Elsevier

Phytochemistry

Volume 170, February 2020, 112200
Phytochemistry

Fatty acids as chemotaxonomic and ecophysiological traits in green microalgae (desmids, Zygnematophyceae, Streptophyta): A discriminant analysis approach

https://doi.org/10.1016/j.phytochem.2019.112200Get rights and content

Highlights

  • Climate at the source site had the highest impact on desmid fatty acid profiles.

  • Stearidonic acid accumulated in desmids cultivated continuously over 35 years.

  • Fatty acids as markers are proposed to define higher ranks in microalgal taxonomy.

Abstract

Desmids (Zygnematophyceae) are a group of poorly studied green microalgae. The aim of the present study was to identify fatty acids (FAs) that could be used as biomarkers in desmids in general, and to determine FAs as traits within different ecophysiological desmid groups. FA profiles of 29 desmid strains were determined and analysed with respect to their geographic origin, trophic preference and age of cultivation. It appeared that merely FAs present in relatively large proportions such as palmitic, linoleic, α-linolenic and hexadecatrienoic acids could be used as biomarkers for reliable categorization of this microalgal group. Linear discriminant analysis applied to three a priori defined groups of desmids, revealed clear strain-specific characteristics regarding FA distribution, influenced by climate and trophic conditions at the source sites as well as by the age of culture and growth phase. Accordingly, when considering FAs for the determination of lower taxonomic ranks we recommend using the term “trait” instead of “biomarker”, as the latter designates unchangeable “fingerprint” of a specific taxon. Furthermore, despite that desmids were regarded as microalgae having stable genomes, long-term cultivation appeared to cause modifications in FA metabolic pathways, evident as a larger proportion of stearidonic acid in desmid strains cultivated over extensive time periods (>35 years).

Graphical abstract

Climate had the highest impact on fatty acid composition in 29 desmid strains investigated, and discriminant analysis significantly separated four clusters of strains according to their geographic origin.

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Introduction

During the last decade numerous studies have investigated the use of microalgae as a new promising source for production of commercially valuable fatty acids (FAs) such as oleic (18:1n-9), palmitic (16:0), linoleic (LA, 18:2n-6), α- and γ-linolenic (ALA and GLA, 18:3n-3 and 18:3n-6), stearidonic (SDA, 18:4n-3), arachidonic (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) (Draaisma et al., 2013). In addition, several investigations have been performed to establish FAs as biomarkers in the determination of taxonomic groups of macro- and microalgae (Viso and Marty, 1993; Lang et al., 2011; Kumari et al., 2013) as well as to determine trophic markers to provide insights into consumer diets (Taipale et al., 2013, 2016). By definition, the term “biomarker” indicates a molecule that allows the detection and isolation of a particular cell type or taxon (Volkman et al., 1998). Among the various biochemical markers, FAs or lipid profiles represent a chemically relatively inert class of compounds that is easy to isolate from biological material, and they represent suitable chemotaxonomic markers (i.e. “fingerprints”) to define taxa of higher rank in algae – phylum, class or order (Volkman et al., 1998; Lang et al., 2011). Although it has been noted that FA profiles of microalgae cannot be used as biomarkers for the determination of lower taxonomic ranks (genera and species) with high certainty, several studies showed that certain microalgal or cyanobacterial genera such as Dunaliella, Nostoc, Anabaena, Microcystis, Scytonema, Bigelowiella, Gymnochlora, and Lotharella, had genus- or species-specific FA profiles which appeared independent on the cultivation conditions (Caudales and Wells, 1992; Viso and Marty, 1993; Krüger et al., 1995; Dembitsky and Srebnik, 2002; Řezanka et al., 2003; Leblond et al., 2005). On the other hand, FA profiles from more than 2000 microalgal strains revealed that closely related species, and even multiple isolates of the same species, could have high variability in FA composition (Lang et al., 2011). Nine Tetraselmis sp. strains were isolated from the same locality and regarded as the same species by the isolator; yet, DHA was found only in two strains while the proportion of ARA and GLA greatly varied among strains (Lang et al., 2011). In addition, changes in FA proportions during cultivation (e.g. a decrease in proportion of polyunsaturated FAs (PUFAs) and an increase in monounsaturated and saturated FAs (MUFAs and SFAs, respectively) as well as influences of cultivation conditions (Hu et al., 2008), may greatly impede the usage of FAs as chemotaxonomical tools in microalgae.

