EFFECT OF GROWTH MEDIUM AND INORGANIC CARBON SOURCE ON GROWTH AND BIOMASS COMPOSITION OF TETRASELMIS STRIATA

Patrinou V.¹*, Patsialou S.¹, Vayenas, D.V.², Aggelis, G.³, Tekerlekopoulou A.G.¹

¹ Department of Sustainable Agriculture, University of Patras, G. Seferi 2, 30100, Agrinio, Greece

² Department of Chemical Engineering, University of Patras, 26500, Rio-Patras, Greece

³ Department of Biology, University of Patras, 26500, Rio-Patras, Greece

Abstract

Tetraselmis striata was cultivated in drilling water with a salinity of 2.8±0.1%. Drilling water was supplemented with two different growth substrates, a ready-to-use commercial fertilizer (Nutri-Leaf 30-10-10) and a modified chemical medium (F/2). Both growth substrates were studied with the addition of NaHCO₃ as the only carbon source, with pure CO₂ (100% v/v) (flow rate of 1 mL CO₂ min⁻¹) or with pure CO₂ (100% v/v) (1 mL CO₂ min⁻¹) and NaHCO₃, simultaneously. The effect of growth medium and inorganic carbon source was then examined on microalga’s growth and biomass composition. The most favorable growth conditions were found to be Nutri-Leaf 30-10-10 and CO₂ flow rate of 1 mL min⁻¹ + NaHCO₃, which lead to a maximum biomass productivity of 154 mg L⁻¹ d⁻¹ (corresponding specific growth rate of 0.477 d⁻¹) and a significant CO₂ biofixation rate (RCO₂) of 293.9 mg L⁻¹ d⁻¹. At the same conditions the biomass of T. striata presented the highest quality (proteins 45.1% d.w., lipids 29.0% d.w., carbohydrates 16.1% d.w. and total chlorophylls 5.1% d.w.), while fatty acid analysis also revealed high percentages of eicosapentaenoic acid (EPA up to 18.2% of the total lipids). Therefore, Nutri-Leaf 30-10-10 and CO₂ flow rate of 1 mL min⁻¹ + NaHCO₃ can be considered a promising strategy for the cultivation of T. striata with the purpose of producing alternative aquafeeds.

Keywords: Growth substrate, inorganic carbon sources, high added value products, fatty acid composition

*Corresponding author: Patrinou Vasiliki (patrinou.v@upatras.gr)

Full text

Poster

Optimization of Cultivation Conditions for Tetraselmis striata and Biomass Quality Evaluation for Fish Feed Production

Feature Paper

Patrinou V, Daskalaki A, Kampantais D, Kanakis DC, Economou CN, Bokas D, Kotzamanis Y, Aggelis G, Vayenas DV, Tekerlekopoulou AG. Optimization of Cultivation Conditions for Tetraselmis striata and Biomass Quality Evaluation for Fish Feed Production. Water. 2022; 14(19):3162. https://doi.org/10.3390/w14193162

Abstract

The marine microalgae Tetraselmis striata was cultivated in drilling waters with different salinities. Growth substrate optimization was performed while the effects of different pH, temperature, photoperiod and CO2 flow rate on biomass productivity and its composition were studied. Results showed that the strain grew better in 2.8% drilling waters employing the fertilizer Nutri-Leaf together with ΝaHCO3. A pH value of 8 resulted in high biomass productivity (79.8 mg L−1 d−1) and biomass composition (proteins 51.2% d.w., carbohydrates 14.6% d.w., lipids 27.8% d.w. and total chlorophylls 5.1% d.w.). The optimum cultivation temperature was found to be 25 ± 1 °C which further enhanced biomass productivity (93.7 mg L−1 d−1) and composition (proteins 38.7% d.w., carbohydrates 20.4% d.w., lipids 30.2% d.w., total chlorophylls 5.1% d.w.). Photoperiod experiments showed that continuous illumination was essential for biomass production. A 10 mL min−1 flow rate of CO2 lead to biomass productivity of 87.5 mg L−1 d−1 and high intracellular content (proteins 44.6% d.w., carbohydrates 10.3% d.w., lipids 27.3% d.w., total chlorophylls 5.2% d.w.). Applying the optimum growth conditions, the produced biomass presented high protein content with adequate amino acids and high percentages of eicosapentaenoic acid (EPA), indicating its suitability for incorporation into conventional fish feeds. In addition, this study analyzed how functional parameters may influence the uptake of nutrients by Tetraselmis.

