**1. Introduction**

One of the most commercially important representatives of the omega-3 fatty acids' (FAs) group is docosahexaenoic acid (DHA), which is a long-chain, highly polyunsaturated omega-3 (n-3) fatty acid (LC-PUFA). DHA is considered one of the most significant and beneficial fatty acids for the health of infants and adults. Numerous research papers have reported that DHA supports the human cardiovascular and nervous systems, prevents the occurrence of inflammatory diseases, alleviates depression, and treats psoriasis and rheumatoid arthritis. Furthermore, DHA plays a key role in the healthy development of the fetal brain and retina, thus it is commonly included in infant-oriented food products and supplements [1,2].

Currently, the main source of DHA is fish oil. However, when compared to microbial DHA, fish-derived PUFAs lack the flexibility of its biosynthetic counterpart, as availability of raw materials (e.g., fish oil for its production) strongly depends on fish resources (e.g.,

**Citation:** Didrihsone, E.;

Dubencovs, K.; Grube, M.; Shvirksts, K.; Suleiko, A.; Suleiko, A.; Vanags, J. *Crypthecodinium cohnii* Growth and Omega Fatty Acid Production in Mediums Supplemented with Extract from Recycled Biomass. *Mar. Drugs* **2022**, *20*, 68. https://doi.org/10.3390/ md20010068

Academic Editors: Maria do Rosário Domingues and Philippe Soudant

Received: 19 December 2021 Accepted: 7 January 2022 Published: 12 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

seasonality and geographical location). The fish oil purification process is also quite difficult, and the resulting product remains unsuitable for all dietary requirements (e.g., vegetarians and vegans) [3–5]. Furthermore, fish oil has a specific odour and taste, which is unpleasant for a noticeable part of people, especially infants. Therefore, omega-3 FA obtainment from fish is suboptimal and poses a negative effect on the environment. Moreover, conventional DHA production currently cannot meet the increasing demand for omega-3 FA for human consumption [5].

A wide misconception is that fish produce DHA themselves through specific metabolic pathways, which are semi-unique to aquatic life forms. Marine organisms, especially different families of fish, in their natural habitats accumulate omega-3 FAs in their organisms through feeding on zooplankton, which in turn consumes the primary omega-3 FAs producers, namely microalgae [2]. However, in fish farms, the use of eicosapentaenoic acid (EPA) and DHA as feed supplements has become a conventional practice. The demand for products containing omega-3 FA has significantly increased during the past decades. However, due to insufficient fish resources, the global market currently is in crisis. As of now, the global fish oil production reaches approximately 1 million metric tonnes per year, of which ~70% is generally used for aquafeeds [6].

Considering all of the above mentioned, direct methods for FAs acquisition from unicellular microorganisms, which have the ability to synthesize DHA on their own, becomes preferable, even though the cost of edible microbial oil is estimated to reach 3000–5000 USD per kg [7], which is considerably higher than conventional fish oil, estimated to exceed 2000 USD per metric ton [6]. Besides, the concentration of DHA in single cell oil (SCO), for example, from *Crypthecodinium cohnii*, can reach much higher concentrations when compared to fish oil (54% and 12% by mass, respectively) [8,9].

Cultivated microalgae-derived oil does not contain heavy metals and cholesterol, and has a neutral taste, which can be easily enhanced depending on the consumer requirements [2]. The average lipid content in microalgae biomass is from 20 to 50% by mass. However, under stress conditions, it can reach even higher levels (up to 85%) [10]. Microalgae species such as *C. cohnii*, *Nannochloropsis gaditana, Isochrysis galbana* and *Phaeodactylum tricornutum* were proven to be suitable for production of PU-FAs on a commercial scale [10]. Multiple commercial scale applications were already previously studied and successfully put into commission (e.g., microalgae cultivation in tubular and flat panel bioreactors [10] and transgenic oilseed plants [11]), which indicates the severity of the DHA shortage that the world is experiencing right now.

