Enzymatic Process for Cystoseira Barbata Valorization: Ethanol Production and Additional By-Products

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Article Enzymatic Process for Cystoseira barbata Valorization: Ethanol Production and Additional By-Products

Doinita-Roxana Cioroiu Tirpan 1,2, Ancaelena Eliza Sterpu 2,*, Claudia Irina Koncsag 2 , Alina Georgiana Ciufu 1 and Tănase Dobre 1

1 Chemical and Biochemical Engineering Department, University “Politehnica” of Bucharest, 1-7 Gheorghe Polizu Str., 011061 Bucharest, Romania; [email protected] (D.-R.C.T.); [email protected] (A.G.C.); [email protected] (T.D.) 2 Chemistry and Chemical Engineering Department, Ovidius University of Constanta, 124 Mamaia Blvd., 900527 Constanta, Romania; [email protected] * Correspondence: [email protected]; Tel.: +40-726-683-163

Abstract: The aim of this study is to evaluate the potential of dried Cystoseira barbata alga for ethanol production through alcoholic fermentation. The inﬂuence of the main factors affecting the fermentation are studied in the frame of a 23 factorial experimental plan. The main factors inﬂuencing the process are the fermentation temperature (t from 25 ◦C to 35 ◦C), the solid to liquid ratio (S/L from 0.040 g/g to 0.080 g/g), and the cellulase ratio (R from 8 U/g d.m to 16 U/g d.m.). The maximum volatile compounds yield of 0.2808 g/g d.m and ethanol yield of 0.0158 g/g d.m were favored by the following experimental conditions: process temperature of 35 ◦C, solid to liquid ratio of 0.0415, and enzyme ratio of 16 U/g d.m. A statistical model was used to correlate the product

yield with the process factors. Additionally, 19 interesting bioactive compounds were found in the enzymatic hydrolysis and alcoholic fermentation broths which seem likely to maintain natural

Citation: Cioroiu Tirpan, D.-R.; defence mechanisms against diseases and physical disorders. Sterpu, A.E.; Koncsag, C.I.; Ciufu, A.G.; Dobre, T. Enzymatic Process for Keywords: Cystoseira barbata; alcoholic fermentation; bioactive compounds; statistical model Cystoseira barbata Valorization: Ethanol Production and Additional By-Products. Processes 2021, 9, 741. https://doi.org/10.3390/pr9050741 1. Introduction Nowadays, the main concern in industry is the depletion of fossil fuels and the exces- Academic Editor: Antonio D. Moreno sive pollution of the environment they bring about; thus, the use of the planet’s renewable resources has become a challenge. However, the production of biofuels such as biodiesel, Received: 26 March 2021 bioethanol, and biogas has certain limitations. In the case of first-generation feedstocks, Accepted: 20 April 2021 there are moral issues regarding the food versus fuel debate in the context of the worldwide Published: 22 April 2021 alimentary crisis. For second-generation feedstocks, there is intense discussion regarding the high costs of biomass pre-treatment in the production process [1]. In this regard, the Publisher’s Note: MDPI stays neutral transition was made to third-generation biomass consisting of microalgae, cyanobacteria, with regard to jurisdictional claims in published maps and institutional affil- and macroalgae, which have multiple advantages, such as their low requirements in terms iations. of cultivation area and water quality (they may grow in seawater, freshwater, or wastewa- ter), as well as their resistance to unfavorable climatic conditions. Additionally, algae have a high growth rate, up to 20 times faster than that of terrestrial plants, and they absorb carbon dioxide from the atmosphere. Seaweeds are recognized as a source of bioethanol [1–6], particularly as the absence of lignin makes polysaccharides saccharification easier [4]. Copyright: © 2021 by the authors. On the other hand, macroalgae are known for their yield of bioactive products, which Licensee MDPI, Basel, Switzerland. can be extracted through different methods [7–11] and used as food supplements or This article is an open access article pharmaceutical products [12–15]. distributed under the terms and conditions of the Creative Commons Brown algae are a phylum of macrophytes with a high content of carbohydrates, con- Attribution (CC BY) license (https:// tributing up to 60% of their dry matter (d.m.) [16,17], but this depends on the species [18], creativecommons.org/licenses/by/ place, harvest time, water temperature, availability of light during the day, salinity, nu- 4.0/). trients concentration, and pre-treatment and extraction methods used. The high con-

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tent of sugars makes them a promising feedstock for bioethanol production by aquaculture [19–21]. Cystoseira barbata (class Phaeophyaceae, order Fucales, family Sargassaceae) is a species that is spread along the Romanian southern coast of the Black Sea [22]. The alginate is the main polysaccharide found in C. barbata in the Black Sea, accounting for 19 ± 1.5% (w/w) of the dry mass [8]. Other polysaccharides present in brown algae are laminaran, mannitol, fucoidan, and cellulose. Selimi et al. [23] demonstrated that the carbohydrate fraction is sulphated (13.81%) and conjugated with proteins (9.86%) and phenolic compounds (4.98%). This is an indication that, in the enzymatic processes leading to bioethanol production, other products are formed, some of them possibly bioactive [24–26]. Additionally, the Cystoseira barbata species is known for its biomedical and pharmaceutical potential, because it contains a variety of bioactive natural substances with antimicrobial, antifungal, antiviral, and antitumor activity [27]. This study focuses on evaluating the potential of the edible brown macroalga Cysto- seira barbata for bioethanol production in parallel with identifying bioactive compounds with therapeutic properties released during enzymatic processes.

