount of arachidonic acid (ARA).
In artificial seawater medium, the highest yield of TFA (473.44 mg/L) was obtained with the aeration rate of 3 L/min and light intensity of 165 µmol/m2s, whilst the highest yield of ARA (115.47 mg/L) was achieved with the aeration rate of 3 L/min and light intensity of 110 µmol/m2s. It was found that higher aeration rate led to more biomass and TFA/ARA production. However, higher light intensity could contribute to biomass accumulation, but it was adverse for TFA and ARA biosynthesis.
By optimizing two operating factors (i.e., light intensity and aeration rate), TFA and ARA production by P. purpureum was significantly improved. This research provides a potential alternative means for producing ARA.
Microalgae, exhibiting promising prospect for nourishment, medicine industry, biofuels production, and many other applications, have attracted global attention in recent decades (Fuentes et al. 2000; Ginzberg et al. 2000; Huo et al. 1997). Particularly, great potential of valuable polyunsaturated fatty acids (PUFAs) produced by photoautotrophic microalgae for large-scale microalgal industries have been found (Thompson 1996; Mendoza et al. 1999; Sukenik 1999). Among PUFAs, arachidonic acid (ARA) and eicosapentaenoic acid (EPA) are two of the most valuable extracts of microalgae. ARA is an important omega-6 polyunsaturated fatty acid (n-6 PUFA) that has been reported as one of the major fatty acids of brain cell phospholipids and a precursor of prostaglandins and leukotrienes (Koletzko and Braun 1991; Kromhout et al. 1985). EPA has been demonstrated effective for preventing and curing thrombosis and arteriosclerosis (Dyerberg 1986; De Bravo et al. 1991), and inhibiting the growth of a human lung carcinoma (Shinmen et al. 1989).
ARA is mainly produced by the microorganism Mortierella fungi and the heterologous expression of the ARA by Escherichia coli (Higashiyama et al. 2002; Bennett et al. 1987; Barclay et al. 1994) due to the fact that Mortierella fungi is more economically feasible, and E. coli is a good gene engineering carrier model and easy to be modified. Microalgae are photosynthetic, thus making them safer and environmentally friendly alternative source for ARA production. Furthermore, challenges, such as odor taste, in the traditional PUFA manufacturing techniques can be obviated using microalgae as raw material for PUFA production (Muradyan et al. 2004). Moreover, microalgae are totally natural compared to E. coli, and they possess other beneficial properties, such as non-toxicity, and less subjected to contamination and environmental fluctuations. In addition, microalgae could also produce other valuable products, such as proteins, pigments, and polysaccharides. In addition, the microalgae residue could be used in many fields, such as health care products and fodder. Therefore, the production of fatty acids by the microalgae is considered more advantageous.
Recently, much effort has been focused on controlling CO2 flow rate to achieve higher fatty acids production considering the potential applications of algae in biodiesel industry (Tang et al. 2011; Cohen et al. 1988). However, although light intensity has been found to improve the fatty acids accumulation in microalgae, few reports have been published on the combined effect of light intensity and aeration rate on total fatty acids (TFA) and ARA productivity by microalgae.
The red microalgae Porphyridium purpureum is one of the very few microalgae species that could accumulate high concentrations of long-chain PUFAs, containing up to 36 % ARA and 17 % EPA of TFA (Ahern et al. 1983; Nichols and Appleby 1969; Jones et al. 1963). Previous efforts in enhancing ARA production by these microalgae usually established at the expense of growth limitation under sub-optimal conditions (Ahern et al. 1983; Nichols and Appleby 1969). The aim of the present study is to establish an efficient, economical, and environmental friendly method for ARA production by P. purpureum. The P. purpureum was cultivated in a simple, open, low-energy, organic carbon, and nitrogen sources free system for the fatty acids production, and the influence of aeration rate and light intensity on the yields of TFA and ARA production is investigated and presented.
The microalgae, P. purpureum, CoE1 was screened and maintained by the authors’ research group. Algae cells were cultivated in 1 L flasks containing 500 mL medium at 25 °C under continuous light illumination in a photoincubator. Four culture media reported to enhance P. cruentum growth, including Jones’ ASW medium (Jones et al. 1963), KOCK medium (Koch 1952), Pringsheim medium II (Ernest and Pringsheim 1949), and F/2 medium (Oh et al. 2009), were screened for biomass production and fatty acids/ARA accumulation. The pH of the mediums was adjusted to 7.6 by Tris–HCl buffer. The medium was sterilized by autoclaving with a pressure of 1 kg/cm2 for 20 min. The light intensities ranged from 110 to 220 μmol/m2s and were provided by cool-white fluorescent lamps. The sterile air was constantly supplied at the aeration rate range of 0.5–3 L/min.