A marine dinoflagellate *C. cohnii* can accumulate PUFAs in significant amounts (up to 25% of DHA or 35% of FAs of dry weight [2,8] )and therefore it has been used previously for industrial production of omega-3 fatty acids [12]. However, in *C. cohnii* cultivation processes, yeast extract (YE) is conventionally used as the nitrogen source, which noticeably affects the cost of the target products [3]. Therefore, the identification of a cheap and renewable substrate for a highly efficient DHA production by *C. cohnii* is necessary. In the literature, suitable carbon sources have been widely studied (e.g., glucose, acetate, glycerol, oleic acid, acetic acid, ethanol, rapeseed meal hydrolysate, crude waste molasses, cheese whey, corn steep liquor, tagatose, carob syrup, date syrup, and galacturonic acid) [3,4,13–16]. The results (see Table 1) show that the highest biomass concentrations were achieved using acetic acid and ethanol in fed-batch fermentations (109 g·L−<sup>1</sup> and 83 g·L<sup>−</sup>1, respectively [17,18]) and in batch fermentations with glucose and acetate (27.7 g·L−<sup>1</sup> and 7.03 g·L−1, respectively [4,19]). The highest DHA concentrations 19 g·L−<sup>1</sup> and 11.7 g·L−<sup>1</sup> were achieved using acetic acid and ethanol, respectively, as carbon sources in fed-batch fermentations [17,18]. In batch fermentations, the highest DHA titres have been achieved with glucose (1.6 g·L−<sup>1</sup> and 1.4 g·L<sup>−</sup>1) [19,20]. However, the effect of nitrogen sources on the cultivation efficiency

has not been studied as extensively as carbon sources. Suitable nitrogen sources for marine protists are tryptone, yeast extract, peptone, soy peptone, urea, monosodium glutamate, nitrate, ammonia, and ammonium chloride [3,13]. It also should be noted that some marine protists (e.g., *Schizochytrium* species) can utilize a wider range of nitrogen sources than others (e.g., *Crypthecodinium* species) [13]. Although nitrogen source variation in *C. cohnii* cultivations has been employed, e.g., urea, yeast extract, meat extract, glutamic acid, ammonium sulphate, ammonium bicarbonate, sodium nitrite, and ammonium nitrate [15,21,22], yet mostly the effect of the nitrogen source on the *C. cohnii* growth has not been the focus of the past studies. The highest DHA titres in microalgae cells—0.99 g·L−1, has been observed when sodium nitrate was utilized [22]. The highest total lipid content, 28.48%, 18.67%, and 18.14% of dry cell weight (DCW), has been observed utilizing threonine, yeast extract, and sodium nitrate, respectively [21–23]. Moreover, to the authors' knowledge, there have been no attempts to use a recycled waste product as a nitrogen source as it will be outlined in the present study.

**Table 1.** *C. cohnii* growth parameters, DHA, and lipid production with different carbon and nitrogen sources.


Where *μ*ma**<sup>x</sup>** is the specific biomass growth rate and Yx/s is the biomass yield from a substrate (carbon source); \* The focus of the study is an effect of nitrogen source on *C. cohnii* growth parameters; \*\* Recalculated values of the results available in the literature.

An alternative way of cultivation could be to use the extraction ethanol (EE), remaining after lipid extraction, as a source of carbon, and extracts from recycled dinoflagellate biomass as a source of carbon, nitrogen, and vitamins. A substitute of conventional nitrogen sources, dinoflagellate extract (DE), is obtained from de-oiled microalgae biomass (i.e., after lipid extraction, by hydrolysis, neutralization with calcium carbonate, sedimentation, separation, evaporation of liquid phase, and drying). The described process can also be called biomass recycling.

The aim of the present study was to evaluate the growth and metabolic response of *C. cohnii* to different carbon and nitrogen sources in growth media including conventional commercially available YE, and two novel extracts (EE, Des). This approach provides more efficient use of the lipid extraction by-products/waste products and circular DHA production process (Scheme 1), and therefore could be beneficial to the bio-economy.

**Scheme 1.** Dinoflagellate extract (DE) and single cell oil (SCO) acquisition process.
