A biorefinery approach to obtain docosahexaenoic acid and docosapentaenoic acid n-6 from Schizochytrium using high performance countercurrent chromatography
Graphical abstract
Introduction
Docosahexaenoic acid (C22:6 n-3, DHA) (Fig. 1) is the major omega-3 polyunsaturated fatty acid in the human brain. It plays a fundamental role in the development and function of human brain and retina in the fetus and infants, and its protective role during aging in neurodegenerative diseases and brain ischemia-reperfusion episodes has been informed [1,2]. More recently, its critical importance in the growth and development of feto-placental unit by augmenting angiogenesis of the first-trimester placenta through stimulating vascular endothelium growth factor (VEGF), angiopoietin-like protein 4 (ANGPTL4), fatty acid-binding proteins (FABPs), or their eicosanoid metabolites, was reviewed [3]. Docosapentaenoic acid (C22:5 n-6, DPA n-6) (Fig. 1) is an omega-6 polyunsaturated fatty acid with a similar structure to DHA, differing in only one double bond [4]. Although DPA n-6 was initially considered undesired, its presence in human brain tissue changed this perception so much that oils containing DHA and DPA n-6 were given full GRAS (generally recognized as safe) status by the Food and Drug Administration (FDA) [5]. DPA n-6 is present at a low concentration in human breast milk [6,7]. This compound exerts beneficial effect against cardiovascular diseases [8,9,10], and has anti-inflammatory effect besides potentiating the same effect of DHA in vivo and in vitro [11]. According to a recent market report [12], the global market size of omega-3 fatty acids accounted for USD 2.49 billion in 2019, and it is foreseen to increase at a composed annual growth rate of 7.7% from 2020 to 2027. To the best of our knowledge, no market report has been issued for DPA n-6. However, given its preventive effect against cardiovascular diseases and ability to potentiate the effect of DHA against inflammation, it may open opportunities for its potential use in the pharmaceutical sector. Fish is the primary source of DHA, and this fatty acid is mainly offered in the form of DHA-rich fish oil [13]. Fish production, as a limited source of polyunsaturated fatty acids, is declining and its global production is expected to decrease in the future [14]. Furthermore, fish oils have undesirable characteristics such as fishy odor and taste, poor oxidative stability, and their quality is variable over different fish species, seasons and location of catching sites [14]. Within this frame of references, microalgae biomass has emerged as a valuable alternative to fish, serving as a source of polyunsaturated fatty acids.
Schizochytrium sp. is a heterotrophic marine microorganism that is currently used as a source for the commercial production of DHA-rich oil. This microorganism exhibits a fast growth rate and high productivity in fermenters [5]. Aside DHA, the Schizochytrium oil also contains DPA n-6, palmitic and myristic acids among other minor fatty acids [15,16]. The methods reported to obtain enriched oil from microalgae include procedures of extraction with organic solvents [17], ionic liquid mixed with a co-solvent (methanol) [18], acid-catalyzed hot-water [19], CO2-expanded ethanol [20], molten-salt/ionic-liquid mixture [21], supercritical CO2 [22] and by enzymatic ethanolysis [23]. The DHA-rich algal oil obtained from Schizochytrium has been mostly established as an active ingredient in infant formula, supplements and fortified food [15,24,25]. DHA demanded in the pharmaceutical sector is for the purified ethyl ester form. Pharmaceutical products containing fish oils-sourced DHA and EPA ethyl esters such as Lovaza, Omtryg, Epanova and Omacor have been approved by the FDA to reduce triglyceride levels in adults suffering from hypertriglyceridemia [26,27]. The reported methods for obtaining pure DHA from microalgae comprise low-temperature crystallization enrichment, purification by the urea inclusion process, followed by solid-support chromatography [28]. Overall, the production of pure DHA from microalgae combines extraction and isolation procedures; but it tends to be a complex and time-consuming operation [28]. Recently, it has been stated that efforts should be focused on the development and optimization of scalable, economically viable, high-efficiency and environmentally friendly extraction and purification methods for obtaining polyunsaturated fatty acids from microalgae [28].
