Lipid production of microalgae isolated from the Ionian Sea of Greece

Authors: Panagiotis Dritsas¹, Elias Asimakis², George Tsiamis², George Aggelis¹

• ¹Greece, University of Patras/Department of Biology
• ²Greece, University of Patras/Department of Sustainable Agriculture

Microalgae are photosynthetic organisms, considered to be a biological material of high importance for basic research and many biotechnological applications. The aim of this study was the isolation of microalgal strains from the Ionian Sea of Greece, their molecular identification and biochemical characterization with emphasis on their ability to accumulate lipid reserves. The lipids which are rich in polyunsaturated fatty acids (PUFAs) are used as additives in human and animal nutrition, in the pharmaceutical and chemical industries, while more saturated lipids towards biodiesel production. 13 strains of microalgae were isolated, belonging to the genera Picochlorum, Nannochloropsis, Tetraselmis, Chlorella and Nephroselmis as revealed by the molecular identification by PCR amplification of the 18S rRNA gene and the ITS (internal transcribed spacer) region. All strains grew satisfactorily when they were cultured in 500cc Erlenmeyer flasks containing 100 mL of artificial seawater, under continuous illumination of 387 μmol m^-2 s^-1 with initial population of 1.5 x 10⁶ cells/mL. Especially, Tetraselmis sp. reached approximately 1,600 mg L^-1 of dry biomass after 450 h. In terms of lipid accumulation, P. costavermella was placed first, exceeding 19% w/w of dry biomass. The major PUFA synthesized by Nannochloropsis strains was eicosapentaenoic acid, while the strains belonging to the genera Picochlorum, Tetraselmis and Chlorella synthesized a-linolenic acid in significant quantities. On the contrary, Nephroselmis pyriformis produced fatty acids which were more suitable for biodiesel manufacture. Additionally, microalgal cell mass of all strains contained proteins and polysaccharides in considerable levels. The biochemical profiles of these new isolates revealed their suitability for use in aquaculture and various industrial applications, highlighting the probability of a more efficient and economical production of high-added value products in large scale applications by the use of newly-isolated microalgae.

2023 AOCS Annual Meeting & Expo – Panagiotis Dritsas.Oral Presentation

Cultivation of marine microalgae, native of the Ionian Sea, in open raceway pond – Production of high-value compounds

Stefania Patsialou^1,4, Panagiotis Dritsas², Yannis Kotzamanis³, George Aggelis², Athanasia G. Tekerlekopoulou¹, Dimitris V. Vayenas⁴

¹ Department of Sustainable Agriculture, School of Agricultural Sciences, University of Patras, 30100 Agrinio, Greece
² Department of Biology, School of Natural Sciences, University of Patras, 26500 Patras, Greece ³ Hellenic Centre for Marine Research/Institute of Marine Biology, Biotechnology and Aquaculture, 19013 Anavyssos, Greece
⁴ Department of Chemical Engineering, School of Engineering, University of Patras, 26500 Patras, Greece

In recent years there is a great interest for high added value bioproducts derived from marine microalgae. These valuable metabolic compounds (e.g. lipids, carbohydrates, pigments, proteins), are well known for their potential use in biotechnological applications in various fields, such as pharmaceuticals, cosmetic products, human and animal nutrition and the production of renewable energy sources. In this work four different marine microalgae species (isolated from coastal areas of the Ionian Sea) were cultivated in a paddle wheel, open, raceway pond photobioreactor (PBR) of 40 L operating volume. Specifically, the strains Nephroselmis pyriformis, Picochlorum costavermella, Picochlorum oklahomense and Nannochloropsis gaditana were examined. Initially, stock cultures of strains were gradually scaled up from 100 ml flasks to 6L aquariums with the aim to inoculate the raceway pond. It should be mentioned that the growth medium was sterile artificial seawater of salinity 33 ‰. In all experiments the pH was remained at the value of about 8.5, the temperature was 23^oC ±1^oC, while continuous illumination (2000-2500 lux) was employed from three LED lamps. Also, the culture was circulated in the pond using a double 4-bladed paddlewheel driven by an electric motor rotating at 35 rpm. Biomass growth was examined for all the above-mentioned microalgae strains, for a period of 19 days. N. pyriformis and P. costavermella presented similar final biomass concentration, 419.1±79.9 mg/L and 405.7±0.2, respectively, while N. gaditana reached a lower value, that of 359.5±42.7 mg/L. An even lower value (212.9±11.4 mg/L) was presented for P. oklahomense. Lipid content was varied greatly by species, with values of 30.7±4.5, 64.7±0.3, 75.2±5.9 and 28.3±3.8 mg/L, for N. pyriformis, N. gaditana, P. costavermella and P. oklahomense, respectively. The final produced biomass will also be characterized for its protein, polysaccharide, amino acid and pigment content, in order to assess the use of biomass in various biotechnological applications.

