CHEMICAL ENGINEERING TRANSACTIONS VOL. 79, 2020 A publication of The Italian Association of Chemical Engineering Online at www.cetjournal.it Guest Editors: Enrico Bardone, Antonio Marzocchella, Marco Bravi Copyright © 2020, AIDIC Servizi S.r.l. ISBN 978-88-95608-77-8; ISSN 2283-9216 Microalgae as Alternative Source of Nutraceutical Polyunsaturated Fatty Acids Antonio Molinoa, Angela Iovinea,b, Gianpaolo Leonec, Giuseppe Di Sanzod, Salvatore Palazzod, Maria Martinod, Paola Sangiorgiod, Tiziana Marinob, Dino Musmarrab,* a ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Territorial and Production System Sustainability Department, CR Portici Piazzale Enrico Fermi, 1 - 80055 Portici (NA), Italy b Department of Engineering, University of Campania “Luigi Vanvitelli”, Via Roma, 29 - 81031 Aversa, Italy. dino.musmarra@unicampania.it c ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Department of Sustainability-CR Casaccia, Via Anguillarese 301, Rome (RM), 00123, Italy d ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Department of Sustainability-CR ENEA Trisaia, SS Jonica 106, km 419+500, 75026 Rotondella (MT), Italy dino.musmarra@unicampania.it The use of sustainable biobased resources is the basis of the European strategy for bioeconomy development. Microalgae represent one of the most interesting biological source, since they are relatively simple microorganisms containing high-value compounds with potential applications in numerous industrial sectors. The global nutraceutical market is well developed and with an enormous potential for growth: suffice to say that it has shown considerable growth with a value in 2016 between 168 and 174 billion euros, with a growth forecast that will allow it to reach between 285 and 313 billion euros in 2024. In accordance with the National Center for Biotechnology Information, by 2050, microalgae will constitute ~18% of protein source among the more diverse market. Healthy properties and excellent nutritional values have been conferred to microalgae, with particular regard to the high content of carbohydrates, vitamins, carotenoids and polyunsaturated fatty acids (PUFAs). The omega-3 PUFAs are extremely important throughout life and are a dietary need found predominantly in Nannochloropsis gaditana oleaginous microalga. In fact, although fish contain long-chain omega-3, they acquire PUFAs mainly from their microalgal diet. Additionally, considering depleting fish supplies, the use of microalgae as alternative raw material might be considered of increasing importance in the near future. Herein we report the assessment of the economic feasibility of an integrated process which allows to extract PUFAs from Nannochloropsis gaditana, by taking into account the main operational parameters able to influence the final products yield. 1. Introduction Microalgae are well known to be a precious source of bioactive compounds with healthy properties and excellent dietary values (Foley et al., 2011). In fact, these micoorganisms are characterized by a high content of nutritional substances such as proteins, carbohydrates, or compounds with important healthy properties such as vitamins, pigments and PUFAs (Foley et al., 2011). Due to the increasing awareness among consumers about beneficial properties of natural compounds and the importance of a diversified diet rich in omega-3 together with a well-balanced ratio between them and omega-6, the global omega-3 market produced sales volume of about 2.99 billion euros in 2018 and it has been estimated it will increase up to 8.91 billion euros at 2025 (www.verifiedmarketresearch.com, 2019). The global demand of Eicosapantenoic/Docosahexaenoic acid (EPA/DHA) in 2013 was estimated to be 124 thousand tonnes, with a predicted increase up to be 241 thousand tonnes by 2020 (van der Voort et al., 2017). Market region analysis for EPA/DHA production highlights a contribution of 22% for Europe, 30% for Asia Pacific, 40% for North America and 3% for other regions (van der Voort et al., 2017). DOI: 10.3303/CET2079047 Paper Received: 11 July 2019; Revised: 9 January 2020; Accepted: 27 February 2020 Please cite this article as: Molino A., Iovine A., Leone G.P., Di Sanzo G., Palazzo S., Martino M., Sangiorgio P., Marino