**1. Introduction**

Lignocellulosic biomass has become the subject of a great deal of attention from researchers in order to mitigate the diminution of fossil reserves stemming from growing energy requirements worldwide. Biomass is considered one of the most low-cost and largest sources of carbon, rendering it the most important raw material with great potential to support the development of a bio-based economy. This renewable feedstock can contribute to the production of versatile and value-added products in either stand-alone processors or biorefineries [1]. Lignocellulose is encountered in hardwood, softwood, grasses, agricultural and forest residues, domestic and municipal solid wastes, as well as food/feed industry residues. The main components of these materials are cellulose, hemicellulose and lignin which form a compact and recalcitrant structure [2]. The present study focuses on agricultural residues and specifically on wheat straw from Greek wheat fields, aiming to exploit the maximum potential of lignocellulosic-derived sugar streams valorization. Towards this direction, the choice of integrated processes for the conversion of all sugar streams is considered to be of paramount importance. Hence, not only the

**Citation:** Asimakopoulou, G.; Karnaouri, A.; Staikos, S.; Stefanidis, S.D.; Kalogiannis, K.G.; Lappas, A.A.; Topakas, E. Production of Omega-3 Fatty Acids from the Microalga *Crypthecodinium cohnii* by Utilizing Both Pentose and Hexose Sugars from Agricultural Residues. *Fermentation* **2021**, *7*, 219. https:// doi.org/10.3390/fermentation7040219

Academic Editor: Giuseppa Di Bella

Received: 2 October 2021 Accepted: 7 October 2021 Published: 8 October 2021

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**Copyright:** © 2021 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/).

pretreatment step of biomass but also the performance of a successful fractionation of biomass to its constituents holds a prominent role in the biorefinery concept.

Due to the recalcitrant structure of lignocellulosic biomass, pretreatment and hydrolysis are deemed necessary, in order not to rate-limit the subsequent fermentation [3]. Therefore, aiming for the adequate disruption of the compact structure of biomass, an initial pretreatment procedure is required in order to remove hemicellulose/lignin and expand the accessible surface area of cellulose for enzymes [4]. Among all pretreatment processes that have been thoroughly examined throughout the recent years, organosolv stands out due to the numerous advantages it offers such as the high purity of cellulose and the isolation of high-quality, sulfur-free lignin [5,6]. The organosolv treatment uses a liquid phase reaction medium of organic solvents or their aqueous solutions and allows for the separation and the simultaneous recovery of three streams, namely a cellulose-rich solid pulp, an aqueous hemicellulose liquid fraction and a dry solid lignin that can be obtained after evaporation of the organic solvent [7]. Moreover, organosolv pretreatment contributes to high lignin dissolution, thus resulting in high delignification rates [8] and preservation of *β-O-4* linkages for downstream lignin utilization [9] while maintaining the formation of sugar degradation products at very low levels. It is worth noting that despite the costs of the chemicals that are used through the organosolv pretreatment, there are many reports in the literature that highlight the recyclability of these solvents, thus reducing the economic constraints as much as possible [7].

Microalgae include both photosynthetic and heterotrophic unicellular organisms that commonly live in freshwater or marine aquatic environments, and they can be of a high value or can be used as a green feedstock for many important products [10]. Nowadays, microalgae are already cultivated for commercial purposes, adding value to the market by producing microalgae-based products, such as health food supplements (nutraceuticals), pharmaceuticals, cosmetics, lubricants and feed for aquaculture hatcheries in agriculture and in many other applications [11]. In this context, the cultivation of heterotrophic microalgae on sustainable, abundant and available carbon sources, such as lignocellulosic residues, not only provides an option to take advantage of these streams but also significantly reduces the cost and carbon footprint compared to autotrophic or conventional microalgae production systems that use pure sugars in the growth medium. This is achieved by converting biomass-derived hydrolysates as low-value resources into value-added bioproducts, including compounds of nutritional value, such as omega-3 fatty acids (FA) and carotenoids [12,13]. Poly-unsaturated fatty acids (PUFAs), especially those with very long chains (LC-PUFAs) such as eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), are bioactive compounds of great importance within the food and nutraceutical industry, where they are used as ingredients in functional foods, fulfilling the appropriate specifications of high value-added products [14]. *Crypthecodinium cohnii* is a heterotrophic microalga widely known for its ability to produce high percentages of DHA [15], a necessary provider for the proper cardiovascular and neural development of humans. This microorganism has been shown to be able to utilize a variety of different carbon sources apart from glucose, including volatile fatty acids from anaerobic digestion [16], ethanol [17], liquor produced after exhausted olive pomace pretreatment [18] as well as sugar-rich hydrolysates obtained after enzymatic hydrolysis of organosolv-pretreated beechwood [19,20]. In this study, we attempted to expand the range of substrates that can support the growth of *C. cohnii* and lead to accumulation of DHA by assessing wheat straw from Greek agricultural residues as a feedstock. Both pentose and hexose streams yielded after organosolv pretreatment were evaluated as carbon sources. The present work reports an efficient holistic approach for the integrated valorization of all sugar-containing fractions of biomass towards the production of this valuable product through fermentation.
