To further investigate the interactions between Ni and Fe within the Ni-Fe/SBA-15 catalyst, H2-TPR analysis of both monometallic Ni/SBA-15, Fe/SBA-15, and bimetallic Ni-Fe/SBA-15 catalysts, was carried out (Figure 3). For the Ni/SBA-15 catalyst, only one peak was found at 430 ◦C, due to the reduction of NiO to metallic Ni. For the monometallic Fe/SBA-15 catalyst, three different reduction peaks were detected, at 370, 550, and 630 ◦C. In this regard, the reduction of Fe2O3 to α-Fe occurs by three consecutive steps: Fe2O3-Fe3O4-FeO-Fe [32]. Therefore, the peak at about 370 ◦C is due to the reduction from Fe2O3 to Fe3O4, whilst that from Fe3O4 to FeO occurs at about 550 ◦C, and that at 630 ◦C corresponds to the reduction from FeO to α-Fe [32]. Regarding the bimetallic Ni-Fe/SBA-15 catalyst, there are two different peaks between 300 and 550 ◦C, whilst no peak was found at 600 ◦C, thus confirming the absence of unalloyed Fe in the synthesized bimetallic catalyst. For 5Ni-1Fe/SBA-15, the reduction peak at 320 ◦C is assigned to the reduction of Fe2O3 to Fe3O4, because Fe2O3 is more easily reducible than NiO [33], whilst that at 440 ◦C is attributed to the reduction of NiO. Compared with the monometallic Ni/SBA-15 catalyst, the reduction peak of the Ni-Fe/SBA-15 catalysts is shifted to higher temperatures, corresponding to the increase of the Fe content, and the H2 consumption

gradually increases. H2 may be more easily dissociated and activated on Ni, and then overflowed to the adjacent iron oxide to reduce it, thus leading to the formation of the Ni-Fe alloy [34].

**Figure 3.** H2-TPR (hydrogen temperature-programmed reduction) profiles of Ni/SBA-15, 5Ni-1Fe/SBA-15, 5Ni-3Fe/SBA-15, and 5Ni-5Fe/SBA-15 catalysts.

H2-TPD profiles of the synthesized catalysts are reported in Figure S2. For Ni/SBA-15, a desorption peak was detected at 100 ◦C, in agreement with the adsorption of hydrogen on the surface of dispersed metal particles Ni in Figure S2. It is remarkable that the hydrogen desorption peak gradually moves to a higher temperature with the corresponding increase of Fe content. Moreover, the corresponding hydrogen desorption peaks for 5Ni-1Fe/SBA-15 and 5Ni-3Fe/SBA-15 are located at 105 and 125 ◦C, respectively, which is higher than that of Ni/SBA-15. However, 5Ni-5Fe/SBA-15 has two hydrogen adsorption sites, a low- (150 ◦C) and high-temperature desorption peak (300 ◦C), the latter deriving from the hydrogen desorption on the surface of Fe or from the poor dispersion of Ni. In addition, the hydrogen desorption peak from Ni/SBA-15 to 5Ni-5Fe/SBA-15 is shifted to a higher temperature, indicating that the adsorption of hydrogen on the active metal surface is enhanced. The desorption peak of Fe/SBA-15 appears at a higher temperature (250 ◦C), which is attributed to the hydrogen desorption from the Fe surface. The adsorption center corresponds to the desorption peak at low temperature, and desorption temperature of Fe/SBA-15 is the highest, meaning that the hydrogen desorption from the Fe species requires more energy than that from the Ni ones. To evaluate the dispersion of the particles, the amount of the hydrogen desorbed from the catalyst surface was calculated (Table 1), and it corresponds to the area of the desorption peak reported in Figure S2. The amount of desorbed hydrogen gradually increases with the area of the desorption peak, indicating the increase of the number of active hydrogen on the catalyst surface. With the increase in Fe content, that is moving from 5Ni-1Fe/SBA-15 to 5Ni-3Fe/SBA-15, the particle dispersion increases. However, for the 5Ni-5Fe/SBA-15 catalyst, the total amount of desorbed H2 does not significantly increase, at the same time showing a worsening of the catalyst dispersion. Moreover, the total acidity of catalysts increases with the amount of Fe. It has been reported that, when Ni is doped with Fe, the unreduced Fe can act as a Lewis acid, thus increasing the acid content of the catalyst [35].

**Table 1.** Chemical properties of the synthesized monometallic Ni/SBA-15, Fe/SBA-15, and bimetallic Ni-Fe/SBA-15 catalysts.

a: Amount of H2 desorption calculated by TPD (Figure S2). b: H/(Ni + Fe) ratio. c: calculated by NH3-TPD.