2. Materials and Methods 2.1. Materials The Cystoseira barbata species was collected on Mangalia beach (N 43◦48036” E 28◦35013”), Romania, immediately after a storm in June 2019. Cellulase from Aspergillus niger, 0.8 U/mg off-white powder, was produced by Sigma Aldrich. Commercial Saccharomyces cerevisiae yeast was procured from SC ROMPACK SRL.

2.2. Equipment and Analysis Methods The determination of water content in the fresh and dried alga was performed with the OHAUS thermo-balance, model MB45. The drying took place in three steps: 7 min at 200 ◦C, 1 min at 150 ◦C, and 12 min at 105 ◦C. For the IR spectrum, the apparatus Nicolet 6700- Thermo Scientific (manufacturer: IKA, location: Watertown, Massachusettes, USA) with the technique ATR-Attenuator Total Reflectance was used. The sample was introduced in the apparatus on a GeSe crystal, and the spectrum was performed in the range of 4000 to 600 cm−1. The final spectrum was the average of 135 scans. The chemical composition of the algae was determined by adapting the separation scheme inspired from [28,29] for the extraction of cellulose: approx. 17 g of dried and ground raw material was placed in a Soxlet apparatus and ethanol extraction of lipids and pigments was performed, then the dry powder was recovered. From this powder, the recovery of alginates in a hydrosoluble form was performed by digestion in ammonium oxalate solution 0.05% (w/w) at boiling point for 1 h. The following step consisted of bleaching the remaining powder in a 2:1 mixture of acetic acid 5% solution and sodium chlorite 2% solution for 3 h at 60 ◦C. Water rinsing was conducted until a neutral pH was reached. After filtration, the cake was soaked in 0.5 M NaOH solution for 12 h at 60 ◦C. After washing and drying, the resulting powder was boiled in 5% (v/v) hydrochloric acid solution for 3 h. This suspension was then filtered, washed to neutral, and dried to obtain the cellulose fibers. The volatile compound concentration in the distillate was determined by adapting the Standard Romanian method SR 184-2/2010 on the oenological apparatus Class-CHEM, Italy, model OH-1. The method was originally intended for the determination of the ethanol concentration in beverages. The relative density of the solution to water was measured with the pycnometer. A total of 200 mL of sample + 60 mL added water was distilled in the 20 apparatus, obtaining 200 mL distillate, then the relative density d20 was correlated with the alcoholic concentration (g/L). Due to the complex composition of the distillate, in this Processes 2021, 9, 741 3 of 14

case the measured concentration was expressed as the volatile compounds concentration 20 equivalent to ethanol d20 = 0.79 . LC-MS analyses were performed on an ultra-precision liquid chromatograph coupled with a liquid-gas (nitrogen) mass spectrometer from Agilent Technologies 6540 UHD Accurate- Mass Q-TOF. The mobile phase was 0.1% (v/v) formic acid. The analysis was performed at 30 ◦C with positive and negative ionization.

2.3. Pre-Treatment Process The algae were washed with seawater in an attempt to remove sand and epiphytes, cleaned from scraps from other algae species such as Ulva lactuca and Cladophora vagabunda, then transported to the laboratory in plastic containers and washed again with distilled water to remove salt. The humidity of a fresh alga sample was measured with the thermo-balance, as described in point 2.2, resulting in 81.36 ± 0.9% (w/w). Then, the fresh algal material was dried in a hot air dehydrator at 45 ◦C until reaching a constant weight and the remaining humidity was determined with the thermo-balance with the same method. The aim of the drying was to preserve the material for a longer time. At 25 ◦C and a relative air humidity of 55%, the remaining humidity in the dry plant was 12.48 ± 0.4% wt. The dried material was milled in a grinder and passed through a certified granulo- metric sieve with a mesh size of 630 µm, thus reducing the raw material size for the more efficient further saccharification of polysaccharides [5].

2.4. Enzymatic Hydrolysis for Polysaccharides Saccharification Enzymatic hydrolysis has a higher potential compared to acid hydrolysis, because it uses less energy to break the walls of cells, gives higher yields in ethanol, and no residual products are formed [30–33]. For the enzymatic hydrolysis, 40 g of algal powder (approximately 35 g d.m.) was boiled in a certain quantity of distilled water (420 mL or 840 mL) for 25 min for sterilization. After the cooling the mixture to approx. 40 ◦C, cellulase was added at different ratios (8 or 16 U/g d.m.) and pH was regulated at 5.5–6 from a natural one of 6.8 with a few drops of 20% (w/w) citric acid solution. It is preferable to use commercial cellulase because traces of other enzymes present in the major enzyme of interest (in this case, cellulase enzyme) will give added advantages during processing [34,35], such as a higher yield in saccharification. Enzymatic hydrolysis was performed at 40 ◦C in a shaking incubator cabinet for 24 h. After hydrolysis, a sample was collected and filtered for the LC-MS analysis. The results and discussion are presented in Section 3.4.

