**3. Results and Discussion**

### *3.1. Composition of HS*

The average composition of HS, listed in Table 1, was the same reported in a previous work performed with the same HS lot [5]. The major structural component of HS was lignin (40.08 wt% in oven-dry basis). Higher lignin contents, in the range 46–51.3 wt%, were reported for the same substrate [7,40,41]. Hemicelluloses accounted for 32.28% of the dry mass of HS, and were mainly constituted by xylan (22.69%), followed by uronic and acetyl substituents (overall content, 8.93%). Minor amounts of arabinan were also detected. Aydinli and Caglar (2012) [42] reported 28.9% of hemicelluloses, a value markedly higher that the 18.7% reported by Surek and Buyukkileci (2017) [7]. The glucan content of HS accounted 26.49% of the raw material, in the range reported by Aydinli and Caglar (2012) [42], and considerably higher than the 18.7% determined by Surek and Buyukkileci (2017) [7].

**Table 1.** Composition of HS expressed as g of component per 100 g of oven-dried raw material. The values are reported as the average of triplicate measurements ± standard deviation.


### *3.2. Hydrothermal Pretreatment*

The hydrothermal pretreatments were carried out under nonisothermal conditions up to reach temperatures in the range 190–225 ◦C. Once the target temperatures were reached, the reaction media were cooled immediately and filtered. The aqueous phases contained hemicellulose-derived oligomers, monosaccharides, sugar decomposition products, and acetic acid. The generation rate of these compounds depended on the severity of the autohydrolysis conditions [20,43,44], here measured by the maximal temperature.

Table 2 shows data regarding the effects of the hydrothermal pretreatments performed at diverse temperatures on both the solid yield and the composition of liquid phases. The solid yield decreased steadily with temperature, a trend that was more marked up to 210 ◦C. This behavior can be explained because increasing temperatures promote the progressive solubilization of xylan and acetyl and uronic groups in hemicelluloses. Although arabinan also makes part of hemicelluloses, its practical importance is limited owing to the low contents. The progressive removal of hemicelluloses from the raw material led to the production of AS with increased glucan and lignin content. At the highest temperature assayed (225 ◦C), the content of both lignin and glucan accounted for 93.13% of AS. The glucan and lignin contents of AS varied in the ranges of 25.47%–38.70% and 41.08%–54.43%, respectively, corresponding to recovery yields of glucan and Klason lignin in solid phase higher than 90% and 85%, respectively. These results are favorable for the subsequent processing of AS by other methods, thus enabling the integral valorization of the raw material [2,13,20].

**Table 2.** Solid yields (measured as g of oven-dry AS/100 g of oven-dry HS) and composition of AS (measured as g component/100 g of oven-dry AS) obtained in experiments performed up to the desired temperatures. The results are reported as the average of triplicate measurements ± standard deviation.


Table 3 lists the results determined for the composition of the reaction media in the same set of experiments. When the hydrothermal pretreatments were carried out at temperatures below 210 ◦C, the concentration of nonvolatile compounds (NVC) increased up to 26.46 g/L. This concentration remained fairly constant in the range 210–220 ◦C, and dropped at the highest temperature assayed. The data are expressed in terms of the identified NVC (INVC), calculated as the joint contributions of OS and monosaccharides. XOS, the most abundant components in AL, reached their highest concentration (16.24 g/L, accounting for 73.67% of the xylan present in the feedstock) at 210 ◦C. Harsher conditions resulted in decreased XOS concentrations, owing to the generation of xylose (and furfural under the most severe conditions assayed). Arabinooligosaccharides (ArOS) were only found (in little concentrations) in assays performed under mild conditions. The corresponding monomer (arabinose) reached its highest concentration (0.45 g/L) at 210 ◦C. Glucooligosaccharides (GOS) and glucose also reached limited concentrations, revealing the solubilization of a small fraction of glucan under the most severe conditions. Concerning the substituents, the concentration of acetyl groups (AG) bound to OS reached concentrations up to 3.55 g/L at 220 ◦C (corresponding to 77.05% of the amount present in the feedstock), whereas the maximal concentrations of uronic groups linked to OS (U) were found at a milder temperature (200 ◦C). From the results shown in Table 3, it can be calculated that the maximum concentration of substituted OS (including GOS, XOS, ArOS, AG, and U) was achieved at 210 ◦C, and reached 20.49 g/L. The concentrations of monosaccharides (maximum value, 2.68 g/L achieved at 220 ◦C) were comparatively low.