XPS analysis was performed to monitor the change of the oxidation state of the bimetallic catalysts. The XPS patterns of Ni 2p and Fe 2p of 5Ni/SBA-15, 5Ni-1Fe/SBA-15, 5Ni-3Fe/SBA-15, 5Ni-5Fe/SBA-15, and 5Fe/SBA-15 are shown in Figure S1, respectively. For the Ni 2p, Ni 2p 3/2 and 2p1/2 spin orbit doublets were deconvoluted into six peaks within the range 845–885 eV (Figure S1a). For the 5Ni/SBA-15 catalyst, the peaks at 853.2, 856.7, and 861.7eV are assigned to the characteristic peaks of Ni0, Ni2+, and satellite peaks of Ni2+ in Ni 2P3/2 orbit, respectively [8]. For 5Ni-1Fe/SBA-15, 5Ni-3Fe/SBA-15, and 5Ni-5Fe/SBA-15 catalysts, the characteristic peaks of Ni0, Ni2+, and satellite peaks of Ni2+ in Ni2P3/2 orbit are progressively shifted to the higher binding energies, respectively, indicating a deeper interaction between Ni and Fe. In detail, the electrons are transferred from the oxophilic site of Fe to Ni, increasing the electron density of the outer layer of Ni, in agreement with the literature [36]. Moreover, the characteristic peaks of 5Ni-3Fe/SBA-15 are shifted greatly (0.6 eV), meaning that the reduction of 5Ni-3Fe/SBA-15 is certainly favored. For the Fe 2p in Figure S1b, two spin orbits are also present as Fe 2p3/2 and Fe 2p1/2. For the Fe/SBA-15 catalyst, the peaks at 707.7, 712.2, and 716.4eV are assigned to Fe0, Fe2+, and Fe3+ on Fe 2p3/2, respectively [37]. For 5Ni-1Fe/SBA-15, 5Ni-3Fe/SBA-15, and 5Ni-5Fe/SBA-15 catalysts, the characteristic peaks of Fe0 and Fe3+ are shifted towards the low binding energies, respectively, indicating that the electrons are indeed obtained by Fe, decreasing the electron density of the outer layer of Fe, and activating the C=O bond. From the XPS results, it can be concluded that the Lewis acid sites of the catalysts derive from the unsaturated coordination of Fe2+ and Fe3+ ions. Moreover, the characteristic peak area of Fe0 in bimetallic Ni-Fe catalyst increases, indicating that the interaction between Ni and Fe promotes the reduction of iron oxide. The above results confirm that 5Ni-3Fe/SBA-15 is the most promising catalyst to test for the activation of the C=O bond of LA.

#### *2.2. LA to GVL Catalytic Tests*

Preliminary catalytic tests were carried out to test the effectiveness of the synthesized catalysts towards GVL production. First, GC-MS analysis was carried out to monitor the progress of the reaction (Figure S4). This analysis confirms that methyl levulinate and GVL represent the main intermediate and target product, respectively, indicating that LA could be completely hydrogenated to GVL, by properly tuning the reaction conditions. In this regard, 4-hydroxyvaleric acid, which is the intermediate of interest for the MPV intermolecular hydrogenation mechanism (path B, Scheme 2), was not detected in the reaction mixture. In principle, LA is firstly adsorbed on the surface of the catalyst and subsequently esterified with methanol (MeOH) to methyl levulinate, benefiting from the Lewis acidity of the catalyst [22]. To investigate the mechanism of LA adsorption on the catalysts, in situ FT-IR analysis was performed. As shown in Figure 4, the LA absorption band in the range 1750–1765 cm−1 is instead absent in Ni/SBA-15 and Ni-Fe/SBA-15, meaning that the physical adsorption of LA on these catalysts is weak. Moreover, in the case of the Ni/SBA-15 catalyst, an absorption band at about 1720 cm−1 was detected, due to the weak chemical adsorption of C=O stretching vibration of LA, indicating an interaction between the cationic or Lewis acid sites on the surface of Ni/SBA-15 catalyst and the lone pair electron of the carbonyl oxygen of LA [38]. In contrast, no absorption

band was found at 1720 cm−1 for 5Ni-3Fe/SBA-15, whilst a blue shift occurs in the range 1650–1680 cm−1, which is due to the strong LA chemisorption. The oxophilic promoter of Fe rendered more Lewis active sites for acid adsorption the C=O bond, in agreement with the results of XPS and NH3-TPD characterization. Moreover, the composition of the gas phase was investigated to highlight possible differences between the catalysts towards the potential production of hydrogen (Figure S3). These analyses confirm that H2 and CO2 are the main reaction products of the gas phase. However, for the Ni/SBA-15 catalyst, the H2 selectivity is only 40 mol %, whilst, in the case of 5Ni-3Fe/SBA-15, the addition of Fe significantly improves the H2 selectivity up to 90 mol %. Instead, regarding the 5Ni-5Fe/SBA-15 catalyst, H2 selectivity decreases, probably as a consequence of the larger particle size of Ni, according to the results of the above-discussed characterization.

**Figure 4.** In situ FT-IR (Fourier transform–infrared spectroscopy) spectrum of LA and LA absorption on Ni/SBA-15 and 5Ni-3Fe/SBA-15 (50 mL·min−1 He, at 200 ◦C, 2 h).