Patrinou et al. (2022)

Optimization of Cultivation Conditions for Tetraselmis striata and Biomass Quality Evaluation for Fish Feed Production

Feature Paper

Patrinou V, Daskalaki A, Kampantais D, Kanakis DC, Economou CN, Bokas D, Kotzamanis Y, Aggelis G, Vayenas DV, Tekerlekopoulou AG. Optimization of Cultivation Conditions for Tetraselmis striata and Biomass Quality Evaluation for Fish Feed Production. Water. 2022; 14(19):3162. https://doi.org/10.3390/w14193162

Abstract

The marine microalgae Tetraselmis striata was cultivated in drilling waters with different salinities. Growth substrate optimization was performed while the effects of different pH, temperature, photoperiod and CO2 flow rate on biomass productivity and its composition were studied. Results showed that the strain grew better in 2.8% drilling waters employing the fertilizer Nutri-Leaf together with ΝaHCO3. A pH value of 8 resulted in high biomass productivity (79.8 mg L−1 d−1) and biomass composition (proteins 51.2% d.w., carbohydrates 14.6% d.w., lipids 27.8% d.w. and total chlorophylls 5.1% d.w.). The optimum cultivation temperature was found to be 25 ± 1 °C which further enhanced biomass productivity (93.7 mg L−1 d−1) and composition (proteins 38.7% d.w., carbohydrates 20.4% d.w., lipids 30.2% d.w., total chlorophylls 5.1% d.w.). Photoperiod experiments showed that continuous illumination was essential for biomass production. A 10 mL min−1 flow rate of CO2 lead to biomass productivity of 87.5 mg L−1 d−1 and high intracellular content (proteins 44.6% d.w., carbohydrates 10.3% d.w., lipids 27.3% d.w., total chlorophylls 5.2% d.w.). Applying the optimum growth conditions, the produced biomass presented high protein content with adequate amino acids and high percentages of eicosapentaenoic acid (EPA), indicating its suitability for incorporation into conventional fish feeds. In addition, this study analyzed how functional parameters may influence the uptake of nutrients by Tetraselmis.

Patrinou et al. (2022)

CAROTENOID PROFILE OF TETRASELMIS STRIATA GROWN UNDER OPTIMAL CULTIVATION CONDITIONS IN A PILOT SCALE BIOREACTOR

Abstract

Microalgae are recognized as a valuable natural source of bioactive compounds, such as proteins, lipids, carotenoids and vitamins, for the aquaculture industry. The carotenoid profile of Tetraselmis striata, laboratory cultivated at different pH, temperature and photoperiod, was analyzed using Ultra High-Pressure Liquid Chromatography (UPLC) coupled with a mass spectrometer (Q-TOF). Identification and quantification of the carotenoid profile were performed using standard solutions (astaxanthin, lutein & zeaxanthin, canthaxanthin, echinenone, lycopene, b-cryptoxanthin, b-carotene), and an internal standard (trans-β-apo-carotenal). A carotenoid extraction protocol was developed and chloroform was selected as the extraction solvent. A saponification protocol was applied for the successful removal of chlorophylls, lipids, and esters, assisting in the quantification of b-carotene, canthaxanthin, and b-cryptoxanthin. However, this method had a notable deterioration effect on the remaining carotenoids. Consequently, a simultaneous extraction protocol without saponification was followed to quantify the rest of the carotenoids. Α scale-up cultivation of T. striata was conducted in a 40 L capacity paddlewheel stainless steel raceway pond (110.5 x 61 x 20 cm, external length, width and height, respectively), by applying the optimal cultivation conditions found in previous laboratory trials (continuous illumination, 25^oC and pH8), in terms of its high biomass productivity (92.5 mg L^-1 d^-1). Carotenoids detected in T. striata were b-carotene (7063.4 mg/kg dry biomass), lutein & zeaxanthin (1692.8 mg/kg), echinenone (190.9 mg/kg), b-cryptoxanthin (40.7 mg/kg), astaxanthin (33.5 mg/kg) and canthaxanthin (2.12 mg/kg), while lycopene was not detected in any of the analyzed samples. The results showed that T. striata can be considered a potential natural source of carotenoid compounds.

