Methods for producing polyunsaturated fatty acids from aquaculture residues

Abstract

The present application relates to valorization of aquaculture residues. More specifically, the present application relates to methods for producing polyunsaturated fatty acids (PUFAs) from aquaculture residues. For example, a method can comprise: fermenting at least one aquaculture residue in the presence of at least one microorganism to produce a biomass comprising PUFAs, optionally isolating the PUFAs, wherein the at least one microorganism is a marine protist microorganism from species selected from Sphaeroforma, Crypthecodinium, Aurantiochytrium, Thraustochytrium and Oblongichytrium. The present application includes biomass produced therefrom, use thereof and kit for producing PUFAs from aquaculture residues.

Description

METHODS FOR PRODUCING POLYUNSATURATED FATTY ACIDS FROM AQUACULTURE RESIDUESCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of priority of co-pending U.S. Provisional Patent Application No. 63 / 710,594 which was filed October 22, 2024, the content of which is incorporated herein by reference in its entirety.FIELD

[0002] The present application is in the field of polyunsaturated fatty acids. More specifically, the present application relates to method for producing the same from aquaculture residues.BACKGROUND

[0003] Polyunsaturated fatty acids (PUFAs) are essential molecules in the human diet that are not naturally produced by human cells. These PUFAs are divided into two main classes: omega-3 and omega-6 fatty acids, each having distinct but vital functions (Kaur et al., 2014). Omega-6 fatty acids participate in pro-inflammatory functions, while omega-3 fatty acids play an anti-inflammatory role and are involved in the production of specialized pro-resolving lipid mediators (Innes & Calder, 2018; Recchiuti et al., 2019; Zuniga-Hernandez et al., 2022). The main sources of omega-3 fatty acids include oils (canola, soybean, walnut) and fatty fish (mackerel, salmon, tuna, sardine, herring). Omega-6 fatty acids are primarily found in oils (sunflower, grape seed, evening primrose, borage) and animal fats (cheese, butter, deli meats, red meat). Although the benefits of omega-3 and omega-6 fatty acids are well-documented and increasingly recognized by the global population, the worldwide availability of VLC-PUFAs (very-long-chain polyunsaturated fatty acids) remains insufficient to meet growing demand (Tocher, 2015).

[0004] To meet the increasing demand for VLC-PUFAs, primarily omega-3 fatty acids, new sources need to be explored. Microalgae have proven to be a sustainable source of high-value molecules due to their rapid growth capacity and low nutrient requirements in both freshwater and marine environments. They also offer a widerange of bioproducts (omega-3 fatty acids, biofuels, pigments) useful in various industrial sectors (food, pharmaceutical, nutraceutical, cosmetic) (Abu-Ghosh et al., 2021; Calijuri et al., 2022). omega-3 fatty acids derived from microalgae are gaining popularity for their therapeutic anti-inflammatory, antifungal, and antibiotic properties (Bule et al., 2018).

[0005] As such, there is need to provide methods for producing PUFAs with improved production yields, utilising simple conditions and nutrients, to address at least some of the drawbacks of known technologies.SUMMARY

[0006] It has been shown herein that processes of the present application provide for improved methods for producing PUFAs from aquaculture residues.

[0007] Accordingly, the present application includes a method for producing polyunsaturated fatty acids (PUFAs) from aquaculture residues, the method comprising:fermenting at least one aquaculture residue in the presence of at least one microorganism to produce a biomass comprising PUFAs,optionally isolating the PUFAs,wherein the at least one microorganism is a marine protist microorganism from species selected from Sphaeroforma, Crypthecodinium, Aurantiochytrium, Thraustochytrium and Oblongichytrium.

[0008] The present application further includes use of at least one marine protists microorganism to produce polyunsaturated fatty acids (PUFAs) from aquaculture residues.

[0009] Also provided is use of a method of the present application, for producing polyunsaturated fatty acids (PUFAs) from aquaculture residues.

[0010] The present application also includes a biomass composition from aquaculture residues, the composition comprising about 0.5% to about 5% of lipids, about 50% to about 65% of proteins and about 15% to about 25% of carbohydrates.15% PUFAs, about 65% of proteins and about 20% of carbohydrates

[0011] The present application further includes a kit for producing polyunsaturated fatty acids (PUFAs) from aquaculture residues, the kit comprising: at least one microorganism being a marine protist microorganism from species selected from Sphaeroforma, Crypthecodinium, Aurantiochytrium, Thraustochytrium and Oblongichytrium;a culture media; andinstructions of use with at least one aquaculture residue.

[0012] Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the application, are given by way of illustration only and the scope of the claims should not be limited by these embodiments but should be given the broadest interpretation consistent with the description as a whole.BRIEF DESCRIPTION OF DRAWINGS

[0013] The embodiments of the application will now be described in greater detail with reference to the attached drawings in which:

[0014] FIG.1 is a graph showing biomass and total lipids of Sphaeroforma arctica at two temperatures (4°C and 12°C), according to exemplary embodiments of the present application.

[0015] FIG.2 is a graph showing sums of omega-3 PUFAs and omega-6 PUFAs with the ratio omega-3 PUFAs on omega-6 PUFAs for the Sphaeroforma arctica at two temperatures (4°C and 12°C), according to exemplary embodiments of the present application.

[0016] FIG.3 is a graph showing biomass and total lipids of Sphaeroforma arctica at media cultures with marine co-products from algae, according to exemplary embodiments of the present application.

[0017] FIG.4 is a graph showing sums of omega-3 PUFAs and omega-6 PUFAs with the ratio omega-3 PUFAs on omega-6 PUFAs for the Sphaeroforma arctica atmedia cultures with marine co-products from algae, according to exemplary embodiments of the present application.

[0018] FIG.5 is a graph showing biomass and total lipids of Sphaeroforma arctica at media cultures with marine co-products from crab, according to exemplary embodiments of the present application.

[0019] FIG.6 is a graph showing sums of omega-3 PUFAs and omega-6 PUFAs with the ratio omega-3 PUFAs on omega-6 PUFAs for the Sphaeroforma arctica at media cultures with marine co-products from crab, according to exemplary embodiments of the present application.

[0020] FIG.7 is a graph showing biomass and total lipids of Sphaeroforma arctica at media cultures with marine co-products from lobster, according to exemplary embodiments of the present application.

[0021] FIG.8 is a graph showing sums of omega-3 PUFAs and omega-6 PUFAs with the ratio omega-3 PUFAs on omega-6 PUFAs for the Sphaeroforma arctica at media cultures with marine co-products from lobster, according to exemplary embodiments of the present application.

[0022] FIG.9 is a graph showing biomass and total lipids of Sphaeroforma arctica at media cultures with marine co-products from algae and crab, according to exemplary embodiments of the present application.

[0023] FIG.10 is a graph showing sums of omega-3 PUFAs and omega-6 PUFAs with the ratio omega-3 PUFAs on omega-6 PUFAs for the Sphaeroforma arctica at media cultures with marine co-products from algae and crab, according to exemplary embodiments of the present application.

[0024] FIG.11 is a graph showing biomass and total lipids of Sphaeroforma arctica at media cultures with marine co-products from algae and lobster, according to exemplary embodiments of the present application.

[0025] FIG.12 is a graph showing sums of omega-3 PUFAs and omega-6 PUFAs with the ratio omega-3 PUFAs on omega-6 PUFAs for the Sphaeroformaarctica at media cultures with marine co-products from algae and lobster, according to exemplary embodiments of the present application.

[0026] FIG.13 is a graph showing biomass and total lipids of Sphaeroforma arctica at different media cultures with a marine co-products flour from lobster, rock crab and whelk, according to exemplary embodiments of the present application. DETAILED DESCRIPTIONI. Definitions

[0027] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art.

[0028] As used in this application and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0029] The term “consisting” and its derivatives as used herein are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0030] The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of these features, elements, components, groups, integers, and / or steps.

[0031] The terms "about", “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate themeaning of the word it modifies or unless the context suggests otherwise to a person skilled in the art.

[0032] As used in the present application, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. For example, an embodiment including “a compound” should be understood to present certain aspects with one compound, or two or more additional compounds.

[0033] In embodiments comprising an “additional” or “second” component, such as an additional or second compound, the second component as used herein is chemically different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0034] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of” or “one or more” of the listed items is used or present.

[0035] The term “suitable” as used herein means that the selection of the particular composition or conditions would depend on the specific steps to be performed, the identity of the components to be transformed and / or the specific use for the compositions, but the selection would be well within the skill of a person trained in the art.II. Methods of the Application

[0036] Protists, unicellular eukaryotes, represent a promising source of VLC-PUFAs. These organisms, particularly marine protists, produce more VLC-PUFAs compared to photosynthetic microalgae and can grow in heterotrophic or mixotrophic conditions, thereby increasing VLC-PUFA concentration (D’lmporzano et al., 2017; Jonasdottir, 2019). Moreover, a property of these microorganisms is their biochemical composition, which can be modified by adapting to various environmental conditions (Sajjadi et al., 2018). The technology for culturing aquatic protists is thus considered an important development for VLC-PUFA production and improving lipid yields. Culture technologies largely depend on the species' metabolism. Autotrophic culture is the oldest method of microorganism culture, and the main industrial technology used inautotrophy is the open pond system (Suparmaniam et al., 2019). Unlike autotrophy, heterotrophic conditions require the addition of an organic carbon source but no light source. Recent studies have shown that using closed bioreactors for VLC-PUFA production is the best method to produce these fatty acids (Li et al., 2019).