Biomass concentration analysis
Algal biomass concentration was determined using the regression Eqs. (1–4) relating the optical density of the culture to the biomass dry weight (DW). The OD604nm was measured with a Shimadzu UV-1750 spectrophotometer every 48 h during the cultivation and the DW was obtained by weighing the algal cells after washing two times with dH2O and subsequently drying in an oven at 75 °C overnight until a constant weight is achieved.
where W medium (g/L) is the dry weight at different culture media, and OD604nm is the absorbance of the suspension at 604 nm.
For the analysis of fatty acid content, the freeze-dried samples of algal biomass were extracted in a chloroform–methanol-water solution according to Bligh and Dyer’s method (Bligh and Dyer 1959). Briefly, ~0.1 g lyophilized algal biomass was added to a solution consisting of 0.8 mL water, 2.0 mL methanol, and 1.0 mL chloroform, and the solution was intensely vibrated for 2 min. Thereafter, an additional 2.0 mL of chloroform and 2.0 mL water were added followed by vibrating for another 2 min. The solution was then centrifuged at 4500 rpm for 10 min. The substratum chloroform phase containing extracted lipids was transferred into a round-bottom flask, while the upper layer was again extracted with 2.0 mL of chloroform for two more times, and the chloroform phases were mixed together and heated in a nitrogen evaporator to remove the chloroform.
Esterification and analysis
In general, the fatty acids are linked to different lipid profiles during the biosynthesis (Merchuk et al. 1998); however, only the fatty acid contents in the form of fatty acid methyl esters (FAMEs) were analyzed, which are enough for the aim of the present study.
FAMEs were prepared by direct esterification of the lipid in 2 mL 1 M KOH–methanol solution following the procedures described by Hartman and Lago (Hartman and Lago 1973) with modifications. Cyclohexane (5 mL, containing 0.15 g/L C17:0 ester as internal standard) was added to the solution, and the mixture was heated at 70 °C for 40 min with a reflux condenser. The mixture was cooled, and then extracted with 2 mL water, and the upper layer was separated for subsequent analysis.
FAME composition was measured utilizing a Shimadzu QP2010SE GC–MS instrument equipped with electron impact ionization (EI) detector and Rtx-5MS column (30 m × 0.25 mm × 0.25 µm). The running temperature was as follows: starting at 120 °C for 1 min, heating at 10 °C/min to 170 °C, maintained for 2 min, heating at 3 °C/min to 260 °C, and maintained for 5 min. The temperature of the injector and detector was kept constant at 260 °C, and the column flow rate and the split ratio were 13.9 mL/min and 5:1, respectively. The sample injection volume was 1 µL. The FAMEs were quantified by the internal standard method and calculated as follows:
Results and discussion
Screening of the growth medium for P. purpureum
Different microalgae strains have different properties, and, therefore, can behave differently vis-à-vis the growth medium. Initially, the effect of culture medium on P. purpureum growth was investigated, and the results are presented in Fig. 1. The highest biomass concentration of 9.95 g/L was achieved after 22 days of cultivation in ASW medium, while a slightly lower biomass concentration was obtained when the algae was cultivated in Pringsheim II and KOCK medium (9.25 and 8.34 g/L, respectively). Unexpectedly, the growth rate of P. purpureum was rather slow in F/2 medium and the final biomass concentration obtained was only 2.58 g/L. However, it was observed that the microalgae growth was accompanied with autoflocculation of cells during cultivation in Pringsheim II medium. As the harvest of lipid requires certain level of biomass, it is imperative to maximize biomass production before lipid extraction to enhance the feasibility of ARA production. An appropriate medium used for algae cultivation to accumulate as much as possible cells is the cornerstone for ARA production. As shown in Fig. 1, the cells of P. purpureum grew fast and stably in the ASW medium, the reason behind the fast and stable growth may be drawn from the fact that the ASW medium provided sufficient nutrition from simulated seawater ingredients providing the microalgae with the primal favorable growth environment. Thus, the ASW medium which contributed to higher biomass production is also believed promising for ARA production by P. purpureum.
