*3.2. Saccharification of Solid Fraction towards the Production of a C6 Sugar-Rich Hydrolysate*

Cellulose-rich solid pulps obtained after pretreatment were enzymatically hydrolyzed with a commercial cellulase cocktail in order to produce a glucose-rich syrup to be utilized as carbon source for the subsequent fermentation. The time course data of hydrolysis and overall % cellulose conversion to glucose are presented in Figure 2, while the TRS released after enzymatic treatment are described in Supplementary Table S2.

reaching 64.9 ± 1.1 mg/mL (721 ± 13 mg/g of pretreated biomass).

**Figure 2.** (**a**) Time course of hydrolysis (solid fraction) as function of incubation time and **(b)** effect of temperature and solvent on the % cellulose conversion. Labels in (**b**) represent the mg of glucose/g of pretreated biomass. **Figure 2.** (**a**) Time course of hydrolysis (solid fraction) as function of incubation time and **(b)** effect of temperature and solvent on the % cellulose conversion. Labels in (**b**) represent the mg of glucose/g of pretreated biomass.

*3.3. Enzymatic Hydrolysis of Liquid Fraction for the Enrichment in C5 Fermentable Sugars* Taking into account that a substantial amount of sugars is required for *C. cohnii* cultivation, L2, L3 and L6 samples were selected to be hydrolyzed and further evaluated as carbon sources, due to the higher concentration of total sugars compared to other samples and due to the presence of xylose. Cellic® HTec2 was used in order to hydrolyze hemicellulose-derived oligosaccharides to monomers that can be utilized by *C. cohnii*. The concentration of monosaccharides was increased after hydrolysis, and the results are presented in Table 3, while the sugar profile is described in Figure 3. The data show that the hydrolysis was efficient, resulting in more than a three-fold increase in the concentration of monosaccharides, especially for the L3 sample (ACO, 175 °C) which reached a hydrolysis yield of 73.4 ± 1.8%. Moreover, all monosaccharides were increased, especially xylose, thus confirming the results from sugar analysis prior to hydrolysis that showed the presence of xylo-oligosaccharides (Figure 1). However, the sample population is rather low to Even though the total number of pretreatment runs was relatively small, a correlation between pretreatment conditions and cellulose conversion to glucose can be extracted. Broadly, wheat straw samples pretreated with ACO showed better hydrolysability that their EtOH counterparts, while there was an upsurge in cellulose conversion as pretreatment temperature increased. More specifically, pretreatment at 150 ◦C with ACO yielded a solid pulp that exhibited 60.2 ± 3.7% cellulose conversion to glucose after 72 h of hydrolysis, while pretreatment with EtOH at 150 ◦C resulted in 50.1 ± 8.0% conversion. The advantageous effect of ACO over EtOH was also profound at higher temperatures (160 and 175 ◦C), while at 175 ◦C, cellulose conversion reached the highest values, namely 76.2 ± 2.6% and 53.9 ± 2.3% for ACO and EtOH pulps, respectively. These values corresponded to 783 ± 26 and 488 ± 21 mg of glucose/g of pretreated biomass. Regarding the amount of TRS, similar trends were observed, with the highest titer achieved at 175 ◦C with ACO, reaching 64.9 ± 1.1 mg/mL (721 ± 13 mg/g of pretreated biomass).

*3.2. Saccharification of Solid Fraction towards the Production of a C6 Sugar-Rich Hydrolysate*

released after enzymatic treatment are described in Supplementary Table S2.

Cellulose-rich solid pulps obtained after pretreatment were enzymatically hydrolyzed with a commercial cellulase cocktail in order to produce a glucose-rich syrup to be utilized as carbon source for the subsequent fermentation. The time course data of hydrolysis and overall % cellulose conversion to glucose are presented in Figure 2, while the TRS

Even though the total number of pretreatment runs was relatively small, a correlation between pretreatment conditions and cellulose conversion to glucose can be extracted. Broadly, wheat straw samples pretreated with ACO showed better hydrolysability that their EtOH counterparts, while there was an upsurge in cellulose conversion as pretreatment temperature increased. More specifically, pretreatment at 150 °C with ACO yielded a solid pulp that exhibited 60.2 ± 3.7% cellulose conversion to glucose after 72 h of hydrolysis, while pretreatment with EtOH at 150 °C resulted in 50.1 ± 8.0% conversion. The advantageous effect of ACO over EtOH was also profound at higher temperatures (160 and 175 °C), while at 175 °C, cellulose conversion reached the highest values, namely 76.2 ± 2.6% and 53.9 ± 2.3% for ACO and EtOH pulps, respectively. These values corresponded to 783 ± 26 and 488 ± 21 mg of glucose/g of pretreated biomass. Regarding the amount of TRS, similar trends were observed, with the highest titer achieved at 175 °C with ACO,