2.5. Bioethanol Production through Alcoholic Fermentation After the release of the fermentable sugars during the enzymatic hydrolysis, the algal broth was filtered and the solution was used for the next step. The alcoholic fermentation started after adding 1 g of commercial yeast Saccharomyces cerevisiae to the filtrate, proceeding from 420 mL or 840 mL initial distilled water, as explained above; anaerobic conditions were secured. Saccharomyces cerevisiae is the most commonly used yeast because it has a high tolerance to unwanted inhibitory compounds and a high glucose fermentation capacity [33,35]. Further, the solution was kept in the dark and at afixed temperature, under mild agitation, providing ethanol formation and carbon dioxide accumulation. The ◦ amount of CO2 produced was measured at room temperature (25 C) with a gasometer during fermentation and related to the bioethanol production. The total concentration of volatile compounds was determined after the distillation of the fermented broth, by correlating the density of the distillate with the content of Organic Matter (OM, equivalent to g/L ethanol). Processes 2021, 9, 741 4 of 14 Processes 2021, 9, x FOR PEER REVIEW 4 of 14

2.6.2.6. Experiment Experiment DesignDesign AA statistical statistical modelmodel accordingaccording to a a 2 233 factorialfactorial plan plan (3 (3 process process factors factors at at2 levels 2 levels each) each) waswas designed. designed. Based Based on on previous previous experimental experimental studies studies [ 5[5,32],,32], the the main main factors factors affecting affecting the productsthe products yields yields in case in ofcase enzymatic of enzymatic hydrolysis hydrolysis followed followed by ethanolic by ethanolic fermentation fermentation are: the proportionare: the proportion between drybetween alga anddry alga water and in wate weight,r in namelyweight, solid-namely to- solid- liquid to- ratio liquid (S/L ratio, g/g ), the(S/L cellulase, g/g), the ratio cellulase (number ratio of (number enzyme unitsof en perzyme gram units of per dry gram matter, of (dryR, U/gmatter, d.m.) (R, and U/g the temperatured.m.) and the of temperature the alcoholic of fermentation the alcoholic fermentation (t, ◦C). (t, °C). TheThe independentindependent variables variables were were set at set two at levels: two levels: S/L = 0.0415S/L = or 0.0415 0.083 g/g, or 0.083R = 8 or g/g, R16=8or16U/gd.m. U/g d.m., and t = ,25 and or 35t = °C. 25 The or35 dependent◦C. The dependentvariables were variables the volatile were compounds the volatile compoundsyield (V, g/g yield d.m.) ( Vand, g/g ethanol d.m.) yield and ( ethanolE, g/g d.m.) yield which (E, g/g were d.m.) considered which werethe process considered re- thesponses. process responses.

3.3. Results Results and and DiscussionDiscussion TheThe results results of of thethe experimentexperiment are presented and and discussed discussed in in the the following. following.

3.1.3.1. FT-IR FT-IR Spectrum Spectrum ofof CystoseiraCystoseira Barbata Powder Powder InIn interpretation interpretation of of the the results, results, the the group group frequencies frequencies characteristic characteristic of of the the main main organic or- substancesganic substances were considered. were considered. TheThe FT-IR FT-IR spectrumspectrum ofof CystoseiraCystoseira barbata barbata powder,powder, presented presented in in Figure Figure 1,1 highlights, highlights severalseveral characteristic characteristic bands—intense,bands—intense, average, average, or or slight—specific slight—specific for for particular particular functional functional groups.groups. TheirTheir identification identification was was conducted conducted in incomparison comparison with with bands bands specified specified in the inlit- the literatureerature [36]. [36].

Figure 1. The FT-IR spectrum of Cystoseira barbata powder. Figure 1. The FT-IR spectrum of Cystoseira barbata powder.

The signal at 3353.25 cm−−1 1corresponds to the -OH group (υOH = 3200–3600 cm−1), but− 1 The signal at 3353.25 cm corresponds to the -OH group (υOH = 3200–3600 cm ), in the same broad band the presence of the amine group is also detected (υNH = 3300–3500 but in the same broad band the presence of the amine group is also detected (υ = 3300– cm−1). The width of the 3353.25 cm−1 band denotes the association of molecules withNH hy- 3500 cm−1). The width of the 3353.25 cm−1 band denotes the association of molecules drogen bonds. The presence of -NH bending at 1608.62 cm−1 also confirms the presence of with hydrogen bonds. The presence of -NH bending at 1608.62 cm−1 also conﬁrms the amine groups. A band of medium intensity at 2925.72 cm−1 shows the valence vibration of presence of amine groups. A band of medium intensity at 2925.72 cm−1 shows the va- C-H bonds in alkane chains (υCH = 2850–2960 cm−1); there is a fairly broad band which may lence vibration of C-H bonds in alkane chains (υ = 2850–2960 cm−1); there is a fairly indicate the presence of a carboxylic acid with moleculesCH strongly associated with hy- broad band which may indicate the presence of a carboxylic acid with molecules strongly OH −1 droxyl groups with hydrogen bonds (υ- = 2500–3200 cm ). Some valence vibration−1 at associated with hydroxyl groups with hydrogen bonds (υ-OH = 2500–3200 cm ). Some valence vibration at 1031.28 cm−1 may indicate the simple bond C-O in ethers and esters −1 (νC-O = 1050–1250 cm ). Alkane chains and ester groups denote the lipids, since C=0,

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C-O, and carboxy groups are present in polysaccharides such as alginates (containing units of mannuronic and guluronic acid) and cellulose. The functional groups -NH2 and -COOH indicate the presence of amino acids, constituents of proteins; the amino acids found in dry alga samples will boost the alcoholic fermentation and inﬂuence the product range found in the fermentation broth. In conclusion, from the FT-IR spectrum the prevalence of polysaccharides in the composition of algae can be observed, with some of the polysaccharides constituting the raw for alcoholic fermentation. Additionally, lipids and proteins in the composition of algae are a source of other compounds produced in the hydrolysis and fermentation processes.

3.2. Fiber Analysis Results A chemical analysis of the algae was performed as described in Section 2.2, and the results are presented in Table1. The main aim was to determine the cellulose content as the raw material for the ethanolic fermentation.

Table 1. Composition of Cystoseira barbata powder.