**Table 3.** Concentrations of products present in the liquid phase of hydrothermal treatments performed at temperatures ranging from 190 to 225 ◦C. Data are expressed in g/L.

GOS: glucooligosaccharides; XOS: xylooligosaccharides; ArOS: arabinooligosaccharides; AG, acetyl groups linked to oligosaccharides; U: uronic acid linked to oligosaccharides; HMF: hydroxymethylfurfural. NVC: total nonvolatile compounds; INVC: identified nonvolatile components; VC: volatile compounds. \* Others: measured as the difference of the total NVC and the INVC.

### *Agronomy* **2020**, *10*, 760

The concentrations of volatile compounds (VC) increased smoothly up to 210 ◦C, and then increased markedly as a result of reactions taking place under severe conditions (for example, cleavage of AG into acetic acid and furfural generation from pentoses).

### *3.3. Refining of Oligosaccharides by Membrane Treatment*

Based on the results discussed above, 210 ◦C was selected as the optimal temperature because this experiment (denoted AL210) led to the highest concentration of substituted OS. However, unwanted compounds (including monosaccharides or nonsaccharide compounds) were also present in the liquid phase, and could limit the application of the NVC fraction in the food, cosmetic, or pharmaceutical industries. OS refining of can be achieved using a number of separation techniques, including solvent extraction [21,45], adsorption [23,33], chromatography [45], and membrane filtration [21,23,25–27]. Membrane processing is currently seen as the most promising technology for the industrial manufacture of high purity, owing to the low energy requirements, easy manipulation of operational variables, and relatively easy scale up [25,45].

Figure 2 shows the scheme of the purification method used in this work, including data regarding the chemical characterization of streams and material balances. The solution AL210 (content of total OS, 20.49 g/L, accounting for 77.41% of the NVC) was employed as a feed. This stream also contained some unwanted components that should be removed, including monosaccharides (2.01 g/L), ONVC (3.97 g/L), and VC (1.11 g/L). The feed solution was refined by DD, which led to a retentate (containing 17.44 g NVC/L) and permeate (NVC concentration, 4.02 g/L). As expected, the retentate showed an increased proportion of nonvolatile solutes corresponding to OS (90.87 g/100 g of NVC, in comparison to 77.41 g OS/100 g of NVC determined for the feed solution AL210). This finding confirmed the suitability of membrane processing for OS purification, keeping a good balance between the concentrations of the target products in retentate (15.85 g/L) and in permeate (1.76 g/L). It can be noted that most unwanted compounds were present in permeate (i.e., 1.63 g ONVC/L and 0.63 g monosaccharides/L).

The available data allow the comparison of the molar ratios of oligomer components (XOS:AG:U) between the feed and the retentate (1:0.47:0.05 and 1:0.43:0.04, respectively). The molar ratio XOS:AG was slightly higher than the one reported in literature for autohydrolysis liquors from hazelnut shells [7]. A comparative molar ratio XOS:AG of 1:0.56 was also reported for membrane processing of autohydrolysis liquors from peanut shells [24].

AL210 also contained VC (1.11 g/L), a fraction mainly made up of acetic acid (0.90 g/L) and minor amounts of HMF and furfural. The VC concentration decreased considerably in the retentate (0.29 g/L) relative to the feed content.

From the above data, it can be concluded that DD of AL210 allowed a selective recovery of substituted OS in retentate, in which the target products accounted for 90.87% of the NVC fraction, while most ONVC, monosaccharides, and VC were removed in the permeate. This finding is in agreement with the results reported in literature [24], with an increasing purity of oligomers from autohydrolysis liquors of peanut shells from 55.70% up to 72.4% using DD. In a related study, Singh et al. (2019) [26] obtained XOS of low degree of polymerization from almond shells using enzymatic treatments and membrane assisted refining. To our knowledge, no previous studies reported on the membrane refining of HS-derived OS.

**Figure 2.** Diagram of membrane processing and composition of the involved streams: AL210, retentate, and permeate. *Agronomy* **2020**, *10*, 760