#### extract a statistically significant correlation between hydrolysis yield and pretreatment *3.3. Enzymatic Hydrolysis of Liquid Fraction for the Enrichment in C5 Fermentable Sugars*

conditions, both in terms of solvents and temperature. The enzymatically treated liquid fractions were further utilized in *C. cohnii* cultures, as described below. Taking into account that a substantial amount of sugars is required for *C. cohnii* cultivation, L2, L3 and L6 samples were selected to be hydrolyzed and further evaluated as carbon sources, due to the higher concentration of total sugars compared to other samples and due to the presence of xylose. Cellic® HTec2 was used in order to hydrolyze hemicellulose-derived oligosaccharides to monomers that can be utilized by *C. cohnii*. The concentration of monosaccharides was increased after hydrolysis, and the results are presented in Table 3, while the sugar profile is described in Figure 3. The data show that the hydrolysis was efficient, resulting in more than a three-fold increase in the concentration of monosaccharides, especially for the L3 sample (ACO, 175 ◦C) which reached a hydrolysis yield of 73.4 ± 1.8%. Moreover, all monosaccharides were increased, especially xylose, thus confirming the results from sugar analysis prior to hydrolysis that showed the presence of xylo-oligosaccharides (Figure 1). However, the sample population is rather low to extract a statistically significant correlation between hydrolysis yield and pretreatment conditions, both in terms of solvents and temperature. The enzymatically treated liquid fractions were further utilized in *C. cohnii* cultures, as described below.

**Figure 3.** Sugar profile of monosaccharide fraction before and after enzymatic hydrolysis of liquid fraction. Standard error is ≤ 2.5% in all measurements. **Figure 3.** Sugar profile of monosaccharide fraction before and after enzymatic hydrolysis of liquid fraction. Standard error is ≤2.5% in all measurements.

**Table 3.** Hydrolysis yield in terms of monosaccharide increase. The fraction of oligosaccharides refers to the sugar streams only (the presence of acetic or uronic acids was not considered). Standard errors are given in parenthesis. **Table 3.** Hydrolysis yield in terms of monosaccharide increase. The fraction of oligosaccharides refers to the sugar streams only (the presence of acetic or uronic acids was not considered). Standard errors are given in parenthesis.


#### *3.4. Lipid Accumulation and DHA Production by C. cohnii Growing on Biomass-Derived Enzymatic Hydrolysates* Hydrolysates after enzymatic treatment of solid and liquid fractions were evaluated *3.4. Lipid Accumulation and DHA Production by C. cohnii Growing on Biomass-Derived Enzymatic Hydrolysates*