Components Concentration, % (wt/wt) Lipids 8.20 Alginates 14.93 Extractibles in NaOH sol. 0.5M 32.08 Extractibles in HCl sol 5% (v/v) 28.97 Cellulose ﬁbers 15.82

3.3. Statistical Model for the Alcoholic Fermentation The experiment design is described in point 2.6. The experiment aimed to obtain a statistical model for the correlation of the product yield with the process factors. As seen in Figure2, in all runs, the alcoholic fermentation started almost immediately after adding the yeast; the CO2 volume (and the corresponding ethanol quantity) increased steadily over time. Most of the batches ceased fermentation after 7 h, as CO2 did not form anymore. The final quantity (g) of CO2 was then calculated with the ideal gas law and correlated with the ethanol quantity through stoechiometric calculations. Additionally, from Figure2 corroborated in Table1, the efficiency of the ethanol production can be deduced. The poorest efficiency is reached at t = 25 ◦C, S/L = 0.083, and U = 8 U/g d.m, and the highest at t = 35 ◦C, S/L = 0.0415, and U = 16 U/g d.m. From Table1, one can observe that the bioethanol yield increases with increasing fermentation temperature and enzyme ratio and decreases with the decrease in the solid to liquid ratio, thus confirming Processes 2021, 9, x FOR PEER REVIEW 6 of 14 the observations of a previous study carried out on dried Ulva lactuca species [5]. The same tendency was observed for the volatile compounds yield.

Figure 2. The CO2 volume released and accumulated during fermentation vs. time. (run#1, run#2,…, run#8 represent the Figure 2. The CO2 volume released and accumulated during fermentation vs. time. (run#1, run#2, ... , run#8 represent the 8 samples from experimental matrix, Table2 2).).

These observations can be quantified by a mathematical correlation. The yields of volatile compounds and ethanol obtained in enzymatic hydrolysis followed by ethanolic fermentation of dry brown algae are summarized in Table 2.

Table 2. Experimental matrix.

Parameter V (g/g d.m.) E (g/g d.m.) Run # t S/L U/mg Experimental Predicted Experimental Predicted (°C) ratio d.m. 1 25 0.083 8 0.1152 0.1089 0.0061 0.0058 2 25 0.083 16 0.1476 0.1452 0.0082 0.0083 3 25 0.0415 8 0.1392 0.1386 0.0076 0.0072 4 25 0.0415 16 0.1656 0.1749 0.0091 0.0096 5 35 0.083 8 0.1992 0.2154 0.0111 0.0110 6 35 0.083 16 0.2592 0.2517 0.0133 0.0135 7 35 0.0415 8 0.2544 0.2451 0.0117 0.0124 8 35 0.0415 16 0.2808 0.2814 0.0158 0.0149

The maximum volatile compounds yield was 0.2808 g/g d.m, while the ethanol maximum yield was 0.0158 g/g d.m or 0.102 g/g cellulose, compared with the theoretical yield of 0.51 g ethanol/g cellulose. In the same conditions, from Ulva lactuca the maximum ethanol yield was 0.0234 g/g DM [37], so the potential for ethanol production is 32.4% lower for Cystoseira barbata. This can be explained by chemical composition differences between the algae. U. lactuca has a higher content in polysaccharides readily saccharificable, such as cellulose, while C. barbata contains a lower content of such polysaccharides, such as laminaran, mannitol, and cellulose. Few studies on bioethanol production from brown algae class Phaeophyaceae have been published. Khan and Noreen [38] reported an ethanol production between 0.0065 and 0.017 g/g d.m for Cystoseira indica and 0.013 and 0.035 g/g d.m for Sargassum tenerrimum, while Hamouda et al. [39] reported an ethanol production from Cystoseira compressa between 0.045 and 0.050 g/g d/m. Alginate from C. barbata is more difficult to destroy with cellulase to obtain fermentable sugars [40]. Metabolically engineered microbes would be more efficiently utilized for the transformation of alginate in fermentable sugars [20]. For the model describing the volatile compounds yield (V) (Equation (1)) and ethanol yield (E) (Equation (2)), linear regression analysis was used to evaluate the correlation between the independent variables and the dependent ones. V = 0.00453 U + 0.01065 t − 0.71566 S − 0.13425, (1) E = 0.00030 U+ 0.00052 t − 0.03313 S − 0.00696. (2)

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Table 2. Experimental matrix.

Parameter V (g/g d.m.) E (g/g d.m.) Run # t S/L U/mg Experimental Predicted Experimental Predicted (◦C) ratio d.m. 1 25 0.083 8 0.1152 0.1089 0.0061 0.0058 2 25 0.083 16 0.1476 0.1452 0.0082 0.0083 3 25 0.0415 8 0.1392 0.1386 0.0076 0.0072 4 25 0.0415 16 0.1656 0.1749 0.0091 0.0096 5 35 0.083 8 0.1992 0.2154 0.0111 0.0110 6 35 0.083 16 0.2592 0.2517 0.0133 0.0135 7 35 0.0415 8 0.2544 0.2451 0.0117 0.0124 8 35 0.0415 16 0.2808 0.2814 0.0158 0.0149