Liquid–liquid chromatography technology such as high performance countercurrent chromatography (HPCCC) has a great potential in this field as a downstream process owing to its proved scalability and high efficiency [29]. For instance, the production of DHA from fish oils has been achieved by countercurrent chromatography and centrifugal partition chromatography, two liquid-liquid chromatography techniques [[30], [31], [32], [33], [34], [35], [36]], however their capacity to isolate simultaneously DHA and DPA n-6 from microalgae has been little explored under an integrated biorefinery approach covering upstream and downstream processes. Furthermore, a single work [37] described the isolation of DHA and other fatty acids in free form (non-esterified fatty acids) from microalgae by countercurrent chromatography, but two steps were necessary to achieve pure DHA and the method was limited only to one injection. More recently, countercurrent chromatography was used as an analytical technique for profiling the fatty acids of the dinoflagellate Alexandrium tamarense [38].
The simultaneous production of DHA and DPA n-6 from Schizochytrium could be an asset to improve the economics of the production of microalgae-sourced DHA. These two fatty acids are chemically similar compounds differing in only one double bond [4]. Therefore, to face the challenge of isolating these two compounds of closely related structures, a separation technique offering a high selectivity is pivotal. In this case, HPCCC may play an important role as it can take advantage of the liquid nature of its stationary phase. In HPCCC, the liquid stationary phase is retained within the column by means of centrifugal force, while the mobile phase is pumped through the column [39]. The separation of the compounds of interest is based on the difference in their partition coefficients between the two immiscible phases; thus, those chemical compounds of a mixture having different partition coefficients between two immiscible phases can be separated by HPCCC. Given that this established technology does not use a solid support as a stationary phase, it therefore offers many advantages over other types of chromatography. For example, it has a unique selectivity, high loading capacity; there is a total recovery of the injected sample, a low risk of sample denaturation and a low solvent consumption [40]. The present study, reports a HPCCC-based downstream process for obtaining sequentially DHA and DPA n-6 ethyl esters from microalgae, under an integrated biorefinery approach.
Section snippets
Microalgae cultivation
In the present investigation, the microalgae Schizochytrium limacinum CO3-H was heterotrophically cultivated in batch mode in a 200 L fermenter (SK group, Črniče, Slovenia) using 150 L of fresh sterile media. The media composition is described in Table S1 and the microalgae growth was monitored as displayed in Fig. S1. Chemicals used in buffers and culture media for microalgae cultivation (Sigma Aldrich, Darmstadt, Germany) as well as yeast extract and glycerol (Condalab, Madrid, Spain and VWR
Preparation and analysis of algal oil
In Schizochytrium, docosahexaenoic acid (DHA) and docosapentaenoic acid n-6 (DPA n-6) are mainly present as storage lipids (triacylglycerols), and at a lower extent structural lipids (phospholipids) [49,50]. The triacylglycerol molecule is composed of three fatty acids esterified to a glycerol backbone, while phospholipids are conformed of two fatty acids esterified to a glycerol unit and a phosphate group which is esterified to choline, ethanolamine or inositol [51]. Hence, different
Conclusion
Most efforts aimed at obtaining fatty acids from microalgae have been addressed separately, either at the level of biomass production or extraction operations. The current study, presents a biorefinery approach based on a sequential separation HPCCC process integrated to heterotrophic cultivation of the microalgae Schizochytrium for obtaining DHA and DPA n-6 ethyl esters from algal oil. Compared to multi-step procedures used so far to obtain pure algal fatty acids, this approach uses only a
Declaration of competing interest
José Cheel conceived and designed the HPCCC experiments and wrote article. Daniela Bárcenas-Pérez and José Cheel performed the extraction and isolation experiments and analyzed data. Martin Lukeš performed the GC-FID analysis, analyzed data, and revised the manuscript. David Kubáč and Petr Kaštánek performed the microalgae cultivation, analyzed data, and revised the manuscript. Jiří Kopecký and Pavel Hrouzek performed qualitative GC–MS analysis, analyzed data, and revised the manuscript.
Acknowledgments
This work was financially supported by the Technology Agency of the Czech Republic (NCK grant, TN010000048/03, JC, DBP, ML, DK, PH, JK) and the National Programme of Sustainability I of the Ministry of Education, Youth and Sports of the Czech Republic (ID: LO1416, JC, PH, JK, DK). Daniela Bárcenas-Pérez gratefully acknowledges the research supervision of Dr. José Cheel (Centre Algatech – Czech Academy of Sciences) during the PhD study as well as the University of South Bohemia for a PhD.
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