Keywords: marine microalgae, Ionian Sea, bioproducts

BIORESTEC 23 – Stefania Patsialou

Isolation of new microalgal strains from the Gulf of Patras (Greece) and investigation of their potential biotechnological applications

Authors: Panagiotis Dritsas¹, Elias Asimakis², George Tsiamis², George Aggelis^1
1Greece, University of Patras/Department of Biology ²Greece, University of Patras/Department of Sustainable Agriculture

Microalgae are phytoplankton, found in all types of aquatic systems. Due to their ability to synthesize metabolites of high-added value in significant quantities, basic research and biotechnology sectors recognize them as biological material of high importance. The goal of this study was the isolation of microalgal strains from the Gulf of Patras (Greece), their molecular identification and biochemical characterization. Emphasis was given on their ability to accumulate lipid reserves, as lipids rich in polyunsaturated fatty acids (PUFAs) are used as additives in food, pharmaceutical and chemical industries, while more saturated lipids can be utilized towards biodiesel production. The molecular identification, by PCR amplification of the 18S rRNA gene and the ITS (internal transcribed spacer) region, revealed that the 6 isolated strains belong to the genera Picochlorum, Nannochloropsis and Nephroselmis. All strains were cultured in 500cc Erlenmeyer flasks containing 0.1 L of artificial seawater and grew satisfactorily. Especially, in terms of dry biomass production, Nannochloropsis sp. from Patras city port marked the highest levels (exceeding 900 mg/L) after 450 h, while P. costavermella was placed first in lipid accumulation (i.e., 19% w/w of dry biomass). Significant quantities of PUFAs were synthesized by the Picochlorum and Nannochloropsis strains (mainly a-linolenic and eicosapentaenoic acid respectively), while the fatty acids of Nephroselmis pyriformis were suitable for biodiesel manufacture. Moreover, all strains accumulated notable amounts of proteins and carbohydrates. Furthermore, the 2 isolated Picochlorum strains were cultured in a Stirred Tank Reactor. In this setup, biomass production was much higher (2-3 times), although slight differences were observed in terms of storage material accumulation between the two types of bioreactors. In conclusion, the biochemical profiles of the isolates showcased their potential suitability in various applications, like aquaculture, which is a sector of crucial importance for the economy of many countries located in the Mediterranean Sea.

BIORESTEC 23 – Panagiotis Dritsas

Απομόνωση μικροφυκών από θαλασσινά νερά του Ιονίου πελάγους και βιοχημικός χαρακτηρισμός τους

Παναγιώτης Δρίτσας1, Ηλίας Ασημάκης2, Γεώργιος Τσιάμης2, Γεώργιος Αγγελής1

1 Εργαστήριο Μικροβιολογίας, Τομέας Γενετικής, Βιολογίας Κυττάρου και Ανάπτυξης, Τμήμα Βιολογίας, Πανεπιστήμιο Πατρών, Ελλάδα

2 Εργαστήριο Μικροβιολογίας Συστημάτων και Εφαρμοσμένης Γονιδιωματικής, Τμήμα Μηχανικών Περιβάλλοντος, Πανεπιστήμιο Πατρών, Ελλάδα

Περίληψη: Σκοπός της εργασίας είναι η απομόνωση στελεχών μικροφυκών από νερά του Ιονίου πελάγους, η μοριακή ταυτοποίηση και ο βιοχημικός χαρακτηρισμός τους. Έμφαση δίνεται στη διερεύνηση της ικανότητάς τους να συσσωρεύουν αποθεματικά λιπίδια. Τα πλούσια σε πολυακόρεστα λιπαρά οξέα λιπίδια των μικροφυκών έχουν ποικίλες εφαρμογές ως πρόσθετα στη διατροφή ανθρώπου και ζώων, στη φαρμακευτική και χημική βιομηχανία, ενώ περισσότερο κορεσμένα λιπίδια χρησιμοποιούνται στη βιομηχανία παραγωγής βιοντήζελ. Έπειτα από δειγματοληψίες κατά μήκος της ακτογραμμής του Ιονίου πελάγους απομονώθηκαν 13 στελέχη μικροφυκών, από τα παράλια των νομών Αχαΐας και Αιτωλοακαρνανίας. Όπως προέκυψε μετά τη μοριακή τους ταυτοποίηση με ενίσχυση PCR του γονιδίου 18S rRNA και της περιοχής ITS (internal transcribed spacer) (περιλαμβάνει τμήμα των 18S rRNA, 28S rRNA, τις μεταγραφόμενες μεταβλητές περιοχές ITS1, ITS2 και το 5,8S rRNA), τα απομονωθέντα στελέχη ανήκουν στα γένη: Picochlorum, Nannochloropsis, Tetraselmis, Chlorella και Nephroselmis. Τα στελέχη καλλιεργήθηκαν για 450 h σε τεχνητό θαλασσινό νερό σε Erlenmeyer φιάλες των 500cc ενεργού όγκου 100 mL, υπό διαρκή φωτισμό έντασης 387 μmol m-2 s-1 και περιοδική ανακίνηση, με αρχικό πληθυσμό 1,5 x 106 κύτταρα/mL. Όλα τα στελέχη αυξήθηκαν ικανοποιητικά, ιδιαίτερα όσα ανήκουν στο γένος Picochlorum. Ειδικότερα, το μεγαλύτερο πληθυσμό έφτασε η καλλιέργεια του Picochlorum oklahomense με 75 x 106 κύτταρα/mL (ξηρή βιομάζα, x = 320,34 ± 55,06 mg/L), απομονωμένο από τον Πατραϊκό κόλπο, όμως τη μεγαλύτερη παραγωγή ξηρής βιομάζας παρουσίασε το Tetraselmis levis (x = 1558,70 ± 75,20 mg/L). Το υψηλότερο επίπεδο συσσώρευσης αποθεματικών λιπιδίων εμφάνισε το Picochlorum costavermella με ποσοστό που υπερέβη το 19% επί της ξηρής βιομάζας.

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