as carbon sources for *C. cohnii* cultures towards the production of a DHA-rich oil. The results are presented in Table 4, showing that both cellulose and hemicellulose-derived streams were able to support the growth of microalgal cells and accumulation of fatty acids. What can be observed is that solid fractions showed a better performance than liquids in terms of growth, TFA accumulation and % DHA concentration. Regarding the solid fractions, EtOH pretreated samples showed higher TFA synthesis (60.5–70.3% of dried cell weight) than their ACO pretreated counterparts (37–39%), while the cell biomass was also slightly higher (Supplementary Figure S1). ACO pretreated samples reached the highest peak for cell biomass productivity at 150 °C pretreatment temperature, where the cell biomass concentration was 6.72 ± 0.67 g/L. On the other hand, EtOHpretreated solid fraction reached a peak at production at 160 °C, namely 6.23 ± 0.25 g/L. The highest TFA concentration observed was 4.38 ± 0.52 and 2.49 ±0.38 g/L of culture medium for EtOH and ACO pretreatment, respectively. No significant differences were observed in the % DHA content of the oil extracted from *C. cohnii* cells in different pretreatment conditions; in fact, only a slight increase could be observed in samples S1–S3 (ACO) compared to S4–S6 (EtOH). As far as the DHA concentration is concerned, the highest values were observed at 150 °C, 120 min with 0.97 ± 0.19 g/L in ACO pretreated samples and at 160 °C, 120 min with 1.41 ± 0.45 g/L % in EtOH pretreated samples, respectively. No correlation was observed between the TFA accumulation, the % DHA and the cellulose content of the initial solid pulps. Moreover, solid fractions pretreated with the same solvent have produced similar levels of fatty acids, indicating that pretreatment temperature was of little significance for % TFA yields. Hydrolysates after enzymatic treatment of solid and liquid fractions were evaluated as carbon sources for *C. cohnii* cultures towards the production of a DHA-rich oil. The results are presented in Table 4, showing that both cellulose and hemicellulose-derived streams were able to support the growth of microalgal cells and accumulation of fatty acids. What can be observed is that solid fractions showed a better performance than liquids in terms of growth, TFA accumulation and % DHA concentration. Regarding the solid fractions, EtOH pretreated samples showed higher TFA synthesis (60.5–70.3% of dried cell weight) than their ACO pretreated counterparts (37–39%), while the cell biomass was also slightly higher (Supplementary Figure S1). ACO pretreated samples reached the highest peak for cell biomass productivity at 150 ◦C pretreatment temperature, where the cell biomass concentration was 6.72 ± 0.67 g/L. On the other hand, EtOH-pretreated solid fraction reached a peak at production at 160 ◦C, namely 6.23 ± 0.25 g/L. The highest TFA concentration observed was 4.38 ± 0.52 and 2.49 ±0.38 g/L of culture medium for EtOH and ACO pretreatment, respectively. No significant differences were observed in the % DHA content of the oil extracted from *C. cohnii* cells in different pretreatment conditions; in fact, only a slight increase could be observed in samples S1–S3 (ACO) compared to S4–S6 (EtOH). As far as the DHA concentration is concerned, the highest values were observed at 150 ◦C, 120 min with 0.97 ± 0.19 g/L in ACO pretreated samples and at 160 ◦C, 120 min with 1.41 ± 0.45 g/L % in EtOH pretreated samples, respectively. No correlation was observed between the TFA accumulation, the % DHA and the cellulose content of the initial solid pulps. Moreover, solid fractions pretreated with the same solvent have produced similar levels of fatty acids, indicating that pretreatment temperature was of little significance for % TFA yields.



All enzymatic hydrolysates from pretreated solid pulps showed higher cell growth, TFA accumulation and % DHA compared to the untreated one, which is indicative of the efficiency of the OxiOrganosolv pretreatment in order to enhance lipid production from biomass. However, it is worth mentioning that *C. cohnii* cell growth on pure glucose resulted in lower biomass production, which was attributed to the high initial concentration of the carbon source (4.5 wt%), which hinders microalga growth [17]. Similar results regarding low cell growth were also observed in samples S3 and S6, where the initial sugar concentration was 42.3 ± 0.8 and 43.2 ± 0.5 g/L, respectively (Supplementary Table S3), which may be attributed to the high concentration of the carbon source. When pure glucose was used as the carbon source, cells accumulated 40.61 ± 3.29% of dry weight TFA, which was comparable to hydrolysates from ACO pretreated samples, resulting in a concentration of 1.08 g/L of culture medium. % DHA was significantly higher (47.2 ± 1.75% of total lipids), which corresponded to 0.51 ± 0.06 g/L. As far as the profile of fatty acids is concerned (Figure 4), one can observe that a low % DHA content is accompanied by a higher accumulation of C18:1, such as in samples S2 and S4, or C16:0 such as in sample S3. *Fermentation* **2021**, *7*, 219 11 of 16

**Figure 4.** Fatty acid profile of oil extracted by *C. cohnii* cells after 120 h of cultivation on biomassderived sugars. **Figure 4.** Fatty acid profile of oil extracted by *C. cohnii* cells after 120 h of cultivation on biomassderived sugars.