The effect of temperature, reaction time, and hydrogen source on the LA hydrogenation to GVL was investigated. Figure 5a shows that the temperature has an important role on the LA conversion and GVL selectivity. Going from 50 to 100 ◦C, both LA conversion and GVL selectivity are low, whilst the catalytic performances significantly improve at 150 ◦C and, even better, at 200 ◦C. LA is not immediately hydrogenated into the corresponding alcohol, and methyl levulinate is the only detected intermediate, whilst GVL is subsequently produced by cyclization and dealcoholation mechanisms [39]. The effect of the reaction time was also considered (Figure 5b). The LA conversion is almost complete after only 30 min, and GVL selectivity rapidly increases from 30 to 120 min, becoming complete after 180 min. Lastly, the effect of the hydrogen source was preliminarily investigated by comparing the effect of molecular hydrogen and MeOH for the GVL production, in the absence/presence of the bimetallic 5Ni-3Fe/SBA-15 catalyst (Figure 5c). The LA conversion in MeOH is slightly higher than that obtained with molecular hydrogen. Remarkably, the GVL selectivity is significantly higher for the catalytic tests carried out in the presence of MeOH, thus confirming the positive synergism achieved thanks to the combined use of the bimetallic Fe-Ni catalyst and MeOH for promoting the selective hydrogenation of the C=O bond. These preliminary data confirm that the catalytic performances of the synthesized catalysts are comparable with those of noble metal catalysts, such as ruthenium, which has been identified as a very efficient catalyst for GVL production. For example, Xiao et al. reported an almost complete LA conversion (99.7 mol %) and a complete selectivity towards GVL, adopting a batch reactor, in the presence of Ru/graphene catalyst (40 bar H2, 200 ◦C, 8 h) [40]. Piskun et al. found a maximum LA conversion of 90 mol %, but a low selectivity (66 mol % of GVL), using Ru/Beta-12.5 (45 Bar H2, 90 ◦C, 2 h) [41]. Luo et al. reported a maximum GVL selectivity of 97.5 mol % at complete LA conversion, adopting Ru/TiO2 as a catalyst, and dioxane as the reaction medium (4 MPa H2, 200 ◦C, 4 h) [42]. However, in all these mentioned cases, pentanoic acid was identified as a reaction by-product, this compound deriving from the GVL ring opening, a reaction catalyzed by highly acidic supports [25]. Instead, in our case, pentanoic acid was not produced, thanks to the milder acidity of the adopted catalyst, leading to a more selective production of the desired GVL.

**Figure 5.** (**A**) The effect of temperature (reaction conditions: 2 g LA, 15 mL MeOH, 0.5 g 5Ni-3Fe/SBA-15, 180 min), (**B**) reaction time (reaction conditions: 2 g LA, 15 mL methanol, 0.5 g 5Ni-3Fe/SBA-15, 200 ◦C) and (**C**) different hydrogen sources (H2 and MeOH) (reaction conditions: 2 g LA, 1 MPa H2 or 15 mL MeOH, 0.5 g 5Ni-3Fe/SBA-15, 200 ◦C, 180 min) on LA conversion and GVL selectivity.

Preliminary recycling tests of the most promising bimetallic catalyst (5Ni-3Fe/SBA-15) were carried out to demonstrate its stability (Figure 6). The 5Ni-3Fe/SBA-15 catalyst was re-used consecutively five times under the best-identified reaction conditions. Before each cycle run, the spent catalyst was simply washed with ethanol in an ultrasonic cleaner and dried in a vacuum oven at 60 ◦C, thus avoiding tedious reactivation procedures. If compared with the performances of the fresh catalyst, those of the spent 5Ni-3Fe/SBA-15 catalyst decrease only slightly, even after five recycling tests, thus confirming the good potential of this catalytic system for GVL production on a larger scale.

**Figure 6.** Recycling tests of 5Ni-3Fe/SBA-15 catalyst.

#### **3. Experimental Section**

#### *3.1. Materials*

Levulinic acid, γ-valerolactone, and methyl levulinate were purchased by Shanghai Aladdin Reagent Company (Aladdin, Shanghai, China). Nickel(II) nitrate hexahydrate [(Ni(NO3)2·6H2O], ferric(III) nitrate hexahydrate [Fe(NO3)3·9H2O], dichloromethane (DCM), and MeOH were purchased from Sigma-Aldrich Trading Co., Ltd. (Sigma-Aldrich, Shanghai, China). SBA-15 was produced by Sinopharm Chemical Reagent Co., Ltd. (Sinopharm, Shanghai, China). Double-distilled H2O was used for the synthesis of catalysts.