ABSTRACT

ABSTRACT (PDF)

POSTER PRESENTATION (PDF)

POSTER PRESENTATION (POWERPOINT)

Cultivation of marine microalgae – Production of biomass and high value added products

Introduction

Marine microalgae are considered versatile cellular factories that produce a plethora of metabolic compounds. The high value added components they produce is a broad category containing mainly lipids, carbohydrates and proteins (Ma et al., 2020). Lipids from marine microalgae are significant as they are a major source of important poly-unsaturated fatty acids (PUFAs) such as omega-3 [EPA (C20:5), DHA (C22:6), α-Linolenic (C18:3(n-3)] and omega-6 (Dammak et al., 2016). Additionally, extracted high value products can be utilized in many different commercial applications including biofuels, health food supplements, aquafeeds, cosmetics, and pharmaceuticals. The addition of microalgal biomass into fish diets is beneficial for fish as it leads to improved growth and fillet quality, increased deposition of proteins in muscle tissue, improved resistance to disease, and higher fatty acid content (Shah et al., 2018).

In this work, four marine microalgae were studied (Nannochloropsis sp., Nannochloropsis oculata, Isochrysis galbana and Tetraselmis striata). Each is currently of high interest for aquafeeds and has the potential to produce important lipids. The aim was to select the most suitable microalga for full-scale production. The strains were cultivated in drilling seawaters, and specific growth rate and biomass productivity were the determining parameters for the selection of the optimum microalga. Growth medium optimization was then performed for the selected strain. Subsequent fatty acid analysis revealed significant EPA and PUFAs contents when the microalga was cultivated in the optimized growth medium.

Materials and methods

Dry cell biomass and lipid content were determined gravimetrically (mg L^-1) as Total Suspended Solids (TSS) according to Standard Methods (1998). Biomass productivity expressed in mg L^-1 d^-1 was calculated following Gonçalves et al. (2016), while maximum specific growth rate (d^-1) was calculated following Tsolcha et al. (2017). Lipid extraction was carried out following Folch (1957) and the fatty acid profile was determined according to AFNOR (1984).

Results and discussion

The microalgal strains were cultivated in 38 ‰ drilling waters originating from the commercial fish farm PLAGTON S.A. (Western Greece). The seawaters had no nutrient load and nutrient supplementation was essential to sustain growth. Only N and P, at a ratio of about 5:1, were added to the drilling waters, with the aim of reducing production costs in potential future full-scale cultivations. Tetraselmis striata, the Chrysophyceae Isochrysis galbana and the two Nannochloropsis species, were cultivated under conditions of continuous illumination of 56 μmol m⁻² s⁻¹, and unregulated temperature and pH. The biomass efficiencies achieved for the different strains are presented in Table 1.

Table 1. Biomass productivities and specific growth rates obtained from the four marine microalgae.

The Nannochloropsis strains presented the lowest biomass productivities and specific growth rates. Although Isochrysis galbana exhibited the highest biomass productivity it was not selected as the optimum species, because of the difficulty in maintaining the culture’s purity. Microscopic observations showed that the strain was susceptible to contamination even in laboratory-scale experiments and thus it was not considered suitable for full-scale cultivations. Therefore, Tetraselmis striata was selected as the optimum species, as it presented satisfactory biomass yields under conditions of high salinity and low nutrient availability.

Substrate optimization was performed to further enhance growth. A medium of double N:P ratio (12:1) was studied in 38 ‰ and 29 ‰ seawaters. The highest biomass productivity (47.6 mg L^-1d^-1) was recorded in the 29 ‰ medium indicating that T. striata prefers lower salinities. The effect of medium composition on growth was also evaluated in 29 ‰ seawater by further enriching the medium with the commercial fertilizer Nutri-Leaf (30% TN, 10% P, 10% K) together with NaHCO₃. Using Nutri-Leaf significantly enhanced the recorded biomass productivity (79.8 mg L^-1d^-1 with a corresponding growth rate of 0.266 d^-1), while the produced biomass also contained high lipid contents of up to 27.8%  dry weight.

Finally, fatty acid analysis of the biomass generated under the optimum cultivation conditions showed that T. striata produced high EPA (27.6%) and PUFAs (33.2%) contents.

Conclusions

The results of this study indicate that the marine microalgae T. striata is suitable for full-scale applications and can achieve significant biomass yields. Additionally, the strain is an important Pufa producer and its high nutritional value shows its suitability for incorporation into aquafeeds.

