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Growth and lipid content of two freshwater microalgae: Asterococcus korschikoffii Ettl and Vischeria helvetica (Vischer & Pascher) Hibberd

Maria J. Correia1*, Mariana Assunção1, Raquel Amaral1, Jorge M. Varejão2 and Lília M. Santos1
  • 1 University of Coimbra, Department of Life Sciences, Portugal
  • 2 Instituto Politécnico de Coimbra, ESAC, CERNAS, Portugal

Microalgae are increasingly considered as a valuable source of biomolecules currently under study for several applications in nutrition, aquaculture and pharmaceutical industries (Koller et al 2014). These organisms can accumulate large amounts of lipids, ranging from 1.5% to 75% per dry weight (D’Alessandro et al 2016) as a response to stimulating environmental conditions. Microalgae contain two types of lipids, the polar structural membrane phospholipids and glycolipids and the neutral storage lipids including triacylglycerols (TAGs). TAGs are mainly stored in vacuoles and may account for 80% of the total lipid content in the cell. Lipid content for a given species depends on the growth phase. In the exponential phase most microalgae accumulate very few TAGs and the lipid content is mostly phospholipids which function to maintain cell structure (Hu et al 2008). In the stationary phase or under adverse environmental conditions, the cessation of cell division is induced and photosynthetic energy is diverted for substantial TAG production and accumulation (Huerlimann et al 2010). TAGs are considered the most interesting lipids for biotechnology sectors (Pignolet et al 2013). Vischeria helvetica (Vischer & Pascher) Hibberd is a spherical to polyhedral microalga with projections raising from the cell wall, with cell diameter ranging from 9 to 11 µm. This species belongs to the Eustigmatophyceae (Hibberd 1981), a class known by its extensively studied oleaginous genus Nannochloropsis. It is expected that more eustigmatophytes may be oleaginous since large amounts of high quality lipids for biodiesel and nutrition purposes were also recently found in Vischeria stellata (Chodat ex Poulton) Pascher (Gao et al 2015). Therefore, other species of Vischeria such as V. helvetica may also reveal abundant lipids with biotechnological interest. Asterococcus korschikoffii Ettl is an understudied green alga that forms 2-4-celled mucilaginous dendroid colonies with fast growth and very large cells up to 42 µm long (Nakazawa et al 2004). It belongs to the Chlorophyceae, a class with ca. 350 known genera (Pulz & Gross 2004) that provides a large source of putative oleaginous microalgae with the advantage that many are unicellular and display high growth rates. These are very relevant characteristics when selecting strains for biomass production and lipid extraction. The studied strains were Vischeria helvetica ACOI 299 and Asterococcus korschikoffii ACOI 326, both isolated from a freshwater lagoon in Serra do Gerês, north of Portugal. Cultures were scaled-up and the growth curves obtained by cell counting and dry weight (DW). For both strains, the total lipid was determined in the middle of the exponential phase (6 and 9 days) and the onset of the stationary phase (15 and 17 days, respectively). For total lipid determination the freeze-dried biomass pellet was extracted with 3 different methods: i) a simple Soxhlet extraction with hexane as solvent, ii) a method described by Gao et al (2015) used for Vischeria stellata and iii) a method described by Matyash et al (2008) in which methyl-tert-butyl ether (MTBE) is used for high recovery of major classes of lipids (Kumar et al 2015). Methods ii) and iii) are characterized by solvent extraction assisted by vortexing and centrifugation in serial steps of decreasing polarity. The addition of water creates 2 phases for assessing the lipidic fraction recovery by pipetting. The total lipid extracts for both strains, in both growth phases, extracted by the 3 methods were first dried in a rotavapor then in a speed-vacuum centrifuge and their dry weight determined. Afterwards, they were fractionated in order to determine the content of neutral lipids (NL), glycolipids (GL) and phospholipids (PL). The fractionation method consisted in a silica column prepared with chloroform, through which the fractions of the extract were eluted using a sequence of solutions with increasing polarity (Christie 1982). Each fraction was evaporated and the weight determined. Growth estimated by DW showed a long exponential phase for both strains, lasting 10 days for V. helvetica and 13 days for A. korschikoffii. During this phase V. helvetica increased 2-fold in DW and 4-fold in cell density. In contrast, A. korschikoffii displayed a lower DW increase of 0.5-fold and a 7-fold increase in cell density. These results indicate that V. helvetica cells may produce large amounts of heavy metabolites, which may be contributing to this effect. The total lipid (TL) estimations varied from 9 mg to 50 mg in the whole study. In A. korschikoffii TL decreased in the stationary phase while in V. helvetica TL increased. This was expected since Vischeria accumulates lipids in old cultures (Gao et al 2015). The decrease in TL in A. korschikoffii may be inversely proportional to the production of large amounts of mucilage. It is known that for mucilaginous species polysaccharide synthesis occurs in the stationary phase, which leads to a decrease in lipid content (Fabregas et al 1998). Neutral lipids are the largest fraction of TL, in comparison to GL and PL, for both strains. It is expected that most NL are TAGs, however for V. helvetica an important portion of the NL may be carotenoids which are produced in large amounts in eustigmatophytes (Gao et al 2015, Ott et al 2015). These results show that the screening of microalgae for interesting lipid content can be biased by the growth phase. It is recommended that strains are evaluated in both exponential and stationary phases in order to avoid the overlooking of potentially interesting oleaginous strains.

References

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Keywords: Microalgae, Eustigmatophyceae, Chlorophyceae, Neutral lipids, Phospholipids, glicolipids

Conference: IMMR | International Meeting on Marine Research 2016, Peniche, Portugal, 14 Jul - 15 Jul, 2016.

Presentation Type: Poster presentation

Topic: Blue Biotechnology

Citation: Correia MJ, Assunção M, Amaral R, Varejão JM and Santos LM (2016). Growth and lipid content of two freshwater microalgae: Asterococcus korschikoffii Ettl and Vischeria helvetica (Vischer & Pascher) Hibberd. Front. Mar. Sci. Conference Abstract: IMMR | International Meeting on Marine Research 2016. doi: 10.3389/conf.FMARS.2016.04.00003

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Received: 26 Apr 2016; Published Online: 11 Jul 2016.

* Correspondence: Miss. Maria J Correia, University of Coimbra, Department of Life Sciences, Coimbra, 3000-456, Portugal, mariajacorreia1@gmail.com