Half-Preparative Scale Synthesis of ( S )-1-Phenylethane-1,2-Diol as a Result of 2-Phenylethanol Hydroxylation with Aspergillus niger (IAFB 2301) Assistance

: Aspergillus niger (IAFB 2301) was employed for bioconversions of 2-phenylethanol as an immobilized or free mycelium and also as a spore suspension. Experiments were conducted on laboratory and half-preparative scale (bioreactor New Brunswick Scientiﬁc, BioFlo Model C32). Thus, A. niger applied as free mycelium, depending on the outcome, supported formation of the mixture of 4-hydroxyphenylacetic acid and hydroxytyrosol (ﬁnal concentration of 13.8 mg / L and 3.7% e ﬃ ciency) or 4-hydroxyphenylacetic acid, as single product (ﬁnal concentration of 140 mg / L and 18% e ﬃ ciency). In case of scaling experiments conducted with ﬂow and batch reactors, accordingly, the following results were achieved: 1. mixture of antioxidants 4-hydroxyphenylacetic acid and hydroxytyrosol formed with ﬁnal concentration of 76 mg / L and 10% e ﬃ ciency (simpliﬁed ﬂow system and immobilized mycelium); 2. ( S )-1-phenylethane-1,2-diol synthesized with a ﬁnal concentration of 447 mg / L and 65% (1.3 L batch reactor). Deutero NMR Spectroscopy; acetonitrile, formic acid, ethanol, ethyl acetate, acetone, methanol, isopropanol, chloroform, hexane, anhydrous magnesium sulfate, glucose from Avantor Performance Materials; potato dextrose broth and potato dextrose agar from Biocorp; polyurethane foams TM25133 (1060–1600 µ m) from Bogmar; FACSFlow, FACSClean, FACSuite CS&T Research Beads (to ﬂow cytometer) from BD Biosciences.


Introduction
Phenolic compounds are of high commercial value, due to their wide application in, e.g., the food industry, medicine and cosmetology. Such polyphenols are plant secondary metabolites and usually occur in quite low concentrations in conjugated form with organic acids or carbohydrates, which makes their isolation and purification difficult and in many cases not effective [1][2][3][4].
Food supplements consist of, among others, antioxidants-free radical neutralizers-which provide a positive effect in the treatment and prevention of many diseases such as tumors, cardiovascular diseases, diabetes, brain strokes, skin diseases and they delay the process of aging. So, the pro-health impact of antioxidants and free radical scavengers constantly focus attention on searching for new compounds and more effective methods of acquiring them [5][6][7].
solid olive residues after the oil production via methods that burden the environment. 4-Hydroxyphenylacetic acid is also a natural ingredient of olive oil, which manifests antioxidant [10,11], anti-inflammatory [12] and skin-whitening activity [13]. Its derivatives are reported as neuroprotectors [14]. Furthermore, 4-hydroxyphenylacetic acid is an important building block for the formation of various drugs, e.g., atenolol, applied in cardiovascular diseases [15]. Another derivative of 2-phenylethanol, chiral-1-phenylethane-1,2-diol, is used for the synthesis of numerous pharmaceuticals of high value. Especially, (S)-1-phenylethane-1,2-diol is an intermediate in the preparation of norfluoxetine and fluoxetine pharmaceuticals, which are applied in the treatment of psychiatric disorders and metabolic problems, and the available methods of synthesis of this compound are limited [16].
Biocatalysis is an alternative way of synthesis of phenylethyl alcohol derivatives. Reported protocols employing fungal mycelia have proved their utility and also are simple, cost-saving and environmentally friendly [17][18][19][20]. Employing free or immobilized forms of biocatalyst or resting spores have allowed manifesting of different enzymatic activities by triggering different metabolic pathways, which have resulted in the synthesis of different phenolic derivatives [17,21,22].
Molds are biocatalysts applied as free or immobilized mycelium for the synthesis of compounds of practical significance [23]. Fungi of the genus Aspergillus are widely applied in the industry because of their various enzymatic activities, including hydroxylation [23][24][25]. Aspergillus niger introduces hydroxyl functionality due to the activity of the Cytochrome P450 (CYP) systems [26]. This complex is responsible for modification of terpenes or aromatic compounds, which leads to fragrances, flavors and pharmaceutical substances [27][28][29][30][31].
The aim of the study was to establish an effective and as simple as possible procedures of biological synthesis of compounds of high value, starting from cheap substrate 2-phenylethyl alcohol and employing fungal biocatalyst Aspergillus niger. As a result, the following structures were obtained: 4-hydroxyphenylacetic acid (1), (S)-1-phenylethane-1,2-diol (2), hydroxytyrosol (2-(3,4-

Microorganism
The fungal biocatalyst used in the present study was Aspergillus niger (IAFB2301), purchased from the Institute of Agricultural and Food Biotechnology, Warsaw, Poland.