These observations can be quantified by a mathematical correlation. The yields of volatile compounds and ethanol obtained in enzymatic hydrolysis followed by ethanolic fermentation of dry brown algae are summarized in Table2. The maximum volatile compounds yield was 0.2808 g/g d.m, while the ethanol maximum yield was 0.0158 g/g d.m or 0.102 g/g cellulose, compared with the theoretical yield of 0.51 g ethanol/g cellulose. In the same conditions, from Ulva lactuca the maximum ethanol yield was 0.0234 g/g DM [37], so the potential for ethanol production is 32.4% lower for Cystoseira barbata. This can be explained by chemical composition differences between the algae. U. lactuca has a higher content in polysaccharides readily saccharificable, such as cellulose, while C. barbata contains a lower content of such polysaccharides, such as laminaran, mannitol, and cellulose. Few studies on bioethanol production from brown algae class Phaeophyaceae have been published. Khan and Noreen [38] reported an ethanol production between 0.0065 and 0.017 g/g d.m for Cystoseira indica and 0.013 and 0.035 g/g d.m for Sargassum tenerrimum, while Hamouda et al. [39] reported an ethanol production from Cystoseira compressa between 0.045 and 0.050 g/g d/m. Alginate from C. barbata is more difficult to destroy with cellulase to obtain fermentable sugars [40]. Metabolically engineered microbes would be more efficiently utilized for the transformation of alginate in fermentable sugars [20]. For the model describing the volatile compounds yield (V) (Equation (1)) and ethanol yield (E) (Equation (2)), linear regression analysis was used to evaluate the correlation between the independent variables and the dependent ones.

V = 0.00453 U + 0.01065 t − 0.71566 S − 0.13425, (1)

E = 0.00030 U+ 0.00052 t − 0.03313 S − 0.00696. (2) The coefficients’ significance was tested using the statistical technique ANOVA (Anal- ysis of Variance). In Tables3 and4, the results of this analysis are presented for the coefficients of Equation (1) and Equation (2), respectively. The ANOVA test shows that all the equation coefficients are significant and indicates that there is strong evidence against the null hypothesis (p < 0.05).

Table 3. ANOVA analysis for the coefﬁcients of Equation (1).

Coefﬁcients Standard Error p-Value Lower 95% Upper 95% Intercept −0.13425 0.03040 0.01155 −0.21866 −0.04983 Variable x1 (t) 0.01065 0.00081 0.00020 0.00837 0.01292 Variable x2 (S) −0.71566 0.19757 0.02231 −1.26422 −0.16710 Variable x3 (U) 0.00453 0.00102 0.01144 0.00169 0.00738 Multiple R = 0.9902; R square = 0.9805; adjusted R square = 0.9659; standard error = 0.0115. Processes 2021, 9, 741 7 of 14

Table 4. ANOVA analysis for the coefﬁcients of Equation (2).

Coefﬁcients Standard Error p-Value Lower 95% Upper 95% Intercept −0.00696 0.00183 0.01929 −0.01206 −0.00186 −5 Variable x1 (t) 0.00052 4 × 10 0.00045 0.00038 0.00066 −5 Variable x2 (S) −0.03313 0.01194 0.05010 −0.06628 2 × 10 −5 Variable x3 (U) 0.00030 6 × 10 0.00752 0.00013 0.00048 Multiple R = 0.9863; R square = 0.9729; adjusted R square = 0.9526; standard error = 0.0007.

In conclusion, the statistical model is accurate in the customary range of process factors. Additionally, the model reﬂects the tendency of the products yields to increase with increasing fermentation temperatures and cellulase ratios, and to decrease with increasing solid to liquid ratio.

3.4. Bioactive Compounds Identification As it is known, brown seaweeds are a significant source of bioactive compounds such as vitamins, minerals, polysaccharides, polyphenols, and fatty acids, which are beneficial in herbivore food [8,41]. In the present study, other compounds were searched for, possibly to be released in the enzymatic processes intended for ethanol production from C. barbata. A batch was processed with the parameters corresponding to Run#8 in Table1, because this run gave the highest yields of volatiles and ethanol, so it was probable we would find more compounds in the broth. A sample (10 mL) was collected after hydrolysis for analysis. Then, the remaining hydrolysate was filtered and submitted to alcoholic fermentation. Afterwards, the broth was distilled, collecting up to 100 mL and aiming not to exceed the temperature of 100 ◦C. The distillate was also analyzed by LC-MS. The chromatogram for the hydrolysate is presented in Figure3. The main peaks were Processes 2021, 9, x FOR PEER REVIEW 8 of 14 identified at retention times in the range of 2.372 to 4.612 min, with another important peak being situated at RT = 21.358 min.

FigureFigure 3. 3.LC-MS LC-MS chromatogram chromatogram of of the the hydrolysate. hydrolysate.

TheThe MS MS spectraspectra were found found with with negative negative an andd then then positive positive ionization, ionization, and, and, for each for eachimportant important peak, peak, the MS the spectra MS spectra were were interpreted. interpreted. The Theapparatus apparatus holds holds a library a library of com- of compounds,pounds, but but some some of ofthe the compounds compounds were were still still unidentified unidentiﬁed and thethe researchersresearchers had had to to proposepropose structures structures in in accordance accordance with with the the molar molar weight, weight, with with the the aid aid of of NIST NIST Chemistry Chemistry Webbook,Webbook, SRD SRD 69 [6942 ],[42], PubChem PubChem [43], [43], and takingand taking into account into account the possible the possible transformations transfor- ofmations polysaccharides. of polysaccharides. In the enzymatic In the enzymatic hydrolysate, hydrolysate, over 43 ions over were 43 ions identiﬁed, were identified, most of themmost with of them a mass-to-charge with a mass-to-charge under 200 under Da, but, 200 as Da, seen but, in Figureas seen4 ,in in Figure the MS 4, spectrum in the MS (negativespectrum ionization) (negative correspondingionization) corresponding to the peak at to RT the = 2.372 peak there at RT are = also 2.372 larger there ions are such also aslargerm/z =ions 827.30 such or as 1151.42. m/z = 827.30 or 1151.42.