ABSTRACT

POSTER PRESENTATION

LABORATORY AND PILOT-SCALE CULTIVATION OF TETRASELMIS STRIATA UNDER OPTIMIZED GROWTH CONDITIONS FOR FISH FEED PRODUCTION

 Abstract

In this work Tetraselmis striata was cultivated in drilling waters (salinity 29 ‰) obtained from the commercial fishery of Plagton S.A.. Previous studies had shown that the microalga displayed optimum growth using 0.2 g L^-1 of the commercial fertilizer Nutri-Leef (30%-TN, 10%-P, 10%-K) together with 0.18 g L^-1 of NaHCO3 at a pH value of 8. The effects of temperature, photoperiod and CO2 flow rate on growth and biomass composition of T. striata were also examined in laboratory conditions. The temperatures of 19±1^oC, 25±1^oC and 28±1^oC were studied under continuous illumination (24:0, L (Light): D (Dark)). The highest biomass productivity of 93.7 mg L^-1 d^-1 was achieved at 25^oC and high protein (49.9%), lipid (23.5%), carbohydrate (19.6%) and pigment contents (5.1%) were also recorded at this temperature. Further experiments were conducted at 25^oC studying the photoperiods of 20:4, 18:6, 12:12 L:D. Results revealed that biomass was significantly affected by light absence and biomass productivity gradually reduced as the dark periods lengthened. The metabolic products exhibited higher accumulation rates under continuous illumination which was selected as optimum. The effect of carbon source was also estimated employing pure CO2 at different flow rates (10 mL min^-1, 20 mL min^-1, 90 mL min^-1). T. striata could not tolerate the high flow rate of 90 mL min^-1 while high biomass productivities (87.5 mg L^-1 d^-1) were recorded at 10 and 20 mL min^-1. Pilot-scale experiments at the optimum growth conditions were conducted in a raceway pond of 40 L capacity. Biomass productivity reached 93.5 mg L^-1 d^-1, while protein, carbohydrate, lipid and pigment contents were 48.8%, 21.6%, 28.1%, 4.8% respectively. In both laboratory- and pilot-scale experiments, analysis of amino acids and fatty acids showed that the produced biomass is suitable for incorporation into conventional fish feeds.

ABSTRACT

POSTER PRESENTATION

ΒΕΛΤΙΣΤΟΠΟΙΗΣΗ ΤΩΝ ΣΥΝΘΗΚΩΝ ΑΝΑΠΤΥΞΗΣ ΤΟΥ ΘΑΛΑΣΣΙΝΟΥ ΜΙΚΡΟΦΥΚΟΥΣ TETRASELMIS
STRIATA KAI ΠΟΙΟΤΙΚΟΣ ΧΑΡΑΚΤΗΡΙΣΜΟΣ ΤΗΣ ΠΑΡΑΓΟΜΕΝΗΣ ΒΙΟΜΑΖΑΣ ΜΕ ΣΚΟΠΟ ΤΗΝ
ΠΑΡΑΓΩΓΗ ΙΧΘΥΟΤΡΟΦΗΣ