T., Musmarra D., 2020, Microalgae as Alternative Source, Chemical Engineering Transactions, 79, 277-282 DOI:10.3303/CET2079047 277 Algae-based EPA/DHA market value corresponds to about 18%, 79% ofwhich extracted from fish oil (mainly anchovy and sardines that, combined, represented over 75% feedstock share for oil extraction) and 3% of EPA/DHA market is covered by krill oil (van der Voort et al., 2017).However, theoil contained in selected microalgae strains, which could substitute both fish and krill EPA/DHA natural sources, manifests several advantages in terms of smell and taste together with other characteristics, such as the vegetarian origin and sustainability, thus becoming a very attracting field in the bioeconomy and sustainability context. In addition, the use of microalgae as unconventional raw material might be considered of growing importance given the near-future depletion of fish supplies. As a confirmation of this, the Food and Agriculture Organization evaluates that the 1000 tons of fish oil supplied in the last few years, will not significantly increase (van der Voort et al., 2017), hence this will confirm as alternative markets can offer enormous opportunities for the microalgae-based EPA/DHA production. EPA and DHA exhibit anti-inflammatory and healthy properties thus contributing to prevent cardiovascular diseases; they are also useful in the treatment of high blood pressure or rheumatoid arthritis (Tomic-Smiljanic et al., 2019). One of their most important ability is the reduction in fasting and post-prandial serum triglycerides; they positively alter lipoprotein particle sizeand contribute to maintain the normal brain function and adequate eye vision (Molino et al., 2019). 1.1 Regulatory issues The use of microalgae for the supply of EPA and DHA is gaining great attention in the food and feed sectors (Molino et al., 2019; Iovine et al., 2019). However, at European level only a few species have been authorized and they are listed in the "novel foods" catalogue, according to the list of new European foods established by theCommission Implementing Regulation (EU) 2017/2470 of 20 December 2017 (www.eur-lex.europa.eu, 2019a). The microalgal biomass/extract/metabolites authorized as novel foods fall into the category of “food products and ingredients consisting of or isolated from microorganisms, fungi or algae” as defined by Regulation (EU) 2015/2283 (www.eur-lex.europa.eu, 2019b). Hence, following the European legislation, actually EPA and DHA can be commercialized in form of oil derived from Ulkenia sp. and Schizochytrium sp. as shown in Table 1. Table 1: Microalgae-based novel food for the production of food supplements. Adapted from Regulation (EU) 2017/2470 (www.eur-lex.europa.eu, 2019b) Authorised Novel Foods Specification Conditions under which the novel food may be used Description/Definition: Specified food category Maximum levels Algal oil from the microalgae Ulkenia sp. Oil from the micro-algae Ulkenia sp. Acid value: ≤ 0.5 mg KOH/g Peroxide value (PV): ≤ 5.0 meq/kg oil Moisture and volatiles: ≤ 0.05 % Unsaponifiables: ≤ 4.5 % Trans-fatty acids: ≤ 1.0 % DHA content: ≥ 32 % Bakery products (breads, rolls and sweet biscuits) 200 mg DHA/100 g Cereal bars 500 mg DHA/100 g Non-alcoholic beverages (including milk based beverages) 60 mg DHA/100 ml Schizochytrium sp. oil rich in DHA and EPA Acid value: ≤ 0.5 mg KOH/g Peroxide value: ≤ 5.0 meq/kg oil Oxidative stability: All food products containing Schizochytrium sp. oil rich in DHA and EPA should demonstrate oxidative stability by appropriate and recognised national/international test methodology (e.g. AOAC) Moisture and volatiles: ≤ 0,05 % Unsaponifiables: ≤ 4.5 % Trans- fatty acids: ≤ 1 % DHA content: ≥ Food Supplements as defined in Directive 2002/46/EC for adult population excluding pregnant and lactating women 3000 mg/day* 278 22.5 % EPA content: ≥ 10 % Food Supplements as defined in Directive 2002/46/EC for pregnant and lactating women 450 mg/day 450 mg/day* Total diet replacement for weight control as defined in Regulation (EU) No 609/2013 and meal replacements for weight control 250 mg/meal In accordance with the particular nutritional requirements of the persons for whom the products are