[0037] Some engineering strategies have been established for the production of VLC-PUFAs from various protists, such as fed-batch fermentations. In fed-batch strategies, the amount of organic carbon is not provided to the culture all at once but is spread overtime according to the species' metabolic rate (Xie et al., 2017). Different strategies aimed at increasing lipid productivity have been developed, the most common being nitrogen deprivation, which induces lipid accumulation but results in a lower biomass growth rate (Sung et al., 2017). However, many challenges remain in producing fatty acids on a large scale, including creating optimal culture conditions to obtain omega-3 fatty acids as well as achieving greater biomass (Topuz, 2016).

[0038] From an economic perspective, heterotrophy offers a significant advantage by not requiring light for growth. Additionally, heterotrophic protists can also grow in dark environments or in the presence of suspended solids that hinder light penetration (Christenson & Sims, 2011). The nutrients required to culture VLC-PUFA-rich microorganisms significantly contribute to the overall costs and carbon footprint of the final product. To overcome this limitation, recycling nutrients from agro-industrial effluents, secondary streams, waste, and by-products appears to be a beneficial approach for VLC-PUFA production from aquatic protists (Ende & Noke, 2019). Byproducts and waste from the food industry (FBW) are characterized by high amounts of organic carbon, proteins, and mineral salts, which could be usefully recovered for biomass culture (Massa et al., 2017; Massa et al., 2019).

[0039] Since most protists rich in VLC-PUFAs originate predominantly from marine environments, using marine co-products from crustaceans, macroalgae, mollusks, or fish could be a better avenue than FBW to increase biomass while maintaining the basic biochemical composition of protists. Indeed, residues from fish processing and aquaculture activities are underutilized. Depending on the species exploited, these residues can account for 10-60% of the harvested biomass,representing thousands of tons of organic waste that are redirected to landfills. Recently, research and initiatives have been developed to exploit these residues, notably as fertilizers or fishing bait.

[0040] Fermentation is a highly versatile process that can be used in numerous commercial applications, including food, nutraceuticals, and cosmetics. Furthermore, the interest in developing fermentation processes with protists is due to the fact that some species can produce oils extremely rich in omega-3 fatty acids. Among obligate heterotrophic protists, it has been reported that phagotrophs such as Pchromonas marina and Gyrodinium dominans produce more EPA and DHA when fed with dried yeast (Yoon et al., 2017), while thrautochrytrides (Aurantochytrium spp., Thraustochrytrium spp., and Schizochytrium spp.) and the dinoflagellate Cryptocodinium cohnii are considered DHA producers (Burja et al., 2006; Chalima et al., 2020). Additionally, Schizochytrium spp. is also used as an industrial producer of docosapentaenoic acid (DPA) (Drouin et al., 2019) as is Sphaeroforma arctica, a marine protist with strong industrial production potential for DPA (Jostensen et al., 2002; Yan et al., 2024).

[0041] To improve the exploitation of microorganisms, research is focusing on soluble residues rich in protein as substitutes for soybean peptone and yeast extracts to introduce them into culture media compositions. Indeed, prior to the present application, no comprehensive study has been conducted to optimize Marine Broth medium, which is traditionally used for culturing these microorganisms. Additionally, the use of fermentation to recycle marine residues on an industrial level has not yet been realized. Current approaches aim to use bacteria or yeasts to produce peptides, oils, antioxidants, or food additives. The use of protist species in these processes has never been done using marine residues. One major constraint of fermenting marine residues is their salt content, which impacts or inhibits microorganism development. Addressing this issue, the use of marine protists avoids a preliminary desalination step of the residues before conditioning them for fermentation.

[0042] Indeed, most heterotrophic marine protists could grow in enriched culture media, including S. arctica, a generalist detritivore capable of recycling organic matterfrom the decomposition of marine organisms. This microorganism offers several advantages in terms of culture conditions (fermentable, saline medium, low temperature) and biochemical composition (peptides, polyunsaturated fatty acids, complex sugars) (Jostensen et al., 2002). A notable characteristic of S. arctica is the high percentage of VLC-PUFAs, representing approximately 50% of total fatty acids (Jostensen et al., 2002), with a total biomass in culture around 0.8 g / L (Yan et al., 2024). Moreover, its biomass is rich in protein (65%) and carbohydrates (20%) as well as atypical peptide structures and complex sugars such as N-acetyl-galactosamine (Jostensen et al., 2002), which are important targets for developing new pharmaceutical, nutraceutical, and cosmetic compounds (Biessen & Van Berkel, 2021).

[0043] Fermentation of S. arctica offers promising prospects both for its biochemical composition and biomass production. Indeed, fermenting S. arctica with the addition of pre-hydrolyzed marine inputs to modify culture media has yielded innovative results in fatty acid production and significantly increased algal biomass at low temperatures. These novel results have demonstrated that the use of marine inputs combined with low temperatures significantly increased biomass production while producing more omega-3 fatty acids compared to traditional culture media. Furthermore, in view of the innovative results obtained with S. arctica other protists such as Sphaeroforma napiecek, Crypthecodinium cohnii, Aurantiochytrium sp. T66, Thraustochytrium aureum GOLDSTEIN, and Oblongichytrium sp. PW19 (P19) should obtain similar results in relation to temperature and the use of marine co-products (algae, crab, lobster, rockfish, shrimp, and whelk).

[0044] Accordingly, the present application provides a method for producing polyunsaturated fatty acids (PUFAs) from aquaculture residues, the method comprising:fermenting at least one aquaculture residue in the presence of at least one microorganism to produce a biomass comprising PUFAs,optionally isolating the PUFAs,wherein the at least one microorganism is a marine protist microorganism from species selected from Sphaeroforma, Crypthecodinium, Aurantiochytrium, Thraustochytrium and Oblongichytrium.

[0045] In some embodiments, the at least one microorganism is selected from Sphaeroforma arctica, Sphaeroforma napiecek, Crypthecodinium cohnii, Aurantiochytrium sp. T66, Thraustochytrium aureum GOLDSTEIN, and Oblongichytrium sp. In some embodiments, the at least one microorganism is Sphaeroforma arctica.

[0046] In some embodiments, the at least one aquaculture residue is from processing of marine species selected from fish, mollusks, Crustacea, macro / microalgae and a combination thereof.

[0047] In some embodiments, the at least one aquaculture residue is from algae, crab, lobster, rockfish, shrimp, whelk or combinations thereof. In some embodiments, the at least one aquaculture residue is from algae, crab, lobster, or combinations thereof.

[0048] In some embodiments, the method further comprises hydrolyzing the at least one aquaculture residue to a hydrolysate form prior to fermenting. In some embodiments, the hydrolyzing comprises subjecting the at least one aquaculture residue to hydrolyzing conditions selected from an acidic ion exchange resin, a basic ion exchange resin, or heating in water.

[0049] In some embodiments, the produced biomass comprises lipids, proteins and carbohydrates. In some embodiments, the produced biomass comprises about 0.5% to about 5% of lipids, about 50% to about 65% of proteins and about 15% to about 25% of carbohydrates. In some embodiments, about 40% to about 60% of the total lipids are PUFAs. In some embodiments, the produced biomass comprises about 1 % to about 5% of lipids, about 55% to about 65% of proteins and about 20% to about 25% of carbohydrates. In some embodiments, about 50% to about 60% of the total lipids are PUFAs.

[0050] In some embodiments, the PUFAs are C to C24 polyunsaturated fatty acids (PUFAs). In some embodiments, the PUFAs are C to C22 PUFAs. In some embodiments, the PUFAs are C20 to C22 PUFAs. In some embodiments, the PUFAs are omega-3 fatty acids, omega-6 fatty acids, or combinations thereof.

[0051] In some embodiments, the PUFAs has a ratio of omega-3 fatty acid to omega-6 fatty acids of about 0.5 to about 3.5, or about 0.6 to about 3.25, or about 0.68 to about 3.15.

[0052] In some embodiments, the fermenting is conducted at a temperature of about 2°C to about 15°C. In some embodiments, the fermenting is conducted at a temperature of about 4°C to about 12°C. In some embodiments, the fermenting is conducted at a temperature of about 4°C to about 10°C. In some embodiments, the fermenting is conducted at a temperature of about 5°C to about 8°C.

[0053] In some embodiments, the fermenting is conducted in a saline culture media. For example, the saline culture media may be seawater or have a salt concentration of about 25 gsait / kgwater to about 35 gsait / kgwater. In some embodiments, the saline culture media has a salt concentration of about 30 gsait / kgwater to about 35 gsait / kgwater.

[0054] In some embodiments, the fermenting is conducted for about 3 to about 14 days. In some embodiments, the fermenting is conducted for about 5 to about 10 days. In some embodiments, the fermenting is conducted for about 6 to about 8 days.III. Uses of the Application

[0055] The present application includes use of at least one marine protists microorganism to produce polyunsaturated fatty acids (PUFAs) from aquaculture residues.