#### *3.2. Preparation of Catalyst*

The metal-based catalysts were prepared by the incipient wetness impregnation method [21]. Solutions of nickel(II) nitrate hexahydrate and ferric(III) nitrate hexahydrate were mixed in the appropriate molar ratio (nominal weight content of 5 wt% Ni, Ni:Fe molar ratio = 5:0, 5:1, 5:2, 5:5). Then, 1 g of SBA-15 was added into the 10 mL volume of the metal precursor solution. The resulting slurry was stirred at room temperature for 2 h, then the catalyst was recovered, and dried overnight at 120 ◦C. The calcination was carried out under N2 atmosphere at 450 ◦C (heating rate 5 ◦C/min) for 1 h and the catalyst was reduced at 450 ◦C (heating rate 5 ◦C/min) for 3 h, under H2 flow.

#### *3.3. Characterization of the Catalysts*

Powder X-ray diffraction (XRD) analysis was carried out by a Rigaku Ultima IV X-ray diffractometer (Rigaku, Tokyo, Japan), utilizing Cu-Kα radiation (λ = 1.5405 Å). The test conditions were tube voltage, 50 kV; tube current, 50 mA; scanning speed, 8◦/min; and scanning range, 10◦–80◦. N2 sorption measurements were carried out at 77 K on a BEL-MAX gas/vapor adsorption instrument (MicrotracBEL Corp., Osaka, Japan). The surface areas were measured by the Brunauer-Emmett-Teller (BET) method. Transmission electron microscopy (TEM) was performed by the JEM-2100 microscope (JEOL, Tokyo, Japan), working at 200 kV. X-ray photoelectron spectroscopy (XPS) characterization was carried out by a Scientific Escalab 250 spectrometer (Thermo, Waltham, MA, USA) with Al*K*α (hν = 1486.6 eV) radiation, at a pressure of about 1 × 10−9 Torr. The binding energy values were corrected by using the C1s peak (284.6 eV). The total acidity was determined by NH3-TPD technique. The calcined samples were characterized using temperature-programmed reduction by a TP-5080 adsorption instrument (xq-instrument, Tianjin, China), which was equipped with a TCD detector. Hydrogen temperature-programmed desorption (H2-TPD) measurements were performed by an Autochem II-2920 (Norcross, GA, USA) instrument. The total H2 desorption was calculated by measuring the areas of the desorption peaks. The FT-IR spectra were acquired by a Nicolet NEXUS 670 (Thermo, Waltham, MA, USA), which was equipped with an in situ IR cell.

#### *3.4. Catalytic Tests*

The catalytic hydrogenation of LA was carried out in a 100-mL stainless steel batch autoclave (Parr, Moline, IL, USA). In a typical run, the autoclave was loaded with the catalyst (0.5 g), LA (2 g), and MeOH (15 mL). Then, the reactor was heated up to the set-point temperature, under a constant stirring rate of 1000 rpm. At the end of the reaction, the slurry was recovered and centrifuged to separate the liquid phase from the spent catalyst, and the former was further analyzed by GC-MS and GC-FID techniques.

#### *3.5. Analysis of the Liquid Phase*

Qualitative analysis of the liquid samples was carried out by a GCMS-QP2010 instrument (Shimadzu, Kyoto, Japan), equipped with an RTX-5MS column (30 m × 0.25 mm × 0.25 μm). The injector temperature was 250 ◦C and a split ratio of 20:1 was employed. The MS operated in EI mode at 70 eV and the ion source temperature was set at 230 ◦C. The temperature program was the following: from 50 ◦C to 250 ◦C at a heating rate of 10 ◦C/min and held for 5 min at 250 ◦C. He was the carrier gas, and a column flow of 1.0 mL/min was used. The quantitative analysis was carried out by a GC-2010 gas-chromatograph (Shimadzu, Kyoto, Japan), equipped with an RTX-50 column (30 m × 0.25 mm × 0.25 μm) and a flame ionization detector. *n*-dodecane was used as the internal standard. Conversion and selectivity parameters were calculated according to the following equations:

$$\text{Conversion of LA (mol }\%) = \left(1 - \frac{n\_{LA \ final}}{n\_{LA \ initial}}\right) \ast 100 \text{ mol }\% \tag{1}$$

$$\text{Selocity to GVL (mol }\%) = \frac{n\_{\text{GVL}}}{n\_{\text{products}}} \ast 100 \text{ mol }\% \tag{2}$$