Microorganism Cultivation Conditions
Aspergillus niger strain was routinely maintained on potato dextrose agar (PDA). Inoculum was prepared as a spore suspension in Triton X-100 water solution (0.05%). Spores were washed off from 5-day sporulating fungal culture (petri dish containing solid PDA medium). The suspension was transferred to a conical flask and diluted up to 20,000 spores/µL (counted by flow cytometry; see Analytical Methods below); 50 µL of such suspension was used to inoculate 100 mL of potato dextrose broth (PDB) in a 250 mL conical flask. Depending on the outcome, the medium was supplemented with an inducer (30 mg of 2-phenylethanol) or not. Cultures were grown on a rotary shaker (135 rpm) at ambient temperature (about 23 • C) for 3 days until the mid-log growth phase was reached.

Immobilization Procedure
The polyurethane foams (1060-1600 µm porosity) were cut into 1 cm cubes, washed with sterile water and autoclaved in 250 mL Erlenmeyer flasks containing 100 mL of potato dextrose broth (20 cubes in each flask); 50 µL of spore suspension was transferred aseptically to sterile medium with polyurethane foams, and cultures of A. niger were incubated under conditions described above.

Procedures of Biotransformation with Free or Immobilized Fungal Mycelium
After cultivation, the biocatalyst biomass (16 g of fresh mycelium or immobilized one) was separated by vacuum filtration (filtration paper 80 g/m 2 ), washed twice with distilled water and transferred to 50 mL of distilled water (conical flasks, 250 mL) and the different amount (depending on the experiment) of the substrate 2-phenylethanol was added: 15 mg (2.5 mM) or 30 mg (5 mM) ( Table 1). Biotransformations were carried out with shaking (135 rpm) for a period of 1 to 9 days at ambient temperature (about 23 • C) in the according number of flasks; for every time interval, three experimental flasks were prepared, because experiments were performed in triplicate. Every 24 h, biotransformations were completed and the contents of particular flasks were taken for analysis and processed separately according to following steps: biocatalyst was separated by vacuum filtration (filtration paper 80 g/m 2 ); supernatant was extracted twice with ethyl acetate. Organic layers were dried over anhydrous MgSO 4 and evaporated under reducing pressure. The final product mixture was dissolved in 0.5 mL of ethanol and analyzed by HPLC.

Biotransformation with Spores of A. niger
Spore suspension 10 mL was used as catalyst directly or was pretreated by temperature impact (stored at the low temperature of 6 or −18 • C for 2.5 weeks or heated at 60 • C for 1 or 3 h). Bioconversion was carried out in conical flasks (250 mL) containing 40 mL of biotransformation medium: glucose solution (4 or 13 mM) and 15 mg (2.5 mM) or 30 mg (5 mM) of 2-phenylethanol solution and spores. The reaction mixtures were incubated on a rotary shaker (135 rpm) at ambient temperature (about 23 • C) from 1 to 9 days. Experiments were performed in triplicate. After an appropriate incubation period, the biocatalyst was removed by vacuum filtration (filtration paper 80 g/m 2 ) and the supernatant was extracted twice   Figure 2) (packed-bed bioreactor, 2 cm:15 cm) was packed with immobilized biocatalyst (1060-1600 µm polyurethane foams, 20 cubes) (16 g of immobilized mycelium), with a continuous flow of biotransformation medium containing 90 mg of the substrate in 150 mL of distilled water (5 mM). The circulation of the medium through the column was forced through application of a peristaltic pump (flow 3 mL/min). Bioconversion was chromatographically monitored after 3 and 6 h and after 1 to 7 days in the same time intervals (24 h). through application of a peristaltic pump (flow 3 mL/min). Bioconversion was chromatographically monitored after 3 and 6 h and after 1 to 7 days in the same time intervals (24 h).