[0056] Also provided is use of a method of the present application, for producing polyunsaturated fatty acids (PUFAs) from aquaculture residues.EXAMPLES

[0057] The following non-limiting examples are illustrative of the present application.General Methods

[0058] Various FAMEs may be quantified by gas chromatography. Table 1 shows some FAMEs with their corresponding retention time and limit of detection, which was used in the following examples.Table 1: Name of FAMEs standard with the time retention and the limit of detection (ng / mL)FAME'S Name RT [min) Limit of detection fng.mL'1) C10:0 Methyl caprate 15.549 1562.5 Cll:0 Methyl undecanoate 16.946 781.25 C12:0 Methyl laurate 18.723 781.25 C13:0 Methyl tridecanoate 21.025 390.63 C14:0 Methyl myristate 24.050 781.25 C14:ln5t Methyl myristelaidate 24.994 781.25 C14:ln5c Methyl myristoleate 25.672 781.25 C15:0 Methyl pentadecanoate 28.101 781.25 C15:ln5t Methyl 10-transpentadecenoate 29.417 781.25 C15:ln5c Methyl 10-pentadecenoate 30.305 781.25 C16:0 Methyl palmitate 33.595 1171.88 C16:ln7t Methyl palmitelaidate 34.897 1562.5 C16:ln7c Methyl palmitoleate 35.702 781.25 C17:0 Methyl heptadecanoate 40.010 1171.88 C17:ln7t Methyl 10-transheptadecenoate 41.239 1562.5 C17:ln7c Methyl 10-heptadecenoate 41.936 1562.5 C18:0 Methyl stearate 45.830 781.25 C18:lnl2t Methyl petroselaidate 46.550 1562.5C18:ln9t Methyl elaidate 46.716 1171.88 C18:ln7t + C18:lnl2c Methyl transvaccenate + petroselinate 47.102 1171.88C18:ln9c Methyl oleate 47.292 781.25 C18:ln7c Methyl vaccenate 47.762 781.25 C18:2n6tt Methyl linoelaidate 48.832 781.25C18:2n6c Methyl linoleate 50.343 1171.88 C18:3n6 Methyl gamma linolenate 52.203 1171.88 C19:lnl2t Methyl 7-transnonadecenoate 52.693 781.25C19:ln9t Methyl 10-transnonadecenoate 52.908 1562.5 C18:3n3 Methyl alpha linolenate 54.330 1171.88 C20:0 Methyl arachidate 58.457 781.25 C20:ln9t Methyl trans 11-eicosenoate 59.541 1562.5 C20:ln9c Methyl 11-eicosenoate 60.247 1562.5 C20:2n6 Methyl 11-14 eicosadienoate 64.026 1171.88 C21:0 Methyl heneicosanoate 65.677 1562.5 C20:3n6 Methyl homogamma linolenate 66.262 1562.5 C20:4n6 Methyl arachidonate 67.662 1171.88 C20:3n3 Methyl 11-14-17 eicosatrienoate 68.941 1562.5 C20:5n3 Methyl eicosapentaenoate 72.864 1562.5 C22:0 Methyl behenate 73.618 1562.5 C22:ln9t Methyl brassidate 75.031 1562.5 C22:ln9c Methyl erucate 75.925 1562.5 C22:2n6 Methyl docosadienoate 80.616 1562.5 C23:0 Methyl tricosanoate 82.304 1562.5 C22:4n6 Methyl docosatetraenoate 85.714 1562.5 C22:5n6 Methyl docosapentaenoate 87.135 1562.5 C24:0 Methyl lignocerate 91.822 3125C22:5n3 Methyl docosapentaenoate 92.176 1562.5 C22:6n3 Methyl docosahexaenoate 93.855 1562.5C24:ln9 Methyl nervonoate 94.920 1562.5Example 1 - Effect of temperature on biomass production and omega-3 synthesis of Sphaeroforma arctica

[0059] S. arctica strains were cultured in Marine Broth (Difco 2216) with added of 10 g / L of NaCI and 20 g / L of soy peptone. Cultures were maintained at 4°C and 12°C and agitated at 100 rpm, kept in darkness with sufficient oxygen supply. Cultures were maintained for 7 days before lipid extraction and fatty acid methyl esters (FAMEs) quantification.

[0060] The direct transesterification method used for fatty acid transesterification of all tissues was adapted from Christen et al., (2020), Ekstrom et al., (2017) and Lepage and Roy (1984). Direct trans-methylation was performed by adding 3 mL of 12% sulfuric acid methanol solution at 90°C for 1 hour in a dry bath. Samples were subsequently cooled to 4°C and 3 mL of nanopure water was added. FAMEs were extracted by adding 800 pL of toluene and 800 pL of hexane followed by centrifugation at 2750 g for 10 min at room temperature. The supernatant was transferred in a vial and frozen at -80°C for further analysis.

[0061] FAMEs were separated and quantified by gas chromatography coupled with a flame ionization detector (GC-FID) (Agilent 8890, G3540A, Agilent Technologies, Inc., Santa Clara, CA, USA) using a 60 m x 0.25 mm i.d. capillary column (DB-23, Agilent Technologies Canada, Mississauga, ON, Canada). Helium was used as carrier gas (230 kPa constant pressure), and temperature vaporization was set at 230°C with a split injection of 50 mL / min. Temperature programming was from 80°C to 130°C (heating rate: 40°C / min), followed by an increase to 170°C (heating rate: 5.5°C / min) at which the temperature was maintained for 25 min, followed by an increase to 195°C (heating rate: 2.5°C / min) which was maintained for 4 min, followed by an increase to 210°C (heating rate: 0.4°C / min) which was maintained for 11 min, followed by a final increase to 225°C (heating rate: 20°C / min) which was maintained for 5 min. Individual methyl esters were identified by comparison with known standards (Table 1 ). The amount of each fatty acid is expressed as a relative percentage for each sample and shown in Table 2.Table 2. Fatty acid composition of omega-6 (n-6) PUFAs and omega-3 (n-3) PUFAs of Sphaeroforma arctica produced at 4° C and 12° CPUFAs 12cC 4°CL'no e'c ac'd (18:2n-6) 0.51 + 0.05 1.74 + 0.18 Gamma- 'no en'c ac'd (18:3 n-6) 0.00 ± 0.00 0.00 + 0.00 E'cosad'eno'c ac'd (20:2 n-6) 4.34 + 0.33 5.18 + 1.26 Ddomo-gamma- 'no eTc ac'd (20:3n-6) 13.45 + 0.34 3.27 + 0.98 Amcn'don'c ac'd (20:4 n-6) 10.38 + 0.45 4.76 + 0.76 Docosad'eno'c ac'd (22:2 n-6) 0.00 + 0.00 0.06 + 0.01 Docosatetmeno'c ac'd (22:4n-6) 0.48 + 0.05 0.22 + 0.05 Docosaaentaeno'c ac'd (22 :5n-6) 0.94 + 0.06 0.40 + 0.02 2 n-6 PUFAs 30.11 + 0.89 15.63 + 3.16 A o na- 'no e'c ac'd (18 :3 n-3) 0.01 + 0.01 0.19 + 0.08 D'nomo-a ana- 'no en'c ac'd (20:3n-3) 0.12 + 0.01 0.20 + 0.03 E'cosa oentaeno'c ac'd (20:5n-3) 9.40 + 0.45 26.93 + 4.41 Docosaoentaeno'c ac'd (22:5n-3) 2.06 + 0.13 3.27 + 0.47 Docosanexaeno'c ac'd (22:6 n-3) 8.93 + 0.39 18.60 + 3.172 n-3 PUFAs 20.52 + 0.79 49.18 + 7.29

[0062] Reducing the temperature during the cultivation of S. arctica did not affect the amount of biomass harvested, as biomass production was comparable at both 12°C and 4°C. However, a significant difference was observed in lipid production within S. arctica cells. Lipid production at 4°C was increased by 65% compared to that at 12°C (FIG.1). This increase in lipid production was also accompanied by an elevation in total polyunsaturated fatty acids (PUFAs). At 12°C, the synthesis of omega-3 and omega-6 fatty acids was equivalent to 20.52% and 30.11 %, respectively, with a PUFAs omega-3 / omega-6 ratio of 0.68, while at 4°C, this ratio was 3.15. This observation suggests that lowering the temperature promotes the synthesis of omega-3 over omega-6 fatty acids (FIG.2 and Table 2). These results demonstrate that reducing the temperature in our culture systems does not primarily reduce biomass quantity but rather modifies the lipid composition of S. arctica by favoring omega-3 synthesis.Example 2 - Effect of the use of co-products from algae on the production of biomass and the synthesis of omega-3 from Sphaeroforma arctica cultures

[0063] S. arctica strains were cultured in Marine Broth (Difco 2216) with added of 10 g / L of NaCI and 10 ml / L of liquid algae. Cultures were maintained at 4°C and agitated at 100 rpm, kept in darkness with sufficient oxygen supply. Cultures were maintained for 7 days before lipid extraction and FAMEs quantification.