#### **4. Conclusions**

γ-valerolactone is an important biomass platform molecule that can be used as a biofuel, pharmaceutical intermediate, food additive, and green solvent. The conversion of levulinic acid into high-value-added γ-valerolactone is an important reaction, and still requires the development of cheap and highly effective catalysts. In this regard, this paper is mainly focused on the synthesis of new Ni-Fe/SBA-15 bimetallic catalysts for improving the hydrogenation of levulinic acid into γ-valerolactone, adopting methanol as the only hydrogen donor. The physicochemical properties of the synthesized bimetallic catalysts were investigated by different techniques, e.g., XRD, TEM, XPS, and H2-TPD. H2-TPD confirmed that the addition of Fe as an oxophilic promoter lowers the reduction temperature of the NiO phase and improves the reducibility of the mixed oxides. At the same time, the presence of Fe favorably increased the Lewis acidity, which is beneficial for the MPV reduction of LA to GVL. Moreover, XPS analysis confirmed an electronic transfer from Fe to Ni, promoting the activation of the C=O bond of LA, and this conclusion is in agreement with the in situ FT-IR characterization. The preliminary catalytic tests have shown that the best bimetallic 5Ni-3Fe/SBA-15 catalyst has promising performances towards GVL synthesis. The effect of temperature and reaction time on GVL production was also considered, achieving complete LA conversion and GVL selectivity, working at 200 ◦C and after 180 min. Lastly, preliminary recycling tests of the best performing Ni-Fe bimetallic catalyst confirmed the possibility of its advantageous re-use, achieving satisfactory performances to γ-valerolactone, even after five catalytic cycles. The good catalytic performances/stability, the low cost, and the easy synthesis/regeneration of our Ni-Fe bimetallic catalysts are key aspects for the real development of γ-valerolactone production on a larger scale, thus filling the still existing gap between the industrial and academic world.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4344/10/9/1096/s1. Figure S1: XPS profiles of reduced catalysts. Figure S2: H2-TPD profiles of the synthesized SBA-15 supported catalysts. Figure S3: Influence of metals supported with SBA-15 on the amount of hydrogen in gas. Figure S4: GC-MS results for hydrogenation of LA with different reaction times.

**Author Contributions:** Conceptualization, L.L.; methodology, Q.Z. and X.H.; analysis, L.L., Q.Z. and X.H.; writing, original draft preparation, X.H. and Q.Z.; writing, review and editing, L.L.; project administration, L.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work is supported by the National Natural Science Foundation of China (Grant No.31801321), and sponsored by Shanghai Sailing Program (Grant No. 19YF1436300) and Natural Science Foundation of Shanghai (Grant No. 18ZR1428100).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Guerbet Reactions for Biofuel Production from ABE Fermentation Using Bifunctional Ni-MgO-Al2O3 Catalysts**

**Zhiyi Wu, Pingzhou Wang, Jie Wang and Tianwei Tan \***

Beijing Key Laboratory of Bioprocess, National Energy R&D Center for Biorefinery, College of Life Science and Technology, Beijing University of Chemical Technology, No. 15 of North Three-ring East Road, Chaoyang District, Beijing 100029, China<EMAIL_ADDRESS>(Z.W<EMAIL_ADDRESS>(P.W<EMAIL_ADDRESS>(J.W.)

**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** To upgrade biomass-derived alcohol mixtures to biofuels under solvent-free conditions, MgO–Al2O3 mixed metal oxides (MMO) decorated with Ni nanoparticles (Ni–MgO–Al2O3) are synthesized and characterized. Based on the result, Ni nanoparticles are highly dispersed on the surface of MgAl MMO. As the Ni loading content varies from 2 to 10 wt.%, there is a slight increase in the mean Ni particle size from 6.7 to 8.5 nm. The effects of Ni loading amount, reducing temperature, and Mg/Al ratio on the conversion and product distribution are investigated. With the increase in both the Ni loading amount and reducing temperature, dehydrogenation (the first step of the entire reaction network) is accelerated. This results in an increase in the conversion process and a higher selectivity for the dialkylated compounds. Due to the higher strength and density of basic sites under high Mg/Al ratios, double alkylation is preferred and more long-chain hydrocarbons are obtained. A conversion of 89.2% coupled with a total yield of 79.9% for C5–C15 compounds is acquired by the as-prepared catalyst (prepared with Ni loading of 6 wt.%, reducing temperature of 700 ◦C, and Mg/Al molar ratio of 3. After four runs, the conversion drops by 17.1%, and this loss in the catalytic activity can be attributed to the decrease in the surface area of the catalyst and the increase in the Ni mean particle size.

**Keywords:** ABE fermentation; Ni-MgO-Al2O3 catalyst; biofuel; catalytic performance

#### **1. Introduction**

The ever-growing concerns with regards to the continual depletion of fossil fuel reserves and the increasing severity of the environmental issues have urged the hastened development of clean energy sources. Biomass has received increasing attention as one of the most promising clean energy sources due to its abundant and renewable nature. In particular, the conversion of biomass into transportation fuels has emerged as a critical research focus in chemistry and engineering-related fields [1–12].