Figure 4. MS spectrum related to the chromatogram in Figure 2, peak at RT = 2.372.

The most abundant bioactive compounds from the hydrolysate, identified in the MS spectra with a negative ionization are listed in Table 5.

Table 5. Possible bioactive compounds identified in the hydrolyzed sample in MS spectra with negative ionization.

Chemical For- Retention Relative Nr. Name m/z mula Time (min) Abundance, % 2.625 29.3 1. Malonic acid 103.00 C3H4O4 2.807 32.0 0.061 30.6 2.625 29.3 2. β-hydroxybutyric acid 113.00 C4H2O4 2.807 32.0 2.906 47.1

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Figure 3. LC-MS chromatogram of the hydrolysate.

The MS spectra were found with negative and then positive ionization, and, for each important peak, the MS spectra were interpreted. The apparatus holds a library of compounds, but some of the compounds were still unidentified and the researchers had to propose structures in accordance with the molar weight, with the aid of NIST Chemistry Webbook, SRD 69 [42], PubChem [43], and taking into account the possible transformations of polysaccharides. In the enzymatic hydrolysate, over 43 ions were identified, Processes 2021, 9, 741 most of them with a mass-to-charge under 200 Da, but, as seen in Figure 4, in the8 ofMS 14 spectrum (negative ionization) corresponding to the peak at RT = 2.372 there are also larger ions such as m/z = 827.30 or 1151.42.

Figure 4. MS spectrum related to the chromatogram in FigureFigure2 2,, peak peak at at RT RT = = 2.372. 2.372.

The most abundant bioactive compounds from the hydrolysate, identiﬁedidentified in the MS spectra with a negative ionization are listed in Table5 5..

Table 5. Possible bioactive compounds identiﬁed in the hydrolyzed sample in MS spectra with negative ionization. Table 5. Possible bioactive compounds identified in the hydrolyzed sample in MS spectra with negative ionization. Retention Time Relative Abundance, Nr. Name m/z Chemical Formula Chemical(min) For- Retention %Relative Nr. Name m/z 2.625mula Time (min) Abundance, 29.3 % 1. Malonic acid 103.00 C3H4O4 2.8072.625 32.0 29.3 1. Malonic acid 103.00 C3H4O4 0.0612.807 30.6 32.0 2.6250.061 29.3 30.6 2.8072.625 32.0 29.3 2. β-hydroxybutyric acid 113.00 C4H2O4 2.9062.807 47.1 32.0 3.142 29.0 2. β-hydroxybutyric acid 113.00 C H O 2.906 47.1 4 2 4 3.247 16.0 3.907 23.9 4.348 26.0 16.812 62.2 21.358 38.8

3. Maltol/Phloroglucinol 125.02 C6H6O3 3.142 29.6

4. Thiophenecarboxilic acid 127.0 C5H4O2S 3.247 15.2

5. Diallyl disulphide 145.01 C6H10S2 2.807 8.7

6. Phenyliminoacetate 147.02 C8H6NO2 2.906 19.5 3.247 17.7 7. Asparagusic acid 149.1 C H O S 4 6 2 2 4.348 48.0 S-Propyl 2- 8. 163.02 C H OS 2.807 11.7 propene-1-sulﬁnothioate 6 12 2

9. 3-Acetylcoumarin 187.11 C11H8O3 3.907 35.4 3.142 11.5 10. Semustine 247.00 C H ClN O 10 18 3 2 3.247 34.8 2-Undecyl-1H-imidazole- 11. 265.16 C H N S 21.358 40.4 carbothioic acid 15 26 2 2

12. Estazolam 293.2 C6H11ClN4 0.061 39.9

The potential beneﬁts of the compounds are presented further: Malonic acid (m/z = 103.00) is a basis substance for the synthesis of drugs with anti- inﬂammatory and antimicrobial activity [44] or for synthesis of valproate, which is used to Processes 2021, 9, 741 9 of 14

treat epilepsy [45] and bipolar disorder. β-hydroxybutyric acid (m/z = 113.00), one of the most abundant compounds in the broth, was obtained in the fragmentation of alginate macro- molecules in a fragment shorter than guluronic acid; β-Hydroxybutyrate can be used as an energy source in the brain when blood glucose is low [46], and it is able to cross the blood– brain barrier into the central nervous system [47]. Maltol (m/z = 125.02) is a protective agent for oxidative stress related to ocular diseases, including glaucoma [48]. Phloroglucinol is another possible compound with m/z = 125.02; it is a commercial drug with antispasmodic properties. Thiophenecarboxilic acid (m/z = 127.00) is an intermediate compound for the synthesis of thiourides: antihistaminic, tuberculostatic, sedative drugs, etc. [49]. Diallyl disulphide (m/z = 145.01) is an organosulfur compound with anti-inflammatory and antioxidative activity in emphysema [50]. Phenyl Iminoacetate (m/z = 147.03) could be an intermediary in the synthesis of isatin derivatives, with potential antibacterial activity [51], or in N-Mannich bases with mitodepressive properties [52]. Ligands of Asparagusic acid (m/z = 149.1) with Pt (II) showed interesting anticancer results in cisplatin-resistant cell lines [53]. S-Propyl 2- propene-1-sulfinothioate (m/z = 165.04) is also a constituent of garlic oil, with anti-neoplasic and antifungal activity. Derivatives of 3-Acetylcoumarin (m/z = 187.11) were tested in vitro for anticancer activity, some of them with good results [54]. Semustine (m/z = 247.00) is a drug used in chemotherapy to treat various types of cancer [55]. The imidazole moiety of 2-Undecyl-1H-imidazole-carbothioic acid (m/z = 265.16) has antifungal activity, and imidazole derivatives synthesized in recent years are drugs used to treat a broad spectrum of diseases [56]. Estazolam (m/z = 293.2) is a benzodiazepine with anticonvulsant, hypnotic, and muscle relaxant activity [57]. Additionally, in the negative ion spectra, at RT = 4.348 and 4.612 min, reducing sugars with m/z = 193,1: Guluronic acid (C6H10O7) and 3-O-methyl-α-D-glucopyranose (C7H14O6) were found, as monomers in the structure of alginate. Their relative abundance was high: 30% and 43%, respectively. In alcoholic fermentation, they are precursors of ethanol. The same compounds were detected also in the MS spectrum, with positive ionization at RT = 4.369 min and a relative abundance of 39.3%. However, in general different compounds were identified in positive ionization spectra, as seen in Table6.