[0064] The direct transesterification method used for fatty acid transesterification of all tissues was adapted from Christen et al., (2020), Ekstrom et al., (2017) and Lepage and Roy (1984). Direct trans-methylation was performed by adding 3 mL of 12% sulfuric acid methanol solution at 90°C for 1 hour in a dry bath. Samples were subsequently cooled to 4°C and 3 mL of nanopure water was added. FAMEs were extracted by adding 800 pL of toluene and 800 pL of hexane followed by centrifugation at 2750 g for 10 min at room temperature. The supernatant was transferred in a vial and frozen at -80°C for further analysis.

[0065] FAMEs were separated and quantified by a GC-FID (Agilent 8890, G3540A, Agilent Technologies, Inc., Santa Clara, CA, USA) using a 60 m x 0.25 mm i.d. capillary column (DB-23, Agilent Technologies Canada, Mississauga, ON, Canada). Helium was used as carrier gas (230 kPa constant pressure), and temperature vaporization was set at 230°C with a split injection of 50 mL / min. Temperature programming was from 80°C to 130°C (heating rate: 40°C / min), followed by an increase to 170°C (heating rate: 5.5°C / min) at which the temperature was maintained for 25 min, followed by an increase to 195°C (heating rate: 2.5°C / min) which was maintained for 4 min, followed by an increase to 210°C (heating rate: 0.4°C / min) which was maintained for 11 min, followed by a final increase to 225°C (heating rate: 20°C / min) which was maintained for 5 min. Individual methyl esters were identified by comparison with known standards (Table 1). The amount of each fatty acid is expressed as a relative percentage for each sample and shown in Table 3.Table 3. Fatty acid composition of omega-6 PUFAs and omega-3 PUFAs of Sphaeroforma arctica produced at media cultures with marine co-products algae^UFAs STD ALL'mo e'c ac'd (IS :2m-6) 2.7S + 0.02 11.29 + 0.07 Gamma- 'mo em'c ac'd (1S:3 m-6) 0.00 + 0.00 0.31 + 0.03 E'cosad'emo'c ac'd (20:2 m-6) 6.24 + 0.15 1.47 + 0.06D'-morio-gamma- 'mo em'c ac'd (20:3m-6) 4.32 + 0.54 0.71 + 0.03 Aracm'dom'c ac'd (20:4m-6) 4.20 + 0.62 S.SS + 0.52 Docosad'emo'c ac'd (22:2m-6; 0.06 + 0.03 0.01 + 0.01 Docosatetraemo'c ac'd (22:4 m-6) 0.21 + 0.17 0.50 + 0.03 Docosa aemtaemo'c ac'd (22:5m-6) 0.67 + 0.01 1.04 + 0.052 m-6 PU FAs 1S.4S + 1.4S 24.20 + 0.67 A oma- 'mo e'c ac'd (lS:3m-3) 0.42 + 0.01 S.10 + 0.10 D'mommo-a oma- 'mo em'c ac'd (20:3m-3) 0.25 + 0.09 0.47 + 0.06 E'cosaoemtaemo'c ac'd (20:5 m-3) 13.6S + 1.S5 IS.49 + 0.4S Docosaoemtaemo'c ac'd (22:5m-3) 1.43 + 0.17 1.6S + 0.01 Docosamexaemo'c ac'd (22:6 m-3) 7.14 + 0.97 14.14 + 0.965 m-3 PU FAs 22.92 + 3. OS 42.S7 + 1.47

[0066] The introduction of marine co-products from algae as a replacement for peptone in standard culture media at 4°C increases biomass by 225% compared to the STD culture medium (FIG.3). In addition to an increase in biomass, it is noted that the addition of algae to the culture media also increases the amount of total lipids by more than 7 times (FIG.3). In terms of lipid composition of omega-6 fatty acids, this is similar between the two-culture media unlike omega-3 fatty acids where these are twice as high in the culture medium containing liquid algae (FIG.4 and Table 3). These results demonstrate that the replacement of peptone by the addition of liquid algae promotes biomass, the amount of total lipids as well as a promotion of omega-3 synthesis.Example 3 - Effect of the use of co-products from crab on the production of biomass and the synthesis of omega-3 from Sphaeroforma arctica cultures

[0067] The marine co-products from crab were hydrolyzed using three different methods. The protocol used was modified from Agustini et al., (2019) and Nurdiyana Husn et al., (2015). The first method involved acid hydrolysis, where a solution of 40 g co-product powder in 200 mL nanopure water was sonicated for 15 minutes, then treated with 16 g acidic ion exchange resin (Amberlyst™ 15(wet) Sigma #216399, CAS = 39389-20-3) at 95°C for 8 hours, followed by filtration (Agustini et al., 2019). The second method used basic hydrolysis under similar conditions but with 30 g basic resin (Sorbtech #PA3065-05L) at 50°C for 8 hours (Nurdiyana Husin et al., 2015). The third method was aqueous extraction, where a solution of 40 g co-product powder in 250 mL water was boiled for 30 minutes and then filtered.

[0068] S. arctica strains were cultured in different growth media as described in Table 4. Cultures were maintained at 4°C and agitated at 100 rpm, kept in darkness with sufficient oxygen supply. Cultures were maintained for 7 days before lipid extraction and FAMEs quantification.Table 4. Compositions of the different media culture used for culture of microalgae strainComposrtionsMedia Culture Abbreviation Marine Broth (g / L) NaCI (g / L) Soy Peotore Ig / L) Algae (mL / Ll Crab (g / L) Lobster (g / L)S“D 13 6 13 Au l_l u l_l - gae A gae 18.6 ID 10 kJ kJ A::d hyd'o ys is c'ab riAC 13.6 13 J 20 kJ Sas'c hyd'o y s i-3 c'ab H3C IS.6 10 kJ £. kJ kJ Aqueous e.c'smon cab AEC 13.6 10 ■"5 0 20 0 Acid hyc-G ysis iobste' HAH IE.6 10 n n u n 3asic hy c 'ci ysi s iobste' n3- 18.6 10 3 u 20 Aqueous ext'acTicn obste' AEH IE.6 10 n n n 7 n Ac d T / d'olys’s c'ab - -!gae n-C - ■i_ 18.6 10 £ 10 23 kJ nyd-olys s zrab ■k Algae HEC - 13 6 13 10 kj -queens eic'sc: or c'ab + A gae AEC - A. 18.6 10 10 £. kJ kJ Ac d b.'d'Cidysis oEste' -- Algae HAH A _ 13 6 10 J 10 U 20 3as c byd'o lysis oh see' + A gap 18.6 10 10 kJ Z. kJAq jeou- ex"8":on I sb see' A gae -r" r 18.6 10 10 U 23

[0069] The direct transesterification method used for fatty acid transesterification of all tissues was adapted from Christen et al., (2020), Ekstrom etal., (2017) and Lepage and Roy (1984). Direct trans-methylation was performed by adding 3 mL of 12% sulfuric acid methanol solution at 90°C for 1 hour in a dry bath. Samples were subsequently cooled to 4°C and 3 mL of nanopure water was added. FAMEs were extracted by adding 800 pL of toluene and 800 pL of hexane followed by centrifugation at 2750 g for 10 min at room temperature. The supernatant was transferred in a vial and frozen at -80°C for further analysis.

[0070] FAMEs were separated and quantified by a GC-FID (Agilent 8890, G3540A, Agilent Technologies, Inc., Santa Clara, CA, USA) using a 60 m x 0.25 mm i.d. capillary column (DB-23, Agilent Technologies Canada, Mississauga, ON, Canada). Helium was used as carrier gas (230 kPa constant pressure), and temperature vaporization was set at 230°C with a split injection of 50 mL / min. Temperature programming was from 80°C to 130°C (heating rate: 40°C / min), followed by an increase to 170°C (heating rate: 5.5°C / min) at which the temperature was maintained for 25 min, followed by an increase to 195°C (heating rate: 2.5°C / min) which was maintained for 4 min, followed by an increase to 210°C (heating rate: 0.4°C / min) which was maintained for 11 min, followed by a final increase to 225°C (heating rate: 20°C / min) which was maintained for 5 min. Individual methyl esters were identified by comparison with known standards (Table 1). The amount of each fatty acid is expressed as a relative percentage for each sample in Table 5.Table 5. Fatty acid composition of omega-6 PUFAs and omega-3 PUFAs of Sphaeroforma arctica produced at media cultures with marine co-products crabPUFAs STD HAC HBC AEG L’no e’c ac’d ( 18 :2n-6) 2.78 ± 0.02 2.02 ±0.45 1.66 ±0.03 1.71 ± 0.29 G a T Ta - ’ n o e Tc ac’d ( 18 : 3 a -6) 0.00 ± 0.00 0.01 ±0.01 0.03 ±0.03 0.58 ± 0.82 E’cesad’eno’c ac’d (20:2'1-6) 6.24 ± 0.15 7.71 ±0.48 3.70 ±0.12 7.97 ± 0.56 D ao Tiu-ga TiTia- ’no en’c ac’d (20:3n-6) 4.32 ± 0.54 4.53 ±0.24 0.91 ±0.01 4.30 ± 0.35 A’acTdoTc ac’d (20:4'1-6) 4.20 ± 0.62 5.64 ±0.89 2.11 ±0.04 4.98 ± 0.86 Docosad’eno’c ac’d (22:2'1-6) 0.06 ± 0.03 0.14 ±0.13 0.06 ±0.04 0.07 ± 0.05 Docosatet'ae'io’c ac’d (22:4'1-6) 0.21 ± 0.17 0.50 ±0.24 0.36 ±0.03 0.54 ± 0.13 Docosa Den ta eno ’c ac’d (22:51-6) 0.67 ± 0.01 0.69 ±0.15 0.46 ±0.03 0.83 ± 0.19 I 1-6 PUFAs 18.48 ± 1.48 21.23 ± 1.12 9.30 ±0.11 20.98 ± 1.06 A Dia- ’io e’c ac’d (18:31-3) 0.42 ± 0.01 0.20 ±0.06 0.20 ±0.01 0.09 ± 0.07 D’i0T0-a oia-’io ei’cac’d (20:31-3) 0.25 ± 0.09 0.18 ±0.12 0.25 ±0.00 0.46 ± 0.16 E’cosaoeitaeio’c ac’d (20:51-3) 13.68 ± 1.85 19.38 ± 3.50 15.64 ±0.10 18.76 ± 2.58 Docosa oeitaeio’c ac’d (22:51-3) 1.43 ± 0.17 2.82 ±0.41 2.67 ±0.03 2.78 ± 0.32 Docosa lexaeio’c ac’d (22:61-3) 7.14 ± 0.97 13.76 ± 3.09 10.21 ±0.03 13.49 ± 2.91I 1-3 PUFAs 22.92 ± 3.08 36.33 ±7.03 28.96 ±0.10 35.57 ± 5.65