Analytical Methods
Products and the substrate were detected by using high-performance liquid chromatography (Beckman System Gold 126 Solvent Module, using Supelcosil LC-18 column, 25 cm × 4.6 mm, 5 µm). The system was operated at a flow rate of 1 mL/min with eluent: 0.1% formic acid/acetonitrile (91:9 v/v). Injections were done in full loop (20 µL). Detection was performed at λ = 276 nm. The products were confirmed by comparison of retention times with commercially available standards (from Sigma Aldrich and Tokyo Chemical Industry) and the amount was determined using calibration curves, which were prepared using the following series of dilutions of standards: for hydroxytyrosol and 4-hydroxyphenylacetic acid, 0.05, 0.07, 0.1, 0.12 and 0.15 mg/mL; for 1-phenylethane-1,2-diol, 6, 7, 8, 9 and 10 mg/mL. The validation parameters are reported in Tables 2-4.  Spore suspensions were prepared with sterile water containing 0.05% Triton X-100, quantified by the mean of flow cytometer (BD FACSVerse) equipped with three lasers (wavelengths 405, 488 and 640 nm) and the system was operated at a low (12 µL/min) flow rate.

Results and Discussion
Antioxidant synthesis via biocatalytic hydroxylation of cheap and commercially available substrates such as 2-phenylethanol [47,48] is an alternative to chemical methods. Fungi of Aspergillus genus are known as appropriate biocatalysts for hydroxylation of aromatic substrates [24][25][26][27][28][29][30][31]. An unquestionable advantage of biocatalysis is flexibility in the meaning of possibility of affecting the reaction effectiveness, course and stereoselectivity by simple medium engineering or modification of form of biocatalyst (immobilization, spores employing instead of mycelium, etc.) [49][50][51].
To establish the most effective protocols of bioconversion of 2-phenylethanol, several efforts were undertaken. Experiments differed in the form of biocatalyst, substrate concentration, time of the biotransformation and biotransformation media and mode of the processes (batch or flow). Products were obtained as a mixture (of different concentrations) or in some cases a single compound was formed, depending on the outcome. Obtained products were defined as 4-hydroxyphenylacetic acid (1), (S)-1-phenylethane-1,2-diol (2) and hydroxytyrosol (3) (Figure 1).
Starting experiments were conducted with the fungal spores, mostly considering the simplicity of the method and the high level of activity of enzymatic systems during germination. Microbial resting forms are much more insusceptible to external factors, e.g., toxicity of chemical compounds, temperature or desiccation [22,52,53]. Therefore, in some cases they can successfully replace other, more sensitive forms of the biocatalyst (vegetative ones) [52,54,55]. Fungal spores are a source of specific, for this form, enzymatic activities [56]. Taking these into account, A. niger spores were applied as a biocatalyst in two ways: with previous temperature activation and directly after culturing [56,57]. Heat shock was meant to stimulate germination and to trigger enzymatic activity to start the bioconversions [53,56,57]. Tested spores were kept at low or high temperatures (6 or −18 • C for 2.5 weeks and 60 • C for one or three hours), and then employed as biocatalysts. However, these approaches resulted in comparably poor results no matter the procedures (data not shown).
Next, experiments with spore suspensions were conducted in biotransformation medium supplemented with different glucose solutions (4 or 13 mM) in combination with various substrate concentrations (Table 1). This approach led to the single product hydroxytyrosol, and the productivity of the conversion increased from 5.4 mg/L (0.7% efficiency) (water media) [58] to 28.4 mg/L (five days, 13 mM of glucose solution and 15 mg/50 mL of substrate solution, 2.5 mM, 7.4% efficiency) ( Table 5). The addition of glucose accelerates the germination of spores and, as a consequence, initiates the bioconversion and improves its effectiveness [56,59,60] (Table 5). As is shown in Table 5, the maximum concentration of product was about 28.4 mg/L (in the presence of 13 mM glucose solution), regardless the starting substrate concentration, which suggests that hydroxylated moieties are finally degraded and utilized by the germinating spores. This can be confirmed by the decrease of the product concentration with extending the process duration and also Symmetry 2020, 12, 989 8 of 14 by the described path of aromatic compound degradation, which is usually initiated by hydroxylation (this will be discussed later in the text body) [61][62][63][64][65].
Fungal spore catalysis was not efficient, only showing that there is a potential for hydroxylation but at very low level. It confirms that during germination hydroxylases are not active (mostly enzymes of secondary pathways) or are not produced at a sufficient level. Next, experiments were focused on the employing of fresh fungal mycelium. Initially, different concentrations of the substrate were applied to estimate the possible toxic impact of phenol derivative towards the viable cells [66,67]. Thus, with increasing of the substrate concentration from 15 mg/50 mL (2.5 mM) to 30 mg/50 mL (5 mM), a significant decrease in the efficiency of the hydroxylation was observed for the mixture of non-isolated products (Figure 1: 1,3), respectively, from 9.5 mg/L (2.6% efficiency) (effectiveness for lower substrate concentration) to 1.8 mg/L (0.3% efficiency) (for higher substrate concentration) on the first day of bioprocess, and from 13.8 mg/L (3.7% efficiency) (effectiveness for lower substrate concentration) to 6.8 mg/L (0.9% efficiency) (for higher substrate concentration), on the second day of bioconversion ( Table 6). For the process with lower concentration of substrate (15 mg/50 mL, 2.5 mM), the level of produced antioxidants, 4-hydroxyphenylacetic acid and hydroxytyrosol, increased until the second day of biotransformation, reaching 13.8 mg/L of the product mixture (3.7% efficiency) and then (after the next three days) the products disappeared (Table 6). These suggest that formed hydroxyl-phenolic compounds were utilized for cell purposes by microbial degradation systems active towards aromatic moieties with one or two hydroxyl functionalities, leading to partial or full mineralization of these molecules [61][62][63][64][65]. Aliphatic side chain modifications are catalyzed by dehydrogenases, namely monooxygenases (cytochrome P450 systems) and/or dioxygenases leading to modifications of the phenyl ring, which is part of the multistage degradation process. These enzymes are also involved in the utilization of the external sources of carbon and energy in microbial cells [61][62][63][64][65]. Table 6. Effect of substrate concentration (2.5 or 5 mM) on biosynthesis effectiveness (a mixture of 4-hydroxyphenylacetic acid and hydroxytyrosol) (fresh mycelium of A. niger). Results are means ± SD over three independent replications. To increase the efficacy of product formation, in the next tests, cultivation medium was supplemented with the substrate (2-phenylethanol) (30 mg/100 mL) to induce particular enzyme activity or to initiate the synthesis of enzymes of desired activity [68]. This approach led to a breakthrough: single product 4-hydroxyphenylacetic acid was formed and the level of hydroxylation increased up to 140 mg/L of the product, starting from 30 mg/50 mL (5 mM) of substrate concentration (18% efficiency), after four days of biotransformation. These proved that such induction not only stimulates the particular enzyme activity but also decreases the sensitivity of biocatalyst towards phenolic molecules, keeping the mycelium active during bioconversion in the presence of 5 mM substrate.