intended* Milk-based drinks and similar products intended for young children 250 mg/meal* Processed cereal based food and baby food for infants and young children as defined in Regulation (EU) No 609/2013 200 mg/100 g* Foods intended to meet the expenditure of intense muscular effort, especially for sportsmen Not specified Foods bearing statements on the absence or reduced presence of gluten in accordance with the requirements of Commission Implementing Regulation (EU) No 828/2014 Not specified Bakery Products (Breads, Rolls and Sweet Biscuits) Not specified Breakfast Cereals 200 mg/100 g* Cooking Fats 500 mg/100 g* Dairy Analogues except drinks 360 mg/100 g* Dairy Products except milk-based drinks 600 mg/100 g for cheese; 200 mg/100 g for soy and imitation milk products (excluding drinks)* Non-alcoholic Beverages (including dairy analogue and milk-based drinks) 600 mg/100 g for cheese; 200 mg/100 g for milk products (including milk, fromage frais and yoghurt products; excluding drinks)* Cereal/Nutrition Bars 80 mg/100 g* Spreadable Fats and Dressings 500 mg/100 g* 600 mg/100 g* * Maximum levels of DHA and EPA combined Nannochloropsis microalga, belonging the Eustigmatophycea estramenopile family, is a specie characterized by the ability to accumulate high content of lipids and fatty acids (Molino et al., 2019; Iovine et al., 2019). Nannochloropsis is a coccoid, unicellular microalga found in fresh, brackish, and seawater sources. In particular, Nannochloropsis gaditana cells show the highest amount of PUFAs and could represent an extremely valuable source of EPA, that can reach values above 4.3% wt/wt; therefore, its biomass and oil extract, represent a promising novel food. Even though Nannochloropsis gaditana and/or its metabolites/extracts have not been authorized by European Commission to be placed on the market, an application has been initiated by a Spanish Company in 2011 and an initial assessment has been carried out 279 by the Agencia española de seguridad alimentaria y nutrición (ES) (Applications under Regulation (EC) N° 258/97 of the European Parliament and of the Council, 2019). This work explores the feasibility of EPA production from Nannochloropsisgaditanaby considering the extraction step as a part of a biorefinery in which every step has been developed with a view of sustainability. 2. Materials and methods The preliminary economic evaluation of EPA extraction from the Nannochloropsis gaditana microalgae takes into consideration the individual contribution of each operation mentioned in Figure 1 and included in the VALUEMAG project (Valuable Products from Algae Using new Magnetic Cultivation and Extraction Techniques, Horizon 2020-Grant Agreement No 745695; www.valuemag.eu, 2019). Figure 1: Process steps taken into consideration for the energetic and economic evaluation The overall process involves microalgae cultivation into ad hoc developed reactor with a conical form (named SOMAC; Savvidou et al., 2019) which might allow to optimize the cells growth, a subsequent biomass harvesting with the concomitant water recovery and recycle. This step mainly consist in a membrane separation by means polymeric hollow fibers having the pore size in the microfiltration range (~0.2 μm) allowing to collect water in the permeate (Marino et al., 2019), and to concentrate algae biomass in the opposite side of the membrane, i.e. into the retentate. The obtained biomass should be pretreated, by a lyophilizer-based drying and ball-mill disruption, and finally used as substrate for EPA extraction. The use of supercritical CO2as green solvent implicates a downstream gas recover and reuse for additional extraction steps. The optimization of the operational conditions for EPA production have been recently reported by Molino et al. (2019) and Iovine et al. (2019) and they have constituted the starting point for the energetic and economic preliminary assessment of the entire VALUEMAG biorefinery (Figure 1). Table 2: Optimized operative conditions for PUFAs extraction from Nannochloropsis gaditana at bench scale Molino et al. (2019) and extract quality evaluation Main operational conditions Biomass Extract Biomass loaded (g) 21.2 Ash 10.12% 2.04% Pressure (bar) 350 Protein 47.37% 