[0071] The introduction of marine co-products from crab to replace peptone in standard culture media at 4°C initially increased biomass by 367%, 127% and 350% compared to STD culture medium for media containing acid crab hydrolysate, basic crab hydrolysate and aqueous extracts crab, respectively (FIG.5). Although most culture media showed similar levels of total lipids compared to STD medium exempt to culture media containing the basic crab hydrolysate where the total lipids are four times higher (FIG.5). Moreover, the lipid composition in terms of omega-3 and omega-6 fatty acids are slightly differed (FIG.6 and Table 5). The increased proportion of omega-3 was more pronounced in cultures containing acid crab hydrolysate and aqueous extracts of crab. These results demonstrate that the replacement of peptone by the addition of crab promotes mostly the biomass by slightly codifying the amount of total lipids as well as a promotion of omega-3 synthesis.Example 4 - Effect of the use of co-products from lobster on the production of biomass and the synthesis of omega-3 from Sphaeroforma arctica cultures

[0072] The marine co-products from lobster were hydrolyzed using three different methods. The protocol used was modified from Agustini et al., (2019) and Nurdiyana Husn et al., (2015). The first method involved acid hydrolysis, where a solution of 40 g co-product powder in 200 mL nanopure water was sonicated for 15 minutes, then treated with 16 g acidic ion exchange resin (Amberlyst 15(wet) Sigma #216399, CAS = 39389-20-3) at 95°C for 8 hours, followed by filtration (Agustini et al., 2019). The second method used basic hydrolysis under similar conditions but with 30 g basic resin (Sorbtech #PA3065-05L) at 50°C for 8 hours (Nurdiyana Husin et al., 2015). The third method was aqueous extraction, where a solution of 40 g co-product powder in 250 mL water was boiled for 30 minutes and then filtered.

[0073] S. arctica strains were cultured in different growth media as described in Table 4. Cultures were maintained at 4°C and agitated at 100 rpm, kept in darkness with sufficient oxygen supply. Cultures were maintained for 7 days before lipid extraction and FAMEs quantification.

[0074] The direct transesterification method used for fatty acid transesterification of all tissues was adapted from Christen et al., (2020), Ekstrom et al., (2017) and Lepage and Roy (1984). Direct trans-methylation was performed by adding 3 mL of 12% sulfuric acid methanol solution at 90°C for 1 hour in a dry bath. Samples were subsequently cooled to 4°C and 3 mL of nanopure water was added. FAMEs were extracted by adding 800 pL of toluene and 800 pL of hexane followed by centrifugation at 2750 g for 10 min at room temperature. The supernatant was transferred in a vial and frozen at -80°C for further analysis.

[0075] FAMEs were separated and quantified by a GC-FID (Agilent 8890, G3540A, Agilent Technologies, Inc., Santa Clara, CA, USA) using a 60 m x 0.25 mm i.d. capillary column (DB-23, Agilent Technologies Canada, Mississauga, ON, Canada). Helium was used as carrier gas (230 kPa constant pressure), and temperature vaporization was set at 230°C with a split injection of 50 mL / min. Temperature programming was from 80°C to 130°C (heating rate: 40°C / min), followed by an increase to 170°C (heating rate: 5.5°C / min) at which the temperature was maintained for 25 min, followed by an increase to 195°C (heating rate: 2.5°C / min)which was maintained for 4 min, followed by an increase to 210°C (heating rate: 0.4°C / min) which was maintained for 11 min, followed by a final increase to 225°C (heating rate: 20°C / min) which was maintained for 5 min. Individual methyl esters were identified by comparison with known standards (Table 1). The amount of each fatty acid is expressed as a relative percentage for each sample, as shown in Table 6. Table 6. Fatty acid composition of omega-6 PUFAs and omega-3 PUFAs of Sphaeroforma arctica produced at media cultures with marine co-products lobster PUFAs STD HAH HBH AEH L’ao e’c ac’d (18:2 a-5) 2.78 ± 0.02 2.01 + 0.61 1.04 ± 0.07 1.77 ± 0.55 Gamma- ’"io ea’cac’d (18:3a-6) 0.00 + 0.00 0.04 ± 0.05 0.10 ± 0.05 0.11 ± 0.13 E’cosad’eao’c ac’d (20:2'1-5) 6.24 ± 0.15 4.80 + 0.47 2.55 ± 0.25 5.45 ± 1.04 D’aoTio-gaaaaaa- ’ao ea’c ac’d (20:3a-6) 4.32 ± 0.54 3.21 ± 0.20 0.51 ± 0.10 3.08 ± 0.32 Araca’doa’c ac’d (20:4a-6) 4.20 ± 0.62 6.25 + 1.50 1.44 ± 0.07 6.12 ± 1.72 Docosad’eao’c ac’d (22:2 a-5) 0.06 ± 0.03 0.09 ± 0.05 0.06 ± 0.00 0.25 ± 0.08 Docosat et 'aeao’c ac’d (22:4 a-5) 0.21 + 0.17 0.38 + 0.14 0.29 ± 0.01 0.50 ± 0.20 Docosaaeataeao’c ac’d (22:5a-5) 0.67 + 0.01 0.64 ± 0.20 0.22 ± 0.00 0.82 ± 0.25 2 a-6 PU FAs 18.48 + 1.48 17.42 ± 0.87 6.22 ± 0.56 18.09 ± 0.61 A ana- ’ ao e’c ac’d (18:3a-3) 0.42 + 0.01 0.32 ± 0.15 0.12 ± 0.01 0.22 ± 0.11 D’aomo-a oaa- ’ao ea’c ac’d (20 :3a-3) 0.25 ± 0.09 0.19 ± 0.10 0.26 ± 0.03 0.27 ± 0.20 E’cosa aeataeao’c ac’d (20:5a-3) 13.68 + 1.85 20.62 ± 3.14 11.52 ± 0.06 21.80 ± 3.23 Docosaaeataeao’c ac’d (22:5a-3) 1.43 + 0.17 2.53 ± 0.15 2.97 ± 0.04 2.35 ± 0.33 Docosaaexaeao’c ac’d (22:6 a-3) 7.14 ± 0.97 16.00 ± 4.78 7.94 ± 0.42 16.97 ± 4.702 a-3 PU FAs 22.92 ± 3.08 39.67 ± 8.00 22.82 ± 0.48 41.61 ± 7.67

[0076] The introduction of marine co-products from lobster to replace peptone in standard culture media at 4°C initially increased biomass by 410%, 259% and 436% compared to STD culture medium for media containing acid lobster hydrolysate, basic lobster hydrolysate and aqueous extracts lobster, respectively (FIG.7). Although most culture media showed similar levels of total lipids compared to STD medium exempt to culture media containing the basic lobster hydrolysate where the total lipids are eight times higher (FIG.7). Moreover, the lipid composition in terms of omega-3 and omega-6 fatty acids are differed (FIG.8 and Table 6). The increased proportion of omega-3 was more pronounced in cultures containing acid lobster hydrolysate and aqueous extracts of lobster which are twice as high. These results demonstrate that thereplacement of peptone by the addition of lobster promotes mostly the biomass and the promotion of omega-3 synthesis.Example 5 - Effect of the use of co-products from algae and crab on the production of biomass and the synthesis of omega-3 from Sphaeroforma arctica cultures

[0077] The marine co-products from crab were hydrolyzed using three different methods. The protocol used was modified from Agustini et al., (2019) and Nurdiyana Husn et al., (2015). The first method involved acid hydrolysis, where a solution of 40 g co-product powder in 200 mL nanopure water was sonicated for 15 minutes, then treated with 16 g acidic ion exchange resin (Amberlyst 15(wet) Sigma #216399, CAS = 39389-20-3) at 95°C for 8 hours, followed by filtration (Agustini et al., 2019). The second method used basic hydrolysis under similar conditions but with 30 g basic resin (Sorbtech #PA3065-05L) at 50°C for 8 hours (Nurdiyana Husin et al., 2015). The third method was aqueous extraction, where a solution of 40 g co-product powder in 250 mL water was boiled for 30 minutes and then filtered.