The effect of aeration rate on biomass production and fatty acids/ARA accumulation by P. purpureum
The effect of aeration rate on P. purpureum growth, TFA content, and fatty acid yield was investigated in ASW culture medium, with the aeration rate range of 0.5–3 L/min. Higher aeration rate promoted the growth of P. purpureum, leading to final biomass yield of 13.07 g/L at the aeration rate of 3 L/min after 18 days of cultivation (Fig. 2). However, the biomass concentration obtained at the aeration rate of 0.5 L/min was only 5.55 g/L, implying that aeration rate has a significant effect on the growth of P. purpureum. These results are in agreement with the previously reported works. Merchuk et al. (1998) reported that the gas flow rate conduced to the accumulation of Porphyridium sp. cells. Obviously, aeration bubbles played an important role on mixing the culture, so that the mass transfer rate would be high enough for cells to exchange nutrients and extracellular metabolites during cultivation. As self-shadowing might occur, aeration bubbles contributed much for cells to get the chance to access photons, which is the prerequisite for photosynthesis. However, higher aeration rate resulted in more intense evaporation and convection of culture medium (data not presented). The shear forces lead to cell disruption easily, which was also observed in the present work (Fig. 3a). The algae cells were bigger than the normal ones (Fig. 3b), and the process of cell disruption is apparently discernible as shown in Fig. 3a. Thus, aeration rate of 3 L/min was considered the highest aeration rate suitable for P. purpureum growth.
The highest TFA content of 43.62 mg/g was accumulated when the aeration rate was set at 1.5 L/min. The maximum TFA yield was 499.84 mg/L, achieved at the aeration rate of 3 L/min (Fig. 4). When P. purpureum was cultivated at the aeration rate of 2 L/min, the TFA yield was 399.76 mg/L, lower than 448.20 mg/L obtained at the aeration rate of 1.5 L/min. When the cells were cultivated at 0.5 and 1 L/min, much lower TFA yields of 136.97 and 216.70 mg/L, respectively, were acquired. Moreover, as the cultivation aeration rate rose from 0.5 to 3 L/min, the ARA production was immensely increased from 33.22 to 125.73 mg/L (Fig. 4), revealing that higher aeration rate played a positive role on ARA biosynthesis. Maximum volumetric content of TFAs and ARA was both obtained at cultivation with high aeration rate conditions possibly due to the constant supply of CO2; which has been proven to play an important role in elevating the lipid content in algal cells (Oh et al. 2009; Akimoto et al. 1998). In addition, CO2 was used for de novo fatty acid synthesis (Jiang et al. 2011). Furthermore, supplying CO2 was favorable for the accumulation of polyunsaturated fatty acids as reported by Tang et al. (2011).
The effect of light intensity on biomass production and fatty acids/ARA accumulation by P. purpureum
It has been reported that light intensity has a significant effect on the growth and lipid production of P. purpureum (Koletzko and Braun 1991; Akimoto et al. 1998). The light intensities of 110, 165, and 220 μmol/m2s with the aeration rates of 1 and 3 L/min were employed in the experiments of the current study. The maximum biomass concentration under high aeration rate (3 L/min) was much higher than that of low aeration rate (1 L/min) for all the light conditions tested (Fig. 5). The final biomass concentration at low aeration rate (1 L/min) was similar for all different light intensities evaluated, but the time for reaching the maximum concentration decreased as light intensity increased. For the algae cultured at high aeration rate (3 L/min), the log phase was shorter than the cases observed for low aeration rate. The highest algae cell yield reached 13.59 g/L at 220 μmol/m2s, while the biomass accumulation at 110 and 165 μmol/m2s was nearly equal but much lower than that achieved at the light intensity of 220 μmol/m2s (Fig. 5).
Simultaneously, the effect of light intensity on TFA and ARA accumulation by P. purpureum was studied and the results are depicted in Fig. 6. In all cases, TFA kept accumulating as cultivation cycle extended, and the content notably increased between 7 and 10 days of cultivation. In earlier culture, the TFA content was almost the same at light intensities of 110 and 165 μmol/m2s, and it was nearly half the level at 220 μmol/m2s. In subsequent periods, the content showed inverse relation with light intensity, reaching 33.66 mg/g at 165 μmol/m2s which was higher than that at low light intensity (110 μmol/m2s). However, TFA accumulation was significantly inhibited by higher light intensity (220 μmol/m2s). The highest TFA yield was obtained at light intensity of 165 μmol/m2s (ca. 308.53 mg/L, Fig. 6a). However, due to higher growth rates (Fig. 5), cultivation at light intensity of 220 μmol/m2s resulted in higher yield of TFA (ca. 219.04 mg/L, Fig. 6c) than that at 110 μmol/m2s.
