Enhancing total fatty acids and arachidonic acid production by the red microalgae Porphyridium purpureum
© The Author(s) 2016
Received: 14 December 2015
Accepted: 15 June 2016
Published: 23 June 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.
KeywordsMicroalgae Porphyridium purpureum Aeration rate Light intensity Total fatty acids Arachidonic acid
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
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.
Results and discussion
Screening of the growth medium for P. purpureum
The effect of aeration rate on biomass production and fatty acids/ARA accumulation by P. purpureum
The effect of light intensity on biomass production and fatty acids/ARA accumulation by P. purpureum
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).
Fatty acid contents in P. purpureum under different light intensities and aeration rates
Light intensity (μmol/m2s)
Fatty acid content (mg/g)
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.
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
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.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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