[0078] S. arctica strains were cultured in different growth media as described in Table 4. Cultures were maintained at 4°C and agitated at 100 rpm, kept in darkness with sufficient oxygen supply. Cultures were maintained for 7 days before lipid extraction and FAMEs quantification.

[0079] The direct transesterification method used for fatty acid transesterification of all tissues was adapted from Christen et al., (2020), Ekstrom et al., (2017) and Lepage and Roy (1984). Direct trans-methylation was performed by adding 3 mL of 12% sulfuric acid methanol solution at 90°C for 1 hour in a dry bath. Samples were subsequently cooled to 4°C and 3 mL of nanopure water was added. FAMEs were extracted by adding 800 pL of toluene and 800 pL of hexane followed by centrifugation at 2750 g for 10 min at room temperature. The supernatant was transferred in a vial and frozen at -80°C for further analysis.

[0080] FAMEs were separated and quantified by a GC-FID (Agilent 8890, G3540A, Agilent Technologies, Inc., Santa Clara, CA, USA) using a 60 m x 0.25 mm i.d. capillary column (DB-23, Agilent Technologies Canada, Mississauga, ON, Canada). Helium was used as carrier gas (230 kPa constant pressure), andtemperature vaporization was set at 230°C with a split injection of 50 mL / min. Temperature programming was from 80°C to 130°C (heating rate: 40°C / min), followed by an increase to 170°C (heating rate: 5.5°C / min) at which the temperature was maintained for 25 min, followed by an increase to 195°C (heating rate: 2.5°C / min) which was maintained for 4 min, followed by an increase to 210°C (heating rate: 0.4°C / min) which was maintained for 11 min, followed by a final increase to 225°C (heating rate: 20°C / min) which was maintained for 5 min. Individual methyl esters were identified by comparison with known standards (Table 1). The amount of each fatty acid is expressed as a relative percentage for each sample and shown in Table 7. Table 7. Fatty acid composition of n-6 PUFAs and n-3 PUFAs of Sphaeroforma arctica produced at different media cultures with a combination the marine co-products from algae and crabPUFAs STD HAH 4 AL HBC + AL AEC - AL .incleic acid (18:2n-6) 2.78 z C.C2 1.62 z C.23 1.8C z C.CC 1.62 z C.2C G arc rra -linolenic acid (183 n-6) C.CC z C.CC C.C1 z C.C2 C.C7 z C.C1 C.17 z C.22 Eicc^adiencic acid (2C :2n-6} 6.24 z C.15 2.4C z C.63 4.72 z C.C1 2.55 z C.72 Dihcrrc-garrrra-linclenic acid (2C:3n-6) 4.32 z C.54 1.12 z C.25 1.75 z C.1C 1.21 z C.18 Arachidonic acid (2C:4n-6) 4.2C z C.62 8.38 z 1.28 4.98 z C.C9 8.CC z 1.79 Docosa dienoic acid (22:2 n-6) C.C6 z C.C3 C.C7 z C.C5 C.C9 z C.C7 C.C3 z C.C2 Dcccsa:e:raencic acid (22:4n-6) C.21 z C.17 C.3C z C.C7 C.59 z C.C4 C.27 z C.C7 Dcccsapen-.aencic acid (22:5n-6) C.67 z C.Cl 1.29 z C.21 C.64 z C.CC 1.C6 z C.23 2 n-63J = As 18.48 z 1.48 15.2C z C.33 14.64 z C.2C 14.92 z C.92 Alpha -linoleic acid (18:3n-3; C.42 z C.Cl C.33 z C.C6 C.26 z C.CC C.26 z C.C9 Dihcrrc-alpha-linclenic acid (2C:3n-3) C.25 z C.C9 C.3C z C.1C C.44 z C.C2 C.32 z C.C5 Eiccsapemaencic acid (2C:5n-3) 13.68 z 1.85 21.93 z 3.24 11.33 z C.27 21. C3 z 4.42 Dcccsapemaencic acid (22:5n-3) 1.43 z C.17 1.18 z C.21 2.38 z C.14 1.C5 z C.C8 Dccc sahexaen ci c acid (22 :6n-3) 7.14 z C.97 19.CC z 4.48 9.63 z C.CC 17.6C z 5.3C1 n-33J=As 22.92 z 3.C8 42.74 z 7.49 24. C4 z C.lC 4C.26 z 9.56

[0081] The introduction of combination of marine co-products from liquid algae and crab to replace peptone in standard culture media at 4°C initially increased biomass by 632%, 406% and 675% compared to STD culture medium for media containing acid crab hydrolysate, basic crab hydrolysate and aqueous extracts crab with liquid algae, respectively (FIG.9). Although most culture media showed high levelsof total lipids compared to STD medium for all culture (FIG.9). Moreover, the lipid composition in terms of omega-3 and omega-6 fatty acids are differed (FIG.10 and Table 7). The increased proportion of omega-3 was more pronounced in cultures containing acid crab hydrolysate and aqueous extracts of crab with liquid algae which are twice as high. These results demonstrate that the replacement of peptone by the combination of crab and liquid algae boosted both biomasses, the amount of total lipids as well as a promotion of omega-3 synthesis.Example 6 - Effect of the use of co-products from algae and lobster on the production of biomass and the synthesis of omega-3 from Sphaeroforma arctica cultures

[0082] The marine co-products from lobster were hydrolyzed using three different methods. The protocol used was modified from Agustini et al., (2019) and Nurdiyana Husn et al., (2015). The first method involved acid hydrolysis, where a solution of 40 g co-product powder in 200 mL nanopure water was sonicated for 15 minutes, then treated with 16 g acidic ion exchange resin (Amberlyst 15(wet) Sigma #216399, CAS = 39389-20-3) at 95°C for 8 hours, followed by filtration (Agustini et al., 2019). The second method used basic hydrolysis under similar conditions but with 30 g basic resin (Sorbtech #PA3065-05L) at 50°C for 8 hours (Nurdiyana Husin et al., 2015). The third method was aqueous extraction, where a solution of 40 g co-product powder in 250 mL water was boiled for 30 minutes and then filtered.

[0083] S. arctica strains were cultured in different growth media as described in Table 4. Cultures were maintained at 4°C and agitated at 100 rpm, kept in darkness with sufficient oxygen supply. Cultures were maintained for 7 days before lipid extraction and FAMEs quantification.

[0084] The direct transesterification method used for fatty acid transesterification of all tissues was adapted from Christen et al., (2020), Ekstrom et al., (2017) and Lepage and Roy (1984). Direct trans-methylation was performed by adding 3 mL of 12% sulfuric acid methanol solution at 90°C for 1 hour in a dry bath. Samples were subsequently cooled to 4°C and 3 mL of nanopure water was added. FAMEs were extracted by adding 800 pL of toluene and 800 pL of hexane followed bycentrifugation at 2750 g for 10 min at room temperature. The supernatant was transferred in a vial and frozen at -80°C for further analysis.

[0085] FAMEs were separated and quantified by a GC-FID (Agilent 8890, G3540A, Agilent Technologies, Inc., Santa Clara, CA, USA) using a 60 m x 0.25 mm i.d. capillary column (DB-23, Agilent Technologies Canada, Mississauga, ON, Canada). Helium was used as carrier gas (230 kPa constant pressure), and temperature vaporization was set at 230°C with a split injection of 50 mL / min. Temperature programming was from 80°C to 130°C (heating rate: 40°C / min), followed by an increase to 170°C (heating rate: 5.5°C / min) at which the temperature was maintained for 25 min, followed by an increase to 195°C (heating rate: 2.5°C / min) which was maintained for 4 min, followed by an increase to 210°C (heating rate: 0.4°C.min-1) which was maintained for 11 min, followed by a final increase to 225°C (heating rate: 20°C / min) which was maintained for 5 min. Individual methyl esters were identified by comparison with known standards (Table 1). The amount of each fatty acid is expressed as a relative percentage for each sample, as shown in Table 8.Table 8. Fatty acid composition of n-6 PUFAs and n-3 PUFAs of Sphaeroforma arctica produced at different media cultures with a combination the marine co-products from algae and lobsterPUFAs STD HAH + AL HBH + AL AEH + AL _ no e'c ac'd (18:2n-6) 2.78 z 0.02 2.03 z 0.29 2.50 z 0.03 1.99 z 0.23 Gamma- no en'c ac'd (18:3n-6) n nr. _ n nr. 0.05 z 0.06 0.16 z 0.03 0.00 z 0.01 E cosad eno c ac d (20:2n-6) 6.24 = 0.15 1.86 z 0.47 2.91 z O.OO 1.76 z 0.59 D'homo -gamma- no on c ac'd (20:3 n-6) 4.32 z 0.54 0.94 z 0.14 0.57 z O.Ol 1.00 z 0.11 A'ach 'don 'c ac d (20:4n-6) 4.20 z 0.62 6.44 z 1.38 3.24 z 0.02 7.87 z 2.55 Docosad eno c ac d (22:2n-6) 0.06 z 0.03 0.10 z 0.05 0.05 z 0.03 0.03 z 0.02 Docosaoeo'aeno c ac'd (22:4n-6) 0.21 z 0.17 0.19 z 0.04 0.37 = 0.04 0.23 z 0.13 Docosapencaeno c ac'd (22:5n-6) 0.67 - 0.01 0.91 z 0.15 0.35 z 0.02 1.03 z 0.28 2 n-6DLFAs 18.48 z 1.48 12.53 z 0.82 10.15 z 0.09 13.92 z 2.03 A pha- no e'c ac'd (18 :3n-3) 0.42 z 0.01 0.38 z 0.05 0.62 z O.OO 0.38 z 0.08 D'homo-a pha- no en'c ac d (2C:3n-3) 0.25 z 0.09 0.23 z 0.12 0.49 z O.OO 0.23 z 0.09 E cosapenoaeno c ac d (20:5n-3) 13.68 z 1.85 24.46 z 4.25 13.25 z 0.09 25.55 z 3.98 Docosapenoaeno c ac'd (22:5n-3) 1.43 z 0.17 1.06 z 0.06 2.64 z 0.04 1.14 z 0.22 Docosahexaeno'c ac d (22:6n-3) 7.14 z 0.97 17.90 z 5.14 9.06 z 0.07 19.83 z 4.73E n-3DLFAs 22.92 z 3.08 44.02 z 9.24 26.07 z 0.12 47.13 z 8.58