Triglyceride, starch-based, and lignocellulosic feedstocks are three types of feedstocks used in the production of biofuel [13–19]. Among these feedstocks, lignocellulosic biomass is considered the most promising candidate due to its high natural abundance [20–22]. To convert lignocellulose into biofuels, two steps are required. Step 1: Solid biomass has to be converted into platform chemicals with better catalytic activity to proceed with further treatments. Step 2: Conversion of platform chemicals into biofuels via C–C coupling reaction and hydrodeoxygenation (HDO), when necessary [23–25]. Various strategies can be implemented during the first step of the conversion process, whereby these strategies can be categorized into two parts: (1) Thermochemical process can be conducted under high temperature and/or pressure, and it can later be combined with chemical upgrading, e.g., Fischer–Tropsch synthesis. (2) The hydrolysis process can be carried out so that small molecules containing oxygen functional groups, such as acetone n-butanol-ethanol (ABE) fermentation products [26], can be obtained using biological or chemical means. After

**Citation:** Wu, Z.; Wang, P.; Wang, J.; Tan, T. Guerbet Reactions for Biofuel Production from ABE Fermentation Using Bifunctional Ni-MgO-Al2O3 Catalysts. *Catalysts* **2021**, *11*, 414. https://doi.org/10.3390/catal11040414

Academic Editor: Domenico Licursi

Received: 3 March 2021 Accepted: 17 March 2021 Published: 24 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

which, these generated small molecules can be upgraded into biofuels via catalytic reactions. Due to the ease of control on the molecular weight of the final hydrocarbons, the hydrolysis strategy has garnered more attention as compared with thermochemical strategy.

The chemical catalytic upgrading of ABE fermentation products to C5–C11 ketones and alcohols in toluene was reported for the first time by Toste and coworkers [27]. The total yield was 86%. Then, the generated C5–C11 ketones and alcohols were deoxygenated into components for the preparation of various products such as gasoline, jet fuel, and diesel fuel. Other than using toluene, Xu and coworkers [28] demonstrated the direct transformation of mimicking ABE fermentation products using water as the solvent, whereby Pd/C was coupled with various bases, e.g., K3PO4, KOH, and K2CO3, as the catalyst. In their report, the type and amount of bases used during the process can play pivotal roles in determining the total yield and product distribution. Furthermore, Xue et al. reported that concentrated ABE mixture could be directly alkylated to C5–C15 or longer chain ketones in a continuous mode using a Pd/C catalyst, with an average conversion rate of >70% [29]. To further improve the recyclability and to avoid the use of alkaline as additives, metal supported on an alkaline substrate, e.g., hydrotalcite (HT) and CaO, has been developed [30,31]. For example, Lee and co-workers used Pd@C and CaO as solid bases to convert ABE mixture in a 180 ◦C batch reactor without any added solvent to produce a mixture of ketones and corresponding alcohols with 78% yield from acetone [32]. Among the various metallic materials (Ru, Pd, Fe, Co, Ni, Cu, and Zn) studied, Pd and Cu demonstrate the best performance with yields of 95% and 92%, respectively. On the other hand, Ni–HT catalyst shows a total yield of 2% [33]. Even though Pd-based and Cu-based catalysts demonstrate high performance, these catalysts still face challenges that require significant attention. For instance, besides the high cost of Pd, significant decarbonylation is observed for Pd–HT catalyst, which can lead to poor selectivity toward the desired products and carbon balance simultaneously [34]. As reported by Onyestyák and coworkers [35,36], a Cu-based catalyst is also unsuitable due to the high production of side products, i.e., esters, via Tishchenko reaction. As such, due to these limitations that plagued Pd-based and Cu-based catalysts, the development of cheap and efficient catalysts is urgently needed. In addition, to achieve green chemistry and simple separation, it is of great significance to convert ABE mixtures under solvent-free conditions. Furthermore, detailed investigations are greatly needed to provide insights into the reaction.

The reaction pathway is mainly comprised of dehydrogenation, aldol condensation, dehydration, and hydrogenation. This leads to clear design considerations when developing the catalysts: catalysts should possess (1) the ability to facilitate dehydrogenation of alcohols and (2) the capacity for aldol condensation. As reported in our previous work [37], Ni nanoparticles are regarded as the most promising catalyst for the upgrading of the ABE mixture due to their high catalytic activity for dehydrogenation/hydrogenation. It is well accepted that aldol condensation takes place at the acid–base site [38–41]. As a result, factors such as Ni loading, morphology of Ni nanoparticles, and the acidity–basicity of the catalyst can exert significant impacts on the catalytic activity of the catalyst.

Herein, a series of Ni–HT catalysts with different Ni loadings and acid–base properties is synthesized via the co-precipitation method. The as-prepared Ni–HT catalysts are characterized using scanning electron microscopy (SEM), high-resolution transmission electron microscope (HRTEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and temperature-programmed desorption (TPD). In this work, various parameters such as Ni loading content, temperature used in the reduction of catalyst, and Mg/Al ratio are systematically investigated for their effects on the total yield and product distribution.