Equally, a step-up in ARA content related to cultivation period was observed, and the highest ARA content was obtained after 14 days under both the light intensities of 110 and 165 μmol/m2s. The accumulation rate was relatively low for the first 10 days at all light conditions evaluated and drastically, an increase in ARA content occurred within the following 4 days (ca. 9.86 mg/g, Fig. 6b) under the light intensity of 165 μmol/m2s. However, it was noticed that prolonged cultivation cycle resulted in gradual decrease in ARA content, which might be due to apoptosis and the very high oxidation rate of ARA. Moderate light condition (165 μmol/m2s) led to the highest ARA content, while high light condition (220 μmol/m2s) provided the minimum. Similar observations were made on the ARA yield, whereby the maximum ARA production was obtained at moderate light intensity (165 μmol/m2s) and lower aeration rate (1 L/min) tested (ca. 82.65 mg/L, Fig. 6d).
Table 1 summarizes the cellular fatty acid contents of P. purpureum cultivated under various light intensities and aeration rates in ASW medium. The major fatty acids found are as follows: C16:0 (Palmitic acid), C18:0 (Stearic acid), C18:2 (Linoleic acid), C20:3 (11, 14, 17-Eicosatrienoic acid), C20:4 n-6 (ARA), and C20:5 n-3 (EPA). Similar results were reported elsewhere. The fatty acids profile of P. cruentum was determined as a function of light intensity (Koletzko and Braun 1991; Akimoto et al. 1998). Cohen’s et al. (1988) reported that there was a positive correlation between light intensity and fatty acid unsaturation. In the case of P. cruentum, the content of 20:5 acid decreased under higher light intensity (Harwood and Russell 1984), whereas the content of 20:4 acid and biomass concentration increased. Meanwhile, the production of unsaturated C-16 and C-18 fatty acids, which are prevalent in glycolipids, have been enhanced by illumination (Rosenberg and Pecker 1964; Constantopoulos and Bloch 1967). Moreover, prolonging illumination time benefited the generation of C20:4 acid, and moderate light/dark cycle was also essential for algae growth and total lipid accumulation (Oh et al. 2009). Merchuk et al. (1998) reported that low gas (air containing 3 % CO2) flow rate had a positive effect on biomass and polysaccharide production by Porphyridium sp. Muradyan et al. (2004) reported that an increase in the CO2 concentration from 2 to 10 % for 1 day was enough to provoke an increase in the total fatty acids on dry weight basis by 30 % in the green algae Dunaliella salina (known to be susceptible to CO2). Feeding microalgae with CO2 enhance the fatty acids’ elongation and desaturation. Furthermore, moderate CO2 level (10 % CO2) enhanced the biomass concentration of both Scenedesmus obliquus SJTU-3 and Chlorella pyrenoidosa SJTU-2; however, high content of TFA and PUFAs was accumulated under high CO2 levels (30–50 %) (Tang et al. 2011).
At lower aeration rate (1 L/min), the cellular content of ARA was always higher than that of EPA (5.57–8.62 mg/g relative to 2.23–3.25 mg/g biomass). The highest cellular TFA and ARA contents were both obtained at 165 μmol/m2s (33.66 and 8.62 mg/g, respectively). When the light intensity was raised from 110 to 165 μmol/m2s, a sharp increase in cellular ARA content by 37 % was achieved. However, it decreased drastically by 35 % at higher light intensity (220 μmol/m2s). On the other hand, a step-up in TFA content by 35 % was obtained, as the light intensity was raised from 110 to 165 μmol/m2s, whereas it decreased by 29 % when light intensity was further raised to 220 μmol/m2s. In the case of high aeration rate (3 L/min), the cellular ARA and TFA contents were higher than that obtained under lower aeration rate (1 L/min). However, slight differences in the EPA content were observed at both aeration rates. Meanwhile, there was a little variation in ARA and TFA contents when the light intensity was raised from 110 to 165 μmol/m2s. The maximum ARA content (9.59 mg/g) was obtained at low light intensity (110 μmol/m2s), whilst the maximum TFA content (39.82 mg/g) was achieved at 165 μmol/m2s. Higher light intensity (220 μmol/m2s) led to marked decline of both ARA and TFA contents, but it took shorter time for the cells to approach the stationary phase (Fig. 4). Thus, all levels of fatty acid contents detected at 18 days were higher than that detected at 14 days of cultivation under light intensity of 220 μmol/m2s. At higher light intensity, the double bond index (DBI) of cellular fatty acids decreased gradually. Whereas, the content ratio of C20:4 acid to C20:5 acid increased with increased light intensity from 110 to 165 μmol/m2s; however, the ratio decreased with further increase of light intensity to 220 μmol/m2s. High aeration rate together with low light intensity conditions contributed to ARA biosynthesis in P. purpureum cells.
