**4. Discussion**

Wheat straw was preferred in this case study since high amounts are made available in Greece as side streams from the agricultural sector. The term "agricultural residues" refers to any kind of by-products or agricultural derivatives without any economic value for the enterprise or their further management or any profitable utilization [29]. In Europe, agricultural waste consists mainly of wheat straw, while its production equals about 32% of the worldwide production [30]. In particular, according to the Centre for Renewable Energy Sources & Saving (CRES) in Greece for the year 2017, wheat straw residues were estimated to be 1,150,738 tonnes of dry matter, thus rendering the valorization of these feedstocks of pivotal importance.

The OxiOrganosolv pretreatment was applied in our previous studies and was proven optimal for the maximum delignification efficiency [6,19,20]. The use of ACO and EtOH as organic solvents in the pretreatment of various lignocellulosic biomass residues has been widely reported in the literature, since they are milder and green solvents that can be easily recovered and reused, making the whole process sustainable and applicable on an industrial scale [31–33]. Moreover, they have the ability not only to cleave the bonds between lignin and hemicellulose during the organosolv pretreatment but also to remove hemicellulose and solubilize the lignin, thereby causing an increase in surface area and pore volume of cellulose, which renders cellulose more accessible to enzymatic hydrolysis [34]. ACO is reported as the most favored ketone used for delignification [7]. However, the prevalence of EtOH is probably linked to its low cost and lack of toxicity, while also leading to the efficient deconstruction of lignocellulosic biomass [33]. The advantageous use of EtOH is supported by recent approaches, such as the integration of first- and secondgeneration ethanol, to enhance the commercialization of cellulose-derived EtOH [34]. In addition, the concentration of EtOH in the reaction medium of organosolv processes has been mainly within 30–60% (*v*/*v*) [35–37], which is in concert with the concentration acquired after the first distillation at first-generation EtOH industries. Both ACO and EtOH have aroused research interest on account of their distinct properties: they dissolve lignin and enable recovery of solid lignin after solvent removal, since the latter is insoluble in aqueous solutions [35]; they enable fractionation of hemicellulose-derived oligosaccharides from cellulose because the former exhibit some solubility in the organic solvent: H2O mixture [36]; they result in cellulose structure swelling, causing the crystal structure of cellulose fibers to be unfolded and thus rendering cellulose more eligible for hydrolysis [38]; and they can be both collected and recycled, thus reducing operating costs. This process seems to be promising for lignocellulose fractionation in order to obtain a solid and a liquid fraction that can be further processed separately, provided that a techno economical study including the solvent recycling is carried out prior to setting up larger scale units.

In our previous works, the advantages of OxiOrganosolv both for the efficient fractionation and increased enzymatic digestibility of hardwoods (beechwood) and softwoods (pine) has been demonstrated [6,20], thus highlighting the advantages of this process compared to the traditional organosolv pretreatment upon addition of sulfuric acid. In the present study, we attempted to further apply the process in crops, showing that it is also efficient for delignification of this type of substrate and the production of sugar-rich streams. Salapa et al. [39] reported the organosolv treatment of wheat straw in the presence of sulfuric acid as catalyst by evaluating five different organic solvents; the results showed that ACO was the most efficient in biomass delignification, achieving 76.4% lignin removal after pretreatment at 180 ◦C for 40 min. However, enzymatic hydrolysis of the produced solid pulp reached only 49.97% cellulose conversion, while in the case of the present study, the yield was 76.2%. In another work, organosolv fractionation of wheat straw upon addition of acid catalyst resulted in 75.8% delignification at 190 ◦C for 60 min, while in the absence of acid at 160 and 170 ◦C (conditions comparable to those in this study), lignin removal was 4.7 and 14.4% respectively, and the enzymatic digestibility of the pulps was only 30.5% and 31.7% [40].