ΠΕΡΙΛΗΨΗ
Στην παρούσα εργασία μελετήθηκε η ανάπτυξη του Tetraselmis striata σε νερά γεώτρησης
αλατότητας 29 ‰, τα οποία προέρχονταν από τις εγκαταστάσεις της ιχθυοκαλλιέργειας PLAGTON
S.A.. Στοχεύοντας στην αύξηση της παραγωγής και στη βελτίωση της ποιότητας της παραγόμενης
βιομάζας μελετήθηκε κατά την ακόλουθη σειρά η επίδραση του υποστρώματος, του pH, της
θερμοκρασίας, της φωτοπεριόδου και της παροχής CO2. Από τα εξεταζόμενα υποστρώματα
(τροποποιημένο F/2, εμπορικό λίπασμα Nutri-Leef (30%-TN, 10%-P, 10%-K) χωρίς και με προσθήκη
NaHCO3,) το Nutri Leef παρουσία NaHCO3 παρουσίασε υψηλές τιμές παραγωγικότητας βιομάζας
(84.2 mg L-1 d-1). Στη συνέχεια εξετάστηκε η επίδραση του pH (7 και 8), με την τιμή του 8 να
χαρακτηρίζεται ως βέλτιστη (παραγωγικότητα βιομάζας 79.8 mg L-1 d-1 με υψηλές συγκεντρώσεις
λιπιδίων, πολυσακχαριτών και χρωστικών) και να χρησιμοποιείται για όλα τα επόμενα πειράματα.
Πειράματα έλαβαν χώρα και σε τρεις διαφορετικές θερμοκρασίες, 19 ± 1, 25 ± 1 και 28 ± 1 oC υπό
συνεχή φωτισμό (24:0, L (Φως): D (Σκοτάδι)). Η θερμοκρασία των 25oC, η οποία κρίθηκε ως
βέλτιστη παρουσίασε την υψηλότερη παραγωγικότητα βιομάζας (93.7 mg L-1 d-1), αλλά και την
υψηλότερη περιεκτικότητα σε βιοενεργά συστατικά. Στην συνέχεια μελετήθηκε η επίδραση της
φωτοπεριόδου στους 25oC εξετάζοντας τις περιόδους των 20:4, 18:6, 12:12 L:D. Τα αποτελέσματα
έδειξαν πως η αύξηση των σκοτεινών περιόδων επέφερε μείωση της παραγωγικότητας της
βιομάζας. Τέλος, μελετήθηκε η επίδραση του καθαρού CO2 στην ανάπτυξη του μικροφύκους
εξετάζοντας τους ρυθμούς παροχής 10, 20 και 90 mL min-1, με την υψηλότερη τιμή
παραγωγικότητας βιομάζας (87.5 mg L-1 d-1) να παρουσιάζεται στην παροχή των 10 mL min-1.
Ανάλυση του προφίλ των αμινοξέων και των λιπαρών οξέων έδειξε επαρκές αμινοξικό προφίλ με
υψηλό πρωτεινικό περιεχόμενο και σημαντικό ποσοστό πολυακόρεστων λιπαρών οξέων
αντιστοίχα, καταλήγοντας στο συμπέρασμα ότι η παραγόμενη βιομάζα είναι κατάλληλη για
ενσωμάτωση σε ιχθυοτροφές.

ΠΕΡΙΛΗΨΗ

ΠΛΗΡΗΣ ΠΕΡΙΛΗΨΗ

ΠΑΡΟΥΣΙΑΣΗ ΣΤΟ ΣΥΝΕΔΡΙΟ

Abstract

Microalgae are an exceptional source of polyunsaturated fatty acids, vitamins, proteins, carbohydrates and amino acids that can improve fish growth. Their biomass nutritional value is determined by these nutrients and therefore, parameters affecting growth and biomass composition (pH, growth media, temperature and photoperiod) are often adjusted.

Tetraselmis striata was cultivated in drilling waters (salinity 29 ‰) obtained from the commercial fish farm Plagton S.A.. Previous studies showed that the microalga displayed optimum growth using 0.2 gr L^-1 of the commercial fertilizer Nutri-Leef (30%-TN, 10%-P, 10%-K) together with an inorganic carbon source (0.18 gr L^-1, NaHCO₃) at a pH value of 8. Under these optimized conditions, the effects of photoperiod and temperature on growth and biomass composition were evaluated. Initially, the photoperiod effect was studied at a temperature of 25 ±1^oC. Tetraselmis was cultivated in constant light (24:0, L(Light):D(Dark)) and then 20:4, 18:6, 12:12 L:D exposure. The results revealed that biomass was significantly affected by light absence. The highest productivity of 93.7 mg L^-1 d^-1 was noted at 24:0 L:D but gradually reduced as the dark periods lengthened. Protein contents ranged between 50.3 to 49.9%, while only at 12:12 L:D were lower at the value of 42.9 %. Lipid contents were 23.5 to 29.7%, while carbohydrate contents were ranged from 14.6-19.7%. Pigments also exhibited higher accumulation rates under 24 h light photoperiod (5.1%) and thus continuous illumination was selected as the optimum growth condition. The growth temperatures of 19±1^oC and 28 ±1^oC were then studied. Recorded biomass productivity values were 69.3 and 55.0 mg L^-1 d^-1 respectively, while protein (43.6-40%) and pigment (3.4-2.7%) contents reduced slightly under these conditions. Thus, optimum growth and biomass composition for Tetraselmis is achieved at constant light and at 25^oC. High quality biomass important for fish growth can be produced under these conditions.

Abstract

Poster Presentation