[0086] The introduction of combination of marine co-products from liquid algae and lobster to replace peptone in standard culture media at 4°C initially increased biomass by 759%, 391% and 713% compared to STD culture medium for media containing acid lobster hydrolysate, basic lobster hydrolysate and aqueous extracts lobster with liquid algae, respectively (FIG.11). Although most culture media showed slightly levels of total lipids compared to STD medium for all culture (FIG.11). Moreover, the lipid composition in terms of omega-3 and omega-6 fatty acids are differed (FIG.12 and Table 8). The increased proportion of omega-3 was more pronounced in cultures containing acid lobster hydrolysate and aqueous extracts of lobster with liquid algae which are twice as high. These results demonstrate that the replacement of peptone by the combination of lobster and liquid algae boosted both biomasses, the amount of total lipids as well as a promotion of omega-3 synthesis. Example 7 - Effect of the use of co-products from algae, lobster flour, rock crab flour and whelk flour on the production of biomass from Sphaeroforma arctica cultures

[0087] The marine co-products flour from lobster, rock crab and whelk were hydrolyzed using aqueous extraction, where a solution of 40 g co-product powder in 250 mL water was boiled for 30 minutes and then filtered.

[0088] S. arctica strains were cultured in different growth media as described in Table 9. Cultures were maintained at 4°C and agitated at 100 rpm, kept in darkness with sufficient oxygen supply. Cultures were maintained for 14 days before biomass and lipid extraction quantification.

[0089] The direct transesterification method used for fatty acid transesterification of all tissues was adapted from Christen et al., (2020), Ekstrom et al., (2017) and Lepage and Roy (1984). Direct trans-methylation was performed by adding 3 mL of 12% sulfuric acid methanol solution at 90°C for 1 hour in a dry bath. Samples were subsequently cooled to 4°C and 3 mL of nanopure water was added. FAMEs were extracted by adding 800 pL of toluene and 800 pL of hexane followed by centrifugation at 2750 g for 10 min at room temperature. The supernatant was transferred in a vial and frozen at -80°C for further analysis.

[0090] The introduction of marine co-products from lobster flour, rock crab flour and whelk flour to replace peptone in standard culture media at 4°C initially increased biomass by 107%, 86% and 2%, respectively compared to STD culture medium (FIG.13). Although all culture media showed similar levels of total lipids compared to STD medium (FIG.13). These results demonstrate that the replacement of peptone by the addition of lobster and rock crab flour promotes mostly the biomass.Table 9: Compositions of the different media culture used for culture of microalgae strainMedia culture Abbreviation Marine Broth (g / L) NaCI (g / L) Soy peptone (g / L) Algae (mL / L) Lobster (g / L) Rock Crab (g / L) Whelk (g / L) Standard STD 186 10 20 0 0 0 0 Algae AL 186 10 0 10 0 0 20 Aqueous extraction whelk AEW 186 10 0 0 0 0 0 Aqueous extraction lobster AEH 186 10 0 0 20 0 0Aqueous extraction rock crab AERC 186 10 0 0 0 20 0

[0091] While the applicant's teachings described herein are in conjunction with various embodiments for illustrative purposes, it is not intended that the applicant's teachings be limited to such embodiments as the embodiments described herein are intended to be examples. On the contrary, the applicant's teachings described and illustrated herein encompass various alternatives, modifications, and equivalents, without departing from the embodiments described herein, the general scope of which is defined in the appended claims.REFERENCES

[0092] Abu-Ghosh, S., Dubinsky, Z., Verdelho, V., & lluz, D. (2021). Unconventional high-value products from microalgae: A review. Bioresource Technology, 329, 124895. https: / / doi.Org / https: / / doi.org / 10.1016 / j.biortech.2021.124895

[0093] Agustini, N., Hidhayati, N., & Wibisono, S. (2019). Effect of hydrolysis time and acid concentration on bioethanol production of microalga Scenedesmus sp. IOP Conference Series: Earth and Environmental Science, 308, 012029. https: / / doi.Org / 10.1088 / 1755-1315 / 308 / 1 / 012029

[0094] Biessen, E. A. L., & Van Berkel, T. J. C. (2021). N-acetyl galactosamine targeting: Paving the way for clinical application of nucleotide medicines in cardiovascular diseases. Arteriosclerosis, Thrombosis, and Vascular Biology, 41( 2), 2855-2865.

[0095] Bule, M. H., Ahmed, I., Maqbool, F., Bilal, M., & Iqbal, H. M. N. (2018). Microalgae as a source of high-value bioactive compounds. Front. Biosci, 10(2), 197-216.

[0096] Burja, A. M., Radianingtyas, H., Windust, A., & Barrow, C. J. (2006). Isolation and characterization of polyunsaturated fatty acid producing Thraustochytrium species: Screening of strains and optimization of omega-3 production. Applied microbiology and biotechnology, 72, 1161-1169.

[0097] Calijuri, M. L., Silva, T. A., Magalhaes, I. B., Pereira, A. S. A. P., Marangon, B. B., Assis, L. R., & Lorentz, J. F. (2022). Bioproducts from microalgae biomass: Technology, sustainability, challenges and opportunities. Chemosphere, 305, 135508. https: / / doi.Org / https: / / doi.org / 10.1016 / j.chemosphere.2022.135508

[0098] Chalima, A., Taxeidis, G., & Topakas, E. (2020). Optimization of the production of docosahexaenoic fatty acid by the heterotrophic microalga Crypthecodinium cohnii utilizing a dark fermentation effluent. Renewable Energy, 152, 102-109.

[0099] Christen, F., Dufresne, F., Leduc, G., Dupont-Cyr, B. A., Vandenberg, G. W., Le Frangois, N. R., Tardif, J.-C., Lamarre, S. G., & Blier, P. U. (2020). Thermal tolerance and fish heart integrity: fatty acids profiles as predictors of species resilience. Conservation Physiology, 8(1), coaa108.

[0100] Christenson, L., & Sims, R. (2011). Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts. Biotechnology advances, 29(6), 686-702.

[0101] D’lmporzano, G., Silvia, S., Davide, V., Barbara, S., & Fabrizio, A. (2017). Microalgae mixotrophic growth: Opportunity for stream depuration and carbon recovery. Prospects and Challenges in Algal Biotechnology, 141-177.

[0102] Drouin, G., Rioux, V., & Legrand, P. (2019). The n-3 docosapentaenoic acid (DPA): A new player in the n-3 long chain polyunsaturated fatty acid family. Biochimie, 159, 36-48.

[0103] Ekstrom, A., Sandblom, E., Blier, P. U., Dupont Cyr, B.-A., Brijs, J., & Pichaud, N. (2017). Thermal sensitivity and phenotypic plasticity of cardiac mitochondrial metabolism in European perch, Perea fluviatilis. Journal of Experimental Biology, 220(3), 386-396.

[0104] Ende, S. S. W., & Noke, A. (2019). Heterotrophic microalgae production on food waste and by-products. Journal of Applied Phycology, 31, 1565-1571.

[0105] Innes, J. K., & Calder, P. C. (2018). Omega-6 fatty acids and inflammation. Prostaglandins, Leukotrienes and Essential Fatty Acids, 132, 41-48. https: / / doi.Org / https: / / doi.org / 10.1016 / j . plefa.2018.03.004

[0106] Jonasdottir, S. H. (2019). Fatty acid profiles and production in marine phytoplankton. Marine drugs, 17(3), 151.

[0107] Jostensen, J.-P., Sperstad, S., Johansen, S., & Landfald, B. (2002). Molecular-phylogenetic, structural and biochemical features of a cold-adapted, marineichthyosporean near the animal-fungal divergence, described from in vitro cultures. European Journal of Protistology, 38(2), 93-104.