78.93% Temperature (°C) 50 Carbohydrates 21.89% 4.44% CO2 flow rate (Kg/min) 0.35 Total dietary fiber (TDF) 4.05% 1.44% Extraction time (min) 135 Lipids 16.57% 13.15% of which FAMEs: 69.84% 89.89% FAME Composition: SFA 28.55% 31.06% MUFA 26.08% 28.25% EPA 36.80% 33.03% Other PUFA 8.57% 7.65% Table 2 shows the main operative conditions for the extraction stage optimized on the basis of the results at bench scale and evidencesthe extract quality obtained after thesupercritical CO2 utilization. The extraction 280 yield of the process has been 19.19% and performing a comparison with the biomass characterization it is possible to observe an increase of proteins and a decrease of ashes content, TDF and carbohydrates. At the end of the extraction process, EPA recovery has been 17.2% by working under a CO2 pressure of 350 bar at 50 °C with a flow rate of 0.35 Kg/min (2229 kgCO2/kgbiomass). Operating potential costs for the VALUEMAG plant has been estimated on the average costs of the installed power (kW), compressed air (l/s), and energy consumption (kWh/day) and have been expressed as euros/day. 3. Results and discussion The cost index for microalgae cultivation as well as for the other steps necessary for producing algal EPA, indicate as ~73.56% is related to the extraction step based on the utilization of supercritical carbon dioxide. The remaining 17.45% cost is attributed to the drying stage which makes use of a lyophilizer. More precisely, by considering that after the dewatering-harvesting step the obtained biomass should be around 10wt% (100 g/l) on the basis of a potential initial production of 5-10 l/day, the energy demand for the Ice Condensing is in the range 30-60 kWh/day for the complete lyophilization and production of 0.5-1.0 kg/day of dry biomass. The mechanical pretreatment which allows to perform cell disruption, should affect the process economy to a lesser extent, i.e. 0.56%, since the energy demand for this operation is estimated to be in the range of 1-2 kWh/day (0.5-1.0 kg/day of dry biomass to treat).The projected cost for growth phase plant, mainly depending on the use of light irradiation source required for the correct microalgae survival and growth,should contribute for ~7.13% and nutrients for around 1.29%. The average cost evaluated for the overall process is around40 euros/day (euros/Kgdry). Figure 2. Operating cost index for microalgae growth and EPA extraction via supercritical CO2 extraction calculated on the basis of the VALUEMAG technology (www.valuemag.eu, 2019). The cost influence related to the growth stage is represented in Figure 3. Figure 2. Operating cost index for microalgae growth step calculated on the basis of the VALUEMAG technology (www.valuemag.eu, 2019). Plant for Growth; 7.13% Nutrients; 1.29% Drying; 17.45% Pretreatment; 0.56%CO2- Extraction; 73.56% Plant for Growth Nutrients Drying Pre-treatment CO2-Extraction Plant for Growth; 27.56% Nutrients; 4.99%Drying; 67.46% Plant for Growth Nutrients Drying 281 Microalgae cultivation planned according theVALUEMAG technology which average cost is estimated to be ~10 euros/day, highlights as ~67.46% of the cost is related to the drying stage, while the cost for the growth plant is around 27.56% and is mainly correlated with the use of LED light irradiation sources. Nutrients consumption cost is estimated to affect the economy for 4.99%. 4. Conclusions The global omega-3 market has promoted sales volume of about 2.99 billion euros at 2018 and it has been estimated it will increase up to 8.91 billion euros at 2025. EPA/DHA market value is correlated to 18% with microalgae as natural source, 79% derives from fish oil (anchovy and sardines combined constitute ˃75% feedstock share for oil extraction) and 3% from krill oil. The oil recovered from algae might be advantageous in view of the pleasant smelll and taste together with the vegetarian origin and sustainability in comparison to fish oil.The supercritical CO2 extractionhas underlined a product composed by 78.9 wt% of proteins and 13.1wt % of lipids, while the rest (about 8%)has been constitutedbycarbohydrated, TDF and ashes. Lipids content in the extracted biomass has an EPA content of about 33%. 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