Amount of Products (mg/L)
However, the main goal of this research was to scale experiments to reasonable, at least half-preparative level. Two approaches of scaling were conducted with simplified models of flow and batch reactors. Some of these procedures required the immobilized form of biocatalyst mycelium to reduce the negative impact of an increased (in reactors) level of substrate towards the fungal Symmetry 2020, 12, 989 9 of 14 cells [17,21,69,70]. Polyurethane foams (1060-1600 µm porosity) were applied as supports for mycelium. To check the activity of the immobilized mycelium, such form of A. niger was applied in a flask system (Table 7) and after three days led to synthesis of 4-hydroxyphenylacetic acid at the level of 25.7 mg/L (3.4% efficiency), and a trace amount of hydroxytyrosol (0.9 mg/L) (0.12% efficiency) was formed (starting from 30 mg/50 mL, 5 mM substrate). In conclusion, this experiment proved the catalytic ability of attached to foams fungal catalysts. This time, with prolongation of the reaction, degradation processes were observed. Table 7. Products (mg/L) (4-hydroxyphenylacetic acid and hydroxytyrosol) synthesized by immobilized cells of A. niger in flask system, starting from 5 mM of substrate concentration. Results are means ± SD over three independent replications.  The above studies allowed us to start experiments with the simplified model of flow bioreactor packed with immobilized fungal mycelium and with 5 mM of water substrate solution (90 mg/150 mL). This time, fungal hydroxylation resulted in the following effectiveness of antioxidant synthesis (Table 8): after three days, 4-hydroxyphenylacetic acid was formed at the level of 75 mg/L (9.8% efficiency) and hydroxytyrosol at 1.1 mg/L (0.14% efficiency). Forced flow of the substrate solution improved the contact between the phenylethanol and immobilized mycelium, while protecting the viable fungal cells against harmful effects of the substrate or product mixture. The concentration of the particular products evolved with time. The hydroxytyrosol level increased with the prolongation of the reaction, whereas the concentration of 4-hydroxyphenylacetic acid decreased, respectively: 4 mg/L of hydroxytyrosol and only 1.7 mg/L of 4-hydroxyphenylacetic acid were obtained on the seventh day of process (Table 8). Such a slow increase of the hydroxytyrosol formation may be the result of the activity of dioxygenases, or alternatively, two stage hydroxylation of 2-phenylethyl alcohol to tyrosol and then to hydroxytyrosol by monooxygenases. Usually dioxygenases catalyze the simultaneous transfer of two oxygen atoms into the substrate [17,26], in contradiction to monooxygenases, which incorporate one oxygen atom from dioxygen into the substrate and the other one is reduced to water moiety [17]. The second way may be confirmed by the presence of a trace amount of tyrosol in the product mixture. Tyrosol is then quickly oxidized to carboxylic acid, which may be the reason for formation of significant quantities of 4-hydroxyphenylacetic acid (4-HPA) after three days of bioprocessing. Liebgott et al. hypothesized that tyrosol oxidation to 4-hydroxyphenylacetic acid is commonly carried out by aryl dehydrogenases, belonging to the superfamily of dehydrogenases, which are found in various viable cells. They demonstrated that the first step is the oxidation of tyrosol to 4-HPA, which then induced 4-hydroxyphenylacetic acid 3-hydroxylase synthesis, which in turn is involved in hydroxytyrosol formation [36]. The induction of 3-hydroxylase activity requires time; therefore the amount of hydroxytyrosol increases slowly. Despite that, a drop in the concentration of products was observed as a progressing degradation [61][62][63][64][65].
In parallel to studies with a simplified flow reactor, hydroxylation of 2-phenylethanol with free A. niger mycelium in the batch bioreactor (1.3 L) was conducted. Thus, 20 times more biomass (320 g) was used for the conversion of 5 mM substrate (458 mg/750 mL). The aeration was maintained at the level of 0.5 L/min and a temperature of 23 • C throughout the process. Under such conditions, the substrate bioconversion was directed to the formation of (S)-1-phenylethane-1,2-diol (2) (447 mg/L, 65% efficiency, after four days) ( Table 9). Better aeration (0.5 L/min) compared to the previous attempts (spores of A. niger, free and immobilized cells of A. niger in flask system or simplified flow system) allowed an increase of the activity of enzymes involved in the process. According to literature data, enzymatic dehydrogenation with the formation of a double bond between carbon atoms in the aliphatic chain is possible. The next step, epoxidation catalyzed by monooxygenase, is followed by the hydrogenation of epoxide [71]. 1-Phenylethane-1,2-diol is formed in two possible ways: via oxidation of 2-phenylethanol and also as an intermediate during the conversion of acetophenone to 2-phenylethanol in fungal cells [72]. Optically pure S-diol is a product of a fungal, highly stereoselective, epoxide ring opening or in the case of non-selective diol formation, it is possible that the one from the enantiomers pair is rapidly metabolized for cell purposes [73][74][75]. The yield of the S-diol formation increases with time until the critical point, which is achieved after the fourth day of biotransformation. As can be seen from Table 9, with time extension, the level of S-diol in the bioconversion media systematically decreased and then was completely mineralized by viable cells; so for this process, the time setting is crucial. Table 9. (S)-1-Phenylethane-1,2-diol synthesis using free cells of A. niger in batch reactor and 5 mM substrate. Results are means ± SD over three independent replications.