Table 6. Possible bioactive compounds identiﬁed in the hydrolyzed sample in MS spectra with positive ionization.

Retention Time Relative Abundance, Nr. Name m/z Chemical Formula (min) %

1. 3-Mercaptopropionic acid 107.09 C6H6O2S 4.473 21.6 33.3 2. Nicotinyl alcohol 110.2 C H NO 2.668 6 7 58.8

3. Furoic acid 113.0 C5H4O3 2.668 25.6

4. Maltol/ Phloroglucinol 127.06 C6H6O3 3.609 32.3 4.088 17.8 4.369 18.0 5. 5-Aminobarbituric acid 144.02 C4H5N3O3 4.473 23.5 4.913 25.4 5.491 24.0

6. Glycerol triformate 177.17 C6H8O6 3.609 32.3

7. Gluconic acid 197.13 C6H12O6 4.088 52.8 4.913 19.5 8. Methoxalen 217.13 C12H8O4 5.491 22.7 10.306 21.8

These are bioactive compounds or bases for other bioactive compounds, as follows: 3-Mercaptopropionic acid (m/z = 107.09) is a building block for glycopeptide synthesis. Nicotinyl alcohol (Piconol) (m/z = 110.2) serves as an intermediate in the synthesis of Processes 2021, 9, x FOR PEER REVIEW 10 of 14

Table 6. Possible bioactive compounds identified in the hydrolyzed sample in MS spectra with positive ionization.

Retention Relative Nr. Name m/z Chemical Formula Time (min) Abundance, % 1. 3-Mercaptopropionic acid 107.09 C6H6O2S 4.473 21.6 33.3 2. Nicotinyl alcohol 110.2 C6H7NO 2.668 58.8 3. Furoic acid 113.0 C5H4O3 2.668 25.6 4. Maltol/ Phloroglucinol 127.06 C6H6O3 3.609 32.3 4.088 17.8 4.369 18.0 5. 5-Aminobarbituric acid 144.02 C4H5N3O3 4.473 23.5 4.913 25.4 5.491 24.0 6. Glycerol triformate 177.17 C6H8O6 3.609 32.3 7. Gluconic acid 197.13 C6H12O6 4.088 52.8 4.913 19.5 8. Methoxalen 217.13 C12H8O4 5.491 22.7 10.306 21.8

Processes 2021, 9, 741 These are bioactive compounds or bases for other bioactive compounds, as follows:10 of 14 3-Mercaptopropionic acid (m/z = 107.09) is a building block for glycopeptide synthesis. Nicotinyl alcohol (Piconol) (m/z = 110.2) serves as an intermediate in the synthesis of ibuprofen piconol, a nonsteroidal anti-inflammatory agent [58]. Furoic acid (m/z = 127.06) was ibuprofen piconol, a nonsteroidal anti-inflammatory agent [58]. Furoic acid (m/z = 127.06) also found in the positive spectrum of the peak RT = 3.247 min (relative abundance, was also found in the positive spectrum of the peak RT = 3.247 min (relative abundance, 27.6%), and its alkyl esters have an anesthetic activity [59]. The bioactivity of compounds 27.6%), and its alkyl esters have an anesthetic activity [59]. The bioactivity of compounds Maltol/Phloroglucinol (m/z = 127.06) was explained before; they were also identified in the Maltol/Phloroglucinol (m/z = 127.06) was explained before; they were also identified in the spectrum of peak RT = 3.142, with negative ionization. 5-Aminobarbituric acid (m/z = 144.02) spectrum of peak RT = 3.142, with negative ionization. 5-Aminobarbituric acid (m/z = 144.02) in a hydantoin rearrangement results in an anticonvulsant agent [60]. Glycerol triformate in a hydantoin rearrangement results in an anticonvulsant agent [60]. Glycerol triformate (Triformin) (m/z = 177.17) is an anti-hyperglycemic agent for the treatment of patients with (Triformin) (m/z = 177.17) is an anti-hyperglycemic agent for the treatment of patients with type 2 diabetes [61]. D-Gluconic acid (m/z = 197.13), C6H12O7, proceeds from glucose under type 2 diabetes [61]. D-Gluconic acid (m/z = 197.13),C6H12O7, proceeds from glucose under the action of cellulase; it is used to maintain the cation–anion balance balance in in electrolyte electrolyte solu- solu- tions [62]; [62]; the calcium salt of gluconic acid is used in calcium therapy and iron salt in the treatment of anemia [[63].63]. Methoxalen (m/z = 217.13) isis sold sold under the name Oxsoralen [[64]64] and is used to treat severe psoriasis, eczema, and vitiligo. Additionally, Glucose (m/z (m/z = = 181.15) 181.15) waswas identified identified in in the the spectrum, spectrum, corresponding corresponding to peakto peak RT RT= 10.306 = 10.306 min min(relative (relative abundance, abundance, 38.6%), 38.6%), and is anda fermentable is a fermentable sugar raw sugar for raweth- anol.for ethanol. After the the alcoholic alcoholic fermentation fermentation and and distillation, distillation, the thedistillate distillate was wassampled sampled and ana- and lyzed.analyzed. The TheLC-MS LC-MS chromatogram chromatogram of the of thedistillate distillate is presented is presented in Figure in Figure 5. 5.