For the development of an efficient organosolv process taking advantage of all sugar streams, valorization of the pentose-rich liquid fraction together with the hexose-rich solid pulp is a prerequisite. Hemicellulose streams have been already studied as a potential substrate for the production of valuable chemicals, such as xylitol produced from corncob [41], biobutanol from birch kraft black liquor [42] and lactic acid from corn stover [43]. Moreover, the produced aqueous fraction contains a high number of xylo-oligosaccharides which have be studied as prebiotic compounds [20,44]. However, no work has yet been made towards the utilization of the liquid fraction for the production of fatty acids from microalga. The great advantage of *C. cohnii* is that this microorganism is able to utilize not only hexoses but also pentoses, accumulating high amounts of DHA-rich oil, as it has been originally reported from Karnaouri et al. using pure xylose and arabinose [19]. In this

study, we further evaluated the ability of the microalga to grow on a true hydrolysate from wheat straw pretreatment. Pentose utilization is still halted because hemicellulose-rich fraction originating from biomass pretreatment may be contaminated with sugar degradation products that act as inhibitors [45]. However, the OxiOrganosolv pretreatment method yields an inhibitor-free solid fraction [6], as was also shown in the present study, which is the great advantage of this process. In the process that is suggested in this work, tuning the pretreatment for the removal of hemicellulose and the release of pentose sugars in the form of monosaccharides would be an option in order to eliminate the necessity for enzymatic treatment. Additionally, concerning wheat straw, no studies have taken place regarding fatty acids production, despite the fact that wheat straw is a widely available agricultural waste.

Regarding the growth of *C. cohnii* and the production of fatty acids, all enzymatic hydrolysates from solid pulps efficiently served as carbon sources in this work. Notably, higher biomass pretreatment temperatures with ACO or EtOH produced very cellulose-rich pulps that progressively lowered the cell biomass productivity when used as carbon source for cell growth. This was attributed to high initial concentration of glucose in the growth medium, which was confirmed with the pure glucose sample tests and is also supported by the literature [46]. Since high initial concentration of carbon source hinders *C. cohnii* growth, employing a fed-batch strategy could improve cell productivity yields and reduce incubation time. As far as the % DHA content is concerned, the results are in accordance with the previous reports in the literature; a variety of different substrates, such as carob pulp syrup, resulting in 48% DHA of total lipids (however with a much lower accumulation of TFA 9.2% of cell biomass) [47], rapeseed meal hydrolysate mixed with crude molasses, yielding 22–34% DHA [48], and cheese whey with corn steep liquor [49] have been used as carbon sources in media formulations for *C. cohnii* fatty acid production. Cultivation on liquid fractions in this work produced far less DHA and more C18:1 when compared to solid fraction hydrolysates, corroborating the idea that the initial sugar concentration and the type of carbon source affected not only the amount of accumulated TFAs but also the quality of the oil and the % DHA content. It has been already reported that when pure xylose and arabinose were used as carbon sources, the oil accumulated in *C. cohnii* cells contained more C18:1 than DHA [19]. Moreover, the initial concentration of sugars in liquid fractions was much lower than that of their solid counterparts (as shown in Table S3), leading to a lower C/N ratio in the culture medium, since the initial concentration of the nitrogen source was constant in all experiments. In addition, although strain-specific, it has been demonstrated that reduced degree of unsaturation and chain-length is related to lower specific growth rate and higher TFA accumulation yields [50].

Cellular stress and nutrient deprivation can affect not only the lipid accumulation but also the fatty acid profile. A lower cell growth has been correlated with a higher lipid content, and this has been observed for many microalgal strains [51]. The differences observed in this study regarding the TFA accumulation and DHA content can be attributed to the fact that *C. cohnii* responds differently to multi-stress factors that are related to the addition of hemicellulosic fraction as carbon source, even after detoxification. Moreover, the difference in the lipid profiles when using either the solid or the liquid fractions might be also correlated with the difference in their sugar profiles; the hydrolysates derived from solid fraction are richer in glucose, whereas the hydrolysis of liquid fractions produced more xylose. Despite the lower yields observed with liquid fractions, this is the first report of the cultivation of *C. cohnii* on the pentose-rich fraction and the valorization of this fraction for the production of DHA.