Time of Biotransformation (day)
Amount of (S)-1-phenylethane-1,2-diol (mg/L) The discussed studies clearly prove that A. niger (KKP 2301) strain can be successfully used for the preparation of various 2-phenylethanol derivatives. The quantity and type of the obtained products are strictly dependent on the form of biocatalyst used as well as the reaction conditions, and these can be set depending on the purpose of the bioconversion.

Conclusions
Aspergillus niger is able to convert 2-phenylethanol to polyphenols of high value: 4-hydroxyphenylacetic acid, hydroxytyrosol and (S)-1-phenylethane-1,2-diol. Formation of the products strongly depend on the conditions of the process, including the mode of the biocatalyst. Considering the Symmetry 2020, 12, 989 11 of 14 results achieved with the spore suspension and immobilized form (flask experiments) of fungal biocatalyst (accordingly, hydroxytyrosol formation with 7.4% efficiency and the mixture of 4-hydroxyphenylacetic acid and hydroxytyrosol with 3.5% efficiency), the conclusion is that, with the exception of saturation (which is obvious in the case of oxidation), the crucial limitation parameter is the possibility of contact between the substrate and the viable cells of the biocatalyst. The better this factor, the better results are achieved as in the case of employing the free mycelium (4-hydroxyphenylacetic acid formation with 18% efficiency) and applying of the flow and batch reactors (accordingly, mixture of antioxidants: 4-hydroxyphenylacetic acid and hydroxytyrosol formed with 10% efficiency and (S)-1-phenylethane-1,2-diol synthesized with 65% efficiency).