#### **2. Results and Discussion**

#### *2.1. Characterization of the as-Prepared Catalyst*

XRD spectrums of Ni-MgO-Al2O3 catalysts with various Ni loading are presented in Figure 1. Distinct peaks located at 2*θ* = 36.1◦, 43.1◦, 62.6◦, and 79.0◦ can be observed, which are consistent with those present in the standard XRD spectrum of MgO (JCPDS

01-075-1525). The diffraction peak located at 35.1◦ can be assigned to Al2O3, which overlaps with the diffraction peak of MgO. The characteristic peaks of Ni are not observed for 0 wt.% Ni loading, which is indicative that the pristine sample does not contain Ni. As the Ni loading increases to 2 wt.%, weak XRD peaks of Ni can be barely observed, which suggests that there is a low percentage of Ni in the catalyst with high dispersity. As the Ni loading increases, three distinct peaks at 44.5◦, 51.8◦, and 76.4◦ can be observed, which correspond to (111), (200), and (220) planes of metallic Ni (JCPDS 03-065-0380), respectively. This XRD result suggests the successful formation of Ni nanoparticles. As shown in Table 1, with the increase in Ni loading from 2 to 10 wt.%, the average crystallite size of Ni nanoparticle increases slightly from 6.7 to 8.5 nm, based on the Scherrer equation.

**Figure 1.** XRD spectrums of the as-prepared catalysts with various Ni loadings.

**Table 1.** Structural properties of Ni-MgO-Al2O3 catalysts with various Ni loadings.

To investigate the morphology of the as-prepared catalyst, SEM, and TEM are employed. Figure S1 shows the SEM images of the as-prepared catalyst. TEM images of various catalysts prepared with different Ni loadings of 2, 6, and 8 wt.% are shown in Figure 2a–c, respectively. Ni nanoparticles are clearly observed as dark spots in the TEM images, and they are highly dispersed on the surface of MgO-Al2O3. By measuring the size of more than 150 nanoparticles observed in the TEM images, corresponding histograms of Ni particle size distributions for catalysts with 2, 6, and 8 wt.% Ni loadings can be derived, and they are presented in Figure 3a–c, respectively. The average particle size of Ni nanoparticles is estimated based on a number-weighted diameter (*d* = ∑ *nidi*/∑ *ni*, *ni* is the number of counted Ni particles with a diameter of *di*) with values of 6.8 and 7.8 nm for catalysts with 2 and 8 wt.% Ni loadings, respectively. This result confirms that the mean particle size of Ni nanoparticles increases slightly with the increase in Ni loading, which is consistent with the XRD results. Based on the high-resolution TEM image shown in Figure 2d, a lattice fringe of 0.203 nm can be clearly observed for the Ni nanoparticle, which corresponds to the (111) plane of Ni [42].

**Figure 2.** TEM images of Ni-MgO-Al2O3 catalysts with Ni loadings of (**a**) 2 wt.%, (**b**) 6 wt.%, and (**c**) 8 wt.% Ni; (**d**) HRTEM image of Ni-MgO-Al2O3 catalyst (6 wt.%).

**Figure 3.** Histograms of the particle size distribution of Ni nanoparticles in the as-prepared catalysts with various Ni loadings: (**a**) 2 wt.%, (**b**) 6 wt.%, and (**c**) 8 wt.%.

To provide greater details to the dispersibility of Ni nanoparticles in the as-prepared catalyst, energy-dispersive X-ray spectroscopy (EDS) elemental mapping is conducted for Ni-MgO-Al2O3 catalyst with 6 wt.% Ni loading. As shown in Figure 4, the elemental distributions of Mg, Al, and Ni in the sample are highly uniform. This result suggests that Ni nanoparticles are homogeneously distributed across the well-mixed MgO-Al2O3 binary oxides.

**Figure 4.** Energy-dispersive X-ray spectroscopy (EDS) elemental mapping of Ni-MgO-Al2O3 catalyst with 6 wt.% Ni loading. (**a**) Mg; (**b**) Al; (**c**) Ni.

BET surface areas, pore volumes, and pore size distributions of Ni-MgO-Al2O3 catalysts with various Ni loading are summarized in Table 1. It can be observed that all catalysts possess high surface areas (larger than 200 m2/g), which is vital in providing a large contact area between the catalyst and the reactant, thus contributing toward high catalytic activity. With the increase in Ni loading, various parameters such as surface area, pore volume, and mean pore diameter exhibit a decreasing trend. Note that as Ni loading increases from 0 to 10 wt.%, the surface area of the sample decreases from 267.1 to 227.2 m2/g. Meanwhile, the pore volume of the sample also decreases from 0.80 to 0.63 cm3/g. This observation may be due to the increased occupancy of Ni nanoparticles in the sample as the Ni loading increases. Pore size distributions and N2 isotherms are provided in Figure 5a,b, respectively. The pore size distribution, determined using the Barrett, Joyner, and Halenda method, illustrates that all the as-prepared catalysts contain mesopores with a mean pore size of approximately 6 nm. The N2 adsorption–desorption isotherms of all the as-prepared catalysts reveal a typical Type IV isotherm with a well-defined N2 hysteresis loop at relative pressures of 0.7–1.0. As such, based on this result, the as-prepared catalysts should possess mesoporous structures.

**Figure 5.** (**a**) Pore size distributions and (**b**) N2 adsorption–desorption isotherms of Ni-MgO-Al2O3 catalysts with various Ni loadings.