Generally, algae exposed to elevated temperatures typically contain higher proportions of SFAs and MUFAs to preserve optimal membrane fluidity (Goss and Wilhelm, 2009). This has been observed as an attribute of various macro- and microalgae and cyanobacteria originating from subtropical and tropical climates (Huerlimann et al., 2010; Kumari et al., 2013; El-Maghraby and Fakhry, 2015; Zea-Obando et al., 2017). In contrast, microalgae inhabiting polar and alpine regions often contain high proportions of PUFAs and oleic acid, and are generally characterized by low growth rates (Ackman et al., 1968; Spijkerman et al., 2012; Torstensson et al., 2013; Pichrtová et al., 2016). Furthermore, studies on FA composition of microalgae have mainly been conducted with isolates from heavily polluted habitats (Abomohra et al., 2016, Abomohra et al., 2017) or using isolates sampled from specific oligotrophic environments, such as high-mountain or polar puddles and snow surfaces (Spijkerman et al., 2012; Lu et al., 2012; Pichrtová et al., 2016; Procházková et al., 2018). To our knowledge, however, no comparative investigations on FAs of strains and/or species within one genus that have different trophic preference have been reported. In addition, the age of microalgal isolates is hypothesized to have a large influence on various metabolic pathways (the phenomenon of the “evolution in a bottle”, Lakeman et al., 2009) and, consequently, may affect FA composition. Certain metabolic changes may occur as an algal culture is established because selection during the isolation, establishment and maintenance drives the phenotype of the culture towards a new optimum (Lakeman et al., 2009). Therefore, all the mentioned factors (climate origin, trophic preference and the age of culture) have to be taken into consideration when using FAs in microalgal/cyanobacterial chemotaxonomy as well as when investigating the commercial utilization of microalgal FAs.

We recently reported that a group of poorly investigated green microalgae, desmids (Zygnematophyceae), contain a remarkably high amount of total FAs (>200 mg g−1 dry weight) (Stamenković et al., 2019). High fractions of linoleic and palmitic acids in Cosmarium crenatum var. boldtianum and C. meneghinii and high proportion of oleic acid in a new isolate of Staurastrum boreale indicated that FA profiles of this microalgal group should be thoroughly investigated in the context of establishing alternative sources for FA production. Considering that the desmids investigated had not been subjected to cryopreservation and conjugation (both of which can insert mutations/gene aberrations, Lakeman et al., 2009), the possibilities for genomic alterations were low. Taking this into account, and that several Cosmarium strains studied exhibited physiological responses in accordance with the climate prevailing at their source sites, it was hypothesized that the genetic composition of desmid strains is stable even after the long-term cultivation (Stamenković and Hanelt, 2013a, 2013b; Stamenković et al., 2014a). Thus, desmids collected from various climatic zones and trophic habitats can be regarded as an ideal group for investigating the potential use of FAs as predictors for chemotaxonomic and ecophysiological attributes.

For assessing FAs as chemotaxonomic and ecophysiological predictors, we herein used linear discriminant analysis (LDA) since it is a multivariate statistical technique commonly used to build a predictive/descriptive model with group discrimination based on observed predictor variables (i.e. FAs) (Hahs-Vaughn, 2016). Hence, LDA was used to examine whether significant differences within three a priori defined groups belonging to one set of desmid strains (climate-origin, trophic-preference, and time-isolation groups) exist based on FA composition. This study aimed to (i) identify specific FAs as chemotaxonomic attributes of desmids in general, taking into account earlier studies and a thorough FA analysis of 29 desmid strains belonging to the genera Cosmarium and Staurastrum; (ii) establish the strength of FAs as ecophysiological predictors in predefined groups of desmids and at different growth phases.

Section snippets

Fatty acid profiling of desmid strains

In total, 27 FAs were quantified in 29 desmid strains investigated (Table S1). The FAs were expressed as molar percentages of total FAs (four samples taken from each culture, n = 4). Unidentified FAs and FAs found in very small fractions were excluded from statistical analyses; altogether, the excluded FAs comprised less than 2 mol % of all quantified FAs. The untransformed means of all FAs are shown in Table S2. The desmid strains investigated were a priori divided into: (1) climate-origin

Conclusions

Along with previous studies, a comprehensive analysis of FA profiles of 29 desmid strains reveals that desmids have a prokaryotic synthesis of glycerogalactolipids which include 18:3 and 16:3 FAs as constituents. LDA applied to the three a priori defined groups of desmids (climate-origin, trophic-preference and time-isolation groups) showed significant differences within these groups at the start and at the end of cultivation merely in the climate-origin group. This indicated that climate had

Algal strains and culture conditions

A total of 29 desmid strains belonging to the genera Cosmarium (19 strains, 14 taxa) and Staurastrum (10 strains, 8 taxa) were purchased from the Microalgae and Zygnematophyceae Collection Hamburg (MZCH, Germany; von Schwartzenberg et al., 2013) and the Coimbra Collection of Algae (ACOI, Portugal; Santos and Santos, 2004) (Table S1). The cultivation conditions and details on the experimental set-up are given in Stamenković et al. (2019). The desmid cultures were grown in a climate chamber at

Acknowledgments

This research project is supported by the University of Gothenburg, and by the research grant of the Swedish Institute provided to M. Stamenković (SI No. 02390/2016). M. Stamenković is also funded by the project No. 173018 of the Ministry of Education, Science and Technological Development of the Republic of Serbia. The authors thank M. Hedblom, J. Pearce and G. Knutsson for valuable support in the laboratory.

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