Figure 5. LC-MS chromatogram of the distilled sample.

Most of the compounds detected in the hydrolyzed sample remained in the fermentation broth, and 16 ions were identified in MS spectra of the distillate with negative ionization, at retention times ranging from 2.525 to 10.922 min, but only two had significant abundance. β-hydroxybutyric acid (m/z = 103.00) was detected at RT: 2.525, 2.822, 6.399, 8.584, and 10.922, with a relative abundance between 50.5% and 61.3%, but its absolute abundance decreased to 1.8 × 104–5.4 × 104 compared with the values in the hydrolysate: 7.8·104–1.6·105. This is an indication that the substance was consumed in alcoholic fermentation. N,N-dimethyl glycine (m/z = 103.04) C4H9NO2, at RT = 3.114 min, with a relative abundance of 47.1% is an α-amino acid [65], a nutritional supplement, and a modulator of ketamine in drugs with antidepressant effects [66]. Additionally, Ethanol (m/z = 45.02) was identified in all the peaks, as a low-mass compound with an absolute abundance up to 1.65 × 105. It was differentiated from the solvent, Formic acid (m/z = 45.00), with an abundance of 5.01–5.48 × 105. In the positive ionization spectra, 17 ions were identified, but only two of them had 4 significant abundance (over 10 ). Leucine (m/z = 217.13),C6H13O2N (RT = 2.789, 3.141, 3.328, 4.511, 10.322, 19.049, 29.949 min), had a relative abundance between 20% and 33.8% and is an essential amino acid important for hemoglobin formation [67]. Curzene (Isogermafurene) (m/z = 217,13),C15H20O, RT = 10.322, with a relative abundance of 33.1% has cytostatic and antitumor activity with limited toxicity and side effects [68]. Unfermented Glucose(m/z = 181.15) was also detected at RT = 10.322 min. It is easy to notice that few volatile compounds were detected in the distillate, among them two amino acids, and the β-hydroxybutyric acid was the most abundant. All these Processes 2021, 9, 741 11 of 14

bioactive compounds have molecular weights very different from that of ethanol, so it is possible for to be separated from it by membrane ﬁltration.

4. Conclusions The ethanol was produced from Cystoseira barbata by the enzymatic hydrolysis of polysaccharides with Aspergillus niger cellulase, followed by alcoholic fermentation with Saccharomyces cerevisiae yeast. An experimental plan according to a 23 factorial was designed. The factors of the process were alcoholic fermentation temperature t, solid to liquid ratio S/L, and cellulase ratio R. Correlations between the three factors and the final process responses, including volatile compounds yield V and ethanol yield E, were established in a statistically sound mathematical model. The optimal working parameters (process temperature of 35 ◦C, solid to liquid ratio of 0.0415, and enzyme ratio of 16 U/g d.m) led to the maximum performance (volatile compounds yield V = 0.2808 g/g d.m. and ethanol yield E = 0.0158 g/g d.m.). The disadvantage of this method in producing ethanol and volatile compounds is the low biomass stock, which makes it impossible to capitalize on this species, but aquaculture under controlled growing conditions can be used. The enzymatic hydrolysate and the distilled fraction from the ethanolic fermentation were analyzed with an ultra-precision liquid chromatograph coupled with a liquid-gas mass spectrometer (LC/MS). A total of 19 active compounds identified in the samples could be used as drugs or as bases for drug synthesis. The challenge is to validate and quantify these compounds, which are presumed to be in significant concentrations, since the yield of volatile compounds is high (between 0.12 and 0.28 g/g d.m.), and to find an efficient separation scheme. The use of an edible alga for bioethanol production and additional by-products seems to be in conflict with the need for food security, but as it represents a dietary supplement and not a base food for animals, the alga could serve for both purposes, provided that it is naturally abundant or cultivated.

Author Contributions: Conceptualization, D.-R.C.T., C.I.K. and T.D.; methodology, D.-R.C.T., A.E.S. and A.G.C.; validation, C.I.K. and T.D.; investigation, D.-R.C.T., A.E.S. and A.G.C.; resources, D.-R.C.T., C.I.K. and T.D.; data curation, D.-R.C.T. and C.I.K.; writing—original draft: D.-R.C.T. and A.G.C.; writing—review and editing: C.I.K., A.E.S. and T.D.; supervision, T.D.; project admin- istration, T.D.; funding acquisition, D.-R.C.T. All authors have read and agreed to the published version of the manuscript. Funding: The work has been funded by the Operational Programme Human Capital of the Ministry of European Funds through the Financial Agreement 51668/09.07.2019, SMIS code 124705. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data used to support the funding of this study are included within the article. Acknowledgments: The chromatograph Agilent Technologies 6540 UHD Accurate-Mass Q-TOF LC/MS has been purchased by University Politehnica of Bucharest, through the Operational Pro- gramme POSSCEA2-O2.2.1.-2013-1 contract No 1970, Code SMIS-CSNR 48652 (2014–2016). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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