Due to similar growth rates (Fig. 5) and cellular fatty acid contents (Table 1), algae cultivation under 110 and 165 μmol/m2s light intensity resulted in similar contents of both TFA (ca. 458.90 mg/L at 110 μmol/m2s; 473.44 mg/L at 165 μmol/m2s, Fig. 6) and ARA (115.47 mg/L at 110 μmol/m2s; 107.49 mg/L at 165 μmol/m2s, Fig. 7), accumulation from total biomass (i.e., volumetric content). However, cultivation under light intensity of 220 μmol/m2s, both TFA and ARA volumetric contents were rather low due to low cellular contents (Table 1) in spite of the high growth rate (Fig. 5).
Discussion: light intensity—one of the key factors affecting the biomass production and fatty acids/ARA accumulation by P. purpureum
In algae cultivation of the current work, higher light intensity resulted in more biomass accumulation, which was in agreement with previous works reported about the effects of light intensity on cell growth and the fatty acid content in algae cells (Koletzko and Braun 1991; Akimoto et al. 1998; Velea et al. 2011). Akimoto et al. (1998) revealed a clear correlation between light intensity and cellular fatty acid content. The authors found that ARA was the main polyunsaturated fatty acid under sub-optimal growth conditions of light intensity, while cellular EPA content decreased sharply. The variations of algal EPA and ARA contents under different growth conditions were also observed in the present study. As presented in Table 1, double bond index (DBI) decreased more obviously as light intensity increased, which indicated that higher light intensity inhibited the synthesis of polyunsaturated fatty acids in P. purpureum cells. More interestingly, the ratio of two main polyunsaturated fatty acids, ARA to EPA peaked in middle light condition (165 μmol/m2s), suggesting that P. purpureum is more favorable for ARA biosynthesis rather than EPA at moderate light condition. Moreover, when P. purpureum was cultured with a moderate aeration rate (1 L/min), light intensity played a prominent role in synthesis of fatty acids. The maximum cellular contents of TFAs were achieved at moderate light condition, while satisfying ARA content was obtained at low light condition under high aeration rate (Table 1). It was reported that high light intensity and low gas flow rates contributed to higher productivities of both biomass and polysaccharides in Porphyridium sp. (Merchuk et al. 1998). Particularly, high light intensity was a key factor for extracellular polysaccharides production by P. purpureum (Liqin et al. 2008). Therefore, the inhibition of fatty acids synthesis caused by high light intensity was likely due to the carbon source consumption for the synthesis of polysaccharides. However, further studies are necessary to reveal this mechanism. Oh et al. (2009) reported that the lipids production was only partially or non-growth related with the cell growth process.
In the present study, the cell growth rate and fatty acids production upon aeration rate indicated that higher cell quantity and large amount of fatty acids could be produced under moderate light conditions, whereas fatty acids synthesis was discontinued once approaching the stationary phase and the content decreased during the apoptosis phase (Table 1; Figs. 6, 7).
This work revealed that high aeration rate with moderate light intensity is promotive factor for biomass and ARA production by P. purpureum; and this provide a promising technical support for the production of ARA. However, additional investigations are needed, including studies on the lipid profile, and highlight tolerance mechanisms by P. purpureum, that may further enhance the production of valuable PUFA by this algae. Furthermore, P. purpureum produces not only fatty acids, but also several other high value biochemicals, such as polysaccharides and phycoerythrin. Comprehensive applications of microalgae biomass, combining several targeted products or services, would be the mainstream of microalgae biotechnology-based industries.
polyunsaturated fatty acid
total fatty acid
fatty acid methyl ester
unsaturated fatty acid
double bond index
electron impact ionization
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GMS and XHZ designed the experiments, GMS, JYC, and XYG performed the experiments, GMS drafted the manuscript, ZL, XHZ, YS, and YHL contributed to the discussion, and ZL and XHZ gave important feedback on draft versions of several sections and improved the manuscript by critical revision. XHZ and LL supervised the research and wrote the final version of the paper. All authors read and approved the final manuscript.
This work was supported by the Special Fund for Fujian Ocean High-Tech Industry Development (No. 2013015), China, and Research Program from the Science and Technology Bureau of Xiamen City in China (3502Z20131016, 3502Z20151254). The authors acknowledge Mr. Theoneste Ndikubwimana in the Department of Chemical and Biochemical Engineering, Xiamen University, for the language refining.
The authors declare that they have no competing interests.