[0108] Kaur, N., Chugh, V., & Gupta, A. K. (2014). Essential fatty acids as functional components of foods - A review. Journal of Food Science and Technology, 57(10), 2289-2303. https: / / doi.org / 10.1007 / s13197-012-0677-0

[0109] Lepage, G., & Roy, C. C. (1984). Improved recovery of fatty acid through direct transesterification without prior extraction or purification. Journal of Lipid research, 25(12), 1391-1396.

[0110] Li, S., Ji, L., Shi, Q., Wu, H., & Fan, J. (2019). Advances in the production of bioactive substances from marine unicellular microalgae Porphyridium spp. Bioresource Technology, 292, 122048.

[0111] Marti-Quijal, F. J., Remize, F., Meca, G., Ferrer, E., Ruiz, M.-J., & Barba, F. J. (2020). Fermentation in fish and by-products processing: An overview of current research and future prospects. Current opinion in food science, 31, 9-16.

[0112] Massa, M., Buono, S., Langellotti, A. L., Castaldo, L., Martello, A., Paduano, A., Sacchi, R., & Fogliano, V. (2017). Evaluation of anaerobic digestates from different feedstocks as growth media for Tetradesmus obliquus, Botryococcus braunii, Phaeodactylum tricornutum and Arthrospira maxima. New biotechnology, 36, 8-16.

[0113] Massa, M., Buono, S., Langellotti, A. L., Martello, A., Russo, G. L., Troise, D. A., Sacchi, R., Vitaglione, P., & Fogliano, V. (2019). Biochemical composition and in vitro digestibility of Galdieria sulphuraria grown on spent cherry-brine liquid. New biotechnology, 53, 9-15.

[0114] Nurdiyana Husin, N. H., Kamal, S., Chuan LingTau, C. L., Nurul Fadzni Muhammad, N. F. M., & Norhani Jusoh, N. J. (2015). Comparison of microbial growth on fish waste peptones from different hydrolysis methods.

[0115] Recchiuti, A., Mattoscio, D., & Isopi, E. (2019). Roles, actions, and therapeutic potential of specialized pro-resolving lipid mediators for the treatment of inflammation in cystic fibrosis [Review], Frontiers in Pharmacology, 10. https: / / doi.Org / 10.3389 / fphar.2019.00252

[0116] Sajjadi, B., Chen, W.-Y., Raman, A. A. A., & Ibrahim, S. (2018). Microalgae lipid and biomass for biofuel production: A comprehensive review on lipid enhancement strategies and their effects on fatty acid composition. Renewable and Sustainable Energy Reviews, 97, 200-232.

[0117] Sung, M. G., Lee, B., Kim, C. W., Nam, K., & Chang, Y. K. (2017). Enhancement of lipid productivity by adopting multi-stage continuous cultivation strategy in Nannochloropsis gaditana.

[0118] Suparmaniam, U., Lam, M. K., Uemura, Y., Lim, J. W., Lee, K. T., & Shuit, S. H. (2019). Insights into the microalgae cultivation technology and harvesting process for biofuel production: A review. Renewable and Sustainable Energy Reviews, 115, 109361.

[0119] Tocher, D. R. (2015). Omega-3 long-chain polyunsaturated fatty acids and aquaculture in perspective. Aquaculture, 449, 94-107. https: / / doi.Org / https: / / doi.org / 10.1016 / j . aquaculture.2015.01.010

[0120] Topuz, O. K. (2016). Algal oil: A novel source of omega-3 fatty acids for human nutrition. Scientific Bulletin Series F. Biotechnologies, 20.

[0121] Xie, D., Miller, E., Sharpe, P., Jackson, E., & Zhu, Q. (2017). Omega-3 production by fermentation of Yarrowia lipolytica From fed-batch to continuous. Biotechnology and bioengineering, 114(4), 798-812.

[0122] Yan, Q. W., Liu, Y.-C., Barrett, C., Haake, K., Seeler, D., May, O., & Zirkle, R. (2024). Accumulation of docosapentaenoic acid (n-3 DPA) in a novel isolate of the marine ichthyosporean Sphaeroforma arctica. Biotechnology Letters, 46(3), 373-383.

[0123] Yoon, E. Y., Park, J., Jeong, H. J., & Rho, J.-R. (2017). Fatty acid composition and docosahexaenoic acid (DHA) content of the heterotrophic dinoflagellate Oxyrrhis marina fed on dried yeast: compared with algal prey. Algae, 32(1), 67-74.

[0124] Zuniga-Hernandez, J., Sambra, V., Echeverria, F., Videla, L. A., & Valenzuela, R. (2022). N-3 PUFAs and their specialized pro-resolving lipid mediators on airway inflammatory response: beneficial effects in the prevention and treatment ofrespiratory diseases [10.1039 / D1F003551G]. Food & Function, 73(8), 4260-4272. https: / / doi.Org / 10.1039 / D1 FO03551 G

Claims

CLAIMS1. A method for producing polyunsaturated fatty acids (PUFAs) from aquaculture residues, the method comprising:fermenting at least one aquaculture residue in the presence of at least one microorganism to produce a biomass comprising PUFAs, optionally isolating the PUFAs,wherein the at least one microorganism is a marine protist microorganism from species selected from Sphaeroforma, Crypthecodinium, Aurantiochytrium, Thraustochytrium and Oblongichytrium.

2. The method of claim 1 , wherein the at least one microorganism is selected from Sphaeroforma arctica, Sphaeroforma napiecek, Crypthecodinium cohnii, Aurantiochytrium sp. T66, Thraustochytrium aureum GOLDSTEIN, and Oblongichytrium sp.

3. The method of claim 1 , wherein the at least one microorganism is Sphaeroforma arctica.

4. The method of any one of claims 1 to 3, wherein the at least one aquaculture residue is from processing of marine species selected from fish, molluscs, Crustacea, macro / microalgae and a combination thereof.

5. The method of any one of claims 1 to 3, wherein the at least one aquaculture residue is from algae, crab, lobster, rockfish, shrimp, whelk or combinations thereof.

6. The method of any one of claims 1 to 5, further comprising hydrolyzing the at least one aquaculture residue to a hydrolysate form prior to fermenting.

7. The method of claim 6, wherein the hydrolyzing comprises subjecting the at least one aquaculture residue to hydrolyzing conditions selected from an acidic ion exchange resin, a basic ion exchange resin, or heating in water.

8. The method of any one of claims 1 to 7, wherein the produced biomass comprises lipids, proteins and carbohydrates.

9. The method of any one of claims 1 to 8, wherein the produced biomass comprises about 0.5% to about 5% of lipids, about 50% to about 65% of proteins and about 15% to about 25% of carbohydrates.

10. The method of claim 9, wherein about 40% to about 60% of the total lipids are PUFAs.

11. The method of any one of claims 1 to 10, wherein the PUFAs are C to C24 polyunsaturated fatty acids (PUFAs).

12. The method of any one of claims 1 to 11 , wherein the PUFAs are omega-3 fatty acids, omega-6 fatty acids, or combinations thereof.

13. The method of any one of claims 1 to 12, wherein the PUFAs has a ratio of omega-3 fatty acid to omega-6 fatty acids of about 0.5 to about 3.5.

14. The method of any one of claims 1 to 13, wherein the fermenting is conducted at a temperature of about 2°C to about 15°C.

15. The method of any one of claims 1 to 14, wherein the fermenting is conducted in a saline culture media having a salt concentration of about 25 gsait / kgwater to about 35 gsait / kgwater.

16. The method of any one of claims 1 to 15, wherein the fermenting is conducted for about 3 to about 14 days.

17. Use of at least one marine protists microorganism to produce polyunsaturated fatty acids (PUFAs) from aquaculture residues.

18. Use of the method of any one of claims 1 to 16, for producing polyunsaturated fatty acids (PUFAs) from aquaculture residues.

19. A biomass composition from aquaculture residues, the composition comprising about 0.5% to about 5% of lipids, about 50% to about 65% of proteins and about 15% to about 25% of carbohydrates.

20. The biomass composition of claim 19, wherein about 40% to about 60% of the lipids are polyunsaturated fatty acids (PUFAs).

21. The biomass composition of claim 20, wherein the lipids comprise PUFAs.

22. The biomass composition of claim 20 or 21 , wherein the PUFAs are C to C24 polyunsaturated fatty acids (PUFAs).

23. The biomass composition of any one of claims 20 to 22, wherein the PUFAs are omega-3 fatty acids, omega-6 fatty acids, or combinations thereof.

24. The biomass composition of any one of claims 20 to 23, wherein the PUFAs has a ratio of omega-3 fatty acid to omega-6 fatty acids of about 0.5 to about 3.5.

25. A kit for producing polyunsaturated fatty acids (PUFAs) from aquaculture residues, the kit comprising:at least one microorganism being a marine protist microorganism from species selected from Sphaeroforma, Crypthecodinium, Aurantiochytrium, Thraustochytrium and Oblongichytriunr,a culture media; andinstructions of use with at least one aquaculture residue.

26. The kit of claim 25, further comprising a container for use to produce a biomass comprising PUFAs.

27. The kit of claim 25, further comprising isolation or purification device for isolating the PUFAs in the biomass.

Images