In an attempt to provide a clear view of the efficiency of the suggested process for the production of TFAs and, more specifically, DHA, the results are expressed in mg/g of *C. cohnii* cultivation using enzymatically treated liquid fractions resulted in lower yields compared to those from the solid fractions, regarding not only the TFA accumulation

pretreated and untreated biomass, as described in Table 5. Regarding the results from the

*cohnii* cell growth, TFA accumulation and % DHA content. When hydrolysate from biomass pretreated with EtOH at 160 °C was used as carbon source (sample S5), 20.3 ± 1.17 mg TFA/g of untreated biomass were produced, corresponding to 7.1 ± 1.2 mg DHA/g of untreated biomass, which was the highest achieved. The above results, when compared to those of the untreated sample (3.8 ± 1.09 mg TFA and 1.1 ± 0.05 mg DHA/g of untreated biomass), were significantly higher, thus demonstrating the efficiency of the pretreatment and the fractionation for the downstream process yields. The liquid fractions resulted in lower yields, with the highest being 10.71 ± 1.08 mg TFA and 1.41 ± 0.39 mg DHA/g of untreated biomass, providing the first documented evidence that hemicellulose-rich

**Table 5.** Summary of total TFA and DHA yields per g of pretreated solid pulp or mL of aqueous liquid fraction and overall yields per g of untreated biomass. Standard errors are given in parenthesis.

> **TFA (mg/g of Untreated Biomass)**

S1 20.75 (3.19) 14.0 (1.09) 8.06 (1.62) 5.4 (1.09) S2 13.76 (0.80) 7.6 (0.35) 4.65 (0.63) 2.6 (0.35) S3 9.97 (1.36) 4.5 (0.22) 3.34 (0.49) 1.5 (0.22) S4 26.74 (3.79) 18.4 (0.74) 5.96 (1.07) 4.1 (0.74) S5 36.48 (4.36) 20.3 (1.17) 11.75 (1.66) 7.1 (1.2) S6 18.93 (5.95) 9.0 (0.93) 5.53 (1.96) 2.6 (0.93) L2 1.95 (0.38) 9.23 (1.77) 0.19 (0.05) 0.90 (0.23) L3 0.94 (0.25) 4.68 (1.23) 0.02 (0.01) 0.10 (0.05) L6 2.97 (0.30) 10.71 (1.08) 0.39 (0.08) 1.41 (0.39) Untreated 3.69 (1.06) 3.8 (1.09) 0.46(0.02) 1.1 (0.05)

**DHA (mg/g of Pretreated Biomass or mg/mL of Liquid Fraction)**

**DHA (mg/g of Untreated Biomass)**

streams can be valorized as carbon sources for *C. cohnii*.

**TFA (mg/g of Pretreated Biomass or mg/mL of Liquid Fraction)**

**Sample**

(with the exception of L2) but also % DHA content; the latter reached up to 13.03 ± 1.41% of total lipids in the L6 sample. One possible explanation would be the presence of sugar degradation products such as furans or phenolic compounds that inhibit the cells biomass; however, after detoxification with activated carbon, the concentration of phenolic compounds was negligible, while no presence of HMF or furfural was detected on HPLC, indicating that the low amounts of DHA could arise from the different sugar profile. The profile of fatty acids, as presented in Figure 4, reveals that hydrolysates from liquid fractions have produced far less DHA and more C18:1 and C16:0 than their solid counterparts. More specifically, C16:0 was 47.8 ± 0.9% and 55.6 ± 2.4% in L2 and L3, respectively, while C18:1 reached 72.7 ± 1.2% of total fatty acids in L6.

In an attempt to provide a clear view of the efficiency of the suggested process for the production of TFAs and, more specifically, DHA, the results are expressed in mg/g of pretreated and untreated biomass, as described in Table 5. Regarding the results from the solid fractions, EtOH pretreatment favored the production of TFA and DHA when considering the pulp recovery yield after pretreatment, the saccharification efficiency, *C. cohnii* cell growth, TFA accumulation and % DHA content. When hydrolysate from biomass pretreated with EtOH at 160 ◦C was used as carbon source (sample S5), 20.3 ± 1.17 mg TFA/g of untreated biomass were produced, corresponding to 7.1 ± 1.2 mg DHA/g of untreated biomass, which was the highest achieved. The above results, when compared to those of the untreated sample (3.8 ± 1.09 mg TFA and 1.1 ± 0.05 mg DHA/g of untreated biomass), were significantly higher, thus demonstrating the efficiency of the pretreatment and the fractionation for the downstream process yields. The liquid fractions resulted in lower yields, with the highest being 10.71 ± 1.08 mg TFA and 1.41 ± 0.39 mg DHA/g of untreated biomass, providing the first documented evidence that hemicellulose-rich streams can be valorized as carbon sources for *C. cohnii*.


**Table 5.** Summary of total TFA and DHA yields per g of pretreated solid pulp or mL of aqueous liquid fraction and overall yields per g of untreated biomass. Standard errors are given in parenthesis.