#### *2.2. Catalytic Upgrading of ABE Mixture*

Scheme 1 shows the illustration of the catalytic upgrading mechanism of ABE mixtures to long-chain compounds. As illustrated in Scheme 1, three main reactions are involved during the catalytic upgrading process. Part A: Alkylation reactions producing ketones. Part B: Guerbet reactions generating alcohols with longer chains. Part C: Selfcondensation of acetones through aldol condensation. Although the detailed mechanism of the Guerbet reaction is still controversial, it is generally believed that both the Guerbet reaction and alkylation reaction involve a series of processes, such as dehydrogenation

of alcohol, aldol condensation between ketone (or aldehyde) and aldehyde, dehydration, and hydrogenation.

**Scheme 1.** Catalytic upgrading mechanism of ABE mixture.

It is commonly acknowledged that nanoparticles can facilitate the dehydrogenation/hydrogenation process. As such, loading amount and dispersibility of nanoparticles can play pivotal roles in influencing the catalytic activity of the catalyst. The growth of carbon chain molecules can be produced by aldol condensation. For example, aldol condensation between acetone and acetaldehyde yields 2-pentanone (2-C5), and with further aldehyde condensation between 2-C5 and acetaldehyde, 4-heptaneone (4-C7) can be obtained. Part D: Alcohols (C5-OH, C7-OH, C9-OH, and C11-OH) can be generated via the hydrogenation of corresponding ketones as shown in Scheme 1. Ketone hydrogenation requires additional hydrogen resources, which may come from two ways. First, it may be released during the aldoesterification process (Scheme 2). However, neither ethyl acetate nor butyl butyrate is observed in our reaction system, and therefore these products can be ruled out. Second, steam reforming of ethanol or butanol may occur with the generation of hydrogen. As mentioned by Fu and Gong [43], nickel nanoparticles possess catalytic activity for the steam reforming of alcohols. The dehydrogenation of alcohols, decarbonylation of aldehydes, water–gas shift reaction, and CH4 conversion are as follows. The activity of alcohol condensation reaction is highly related to the acidity and basicity of the catalyst, which indicates that the optimization of acid/base strength or acid/base amount of catalyst can play a significant role.

**Scheme 2.** Esterification between alcohol and aldehyde.

According to the abovementioned analyses, the effects of Ni amount on the ABE conversion yield and product distribution are firstly investigated. As shown in Figure 6, when ABE conversion is conducted using MMO catalyst (without Ni nanoparticle), an ABE conversion yield of 8.4% with two main products, i.e., 2-C5 and 2-C7 (monoalkylation of acetone with ethanol and butanol), are obtained. This result clearly shows that Mg-Al MMO exhibits low activity toward the dehydrogenation of ethanol and 1-butanol. Interestingly, as Ni nanoparticle is incorporated into Mg-Al MMO, ABE conversion yield is significantly improved as observed in Figure 6. Note that as Ni loading in the catalyst increases from 0 to 2 wt.%, ABE conversion yield increases drastically from 8.4% to 58.8%. After which, as Ni loading increases from 2 wt.% to 6 wt.%, ABE conversion yield continues to increase steadily from 58.8% to 89.2%. When the nickel loading is more than 6 wt.%, the yield decreases from 89.2% to 86.48% with increasing nickel loadings. Furthermore, it can be observed that with the increase in Ni loading, the main product changes from mono-alkylated compounds (C5 and 2-C7) to double-alkylated ones with longer carbon chains (4-C7 to C15). For instance, the total yield of double-alkylated compounds reaches 79.88% when the Ni loading is 6 wt.%. Two key reasons can be used to explain such phenomenon: (1) As Ni nanoparticle exhibits high dehydrogenation activity, increasing Ni loading would translate to the production of more aldehydes, which can then act as reactants for subsequent aldol condensation. This process can lead to a significant improvement in double alkylation. However, over-high Ni loading content is not valuable for the conversion yield. (2) C=C bonds in α, β-unsaturated ketones are kinetically and thermodynamically favored by the Ni site, and therefore saturated ketones are generated [44].

**Figure 6.** ABE conversion using Ni-MgO-Al2O3 catalysts with various Ni loading (reaction conditions: 15 g of ABE mixtures with 1.5 g of Ni-MgO-Al2O3 catalyst, molar ratio of Mg/Al: 3, temperature used in the reduction of catalyst: 700 ◦C, 240 ◦C, 20 h).

Other than the Ni loading contents, the temperature used in the reduction of the catalyst can also influence the catalytic activity of the catalyst. As such, various temperatures are used in the preparation process, and the corresponding catalytic performances of the as-prepared catalysts are shown in Figure 7. It is clearly shown that 7% ABE conversation yield with C5 as the sole product is achieved when catalysts that are reduced at 400 ◦C and 500 ◦C are used. As the temperature used in the reduction of catalyst increases to 600 ◦C, an increase in the ABE conversion yield is observed, with long-chain ketones and alcohols as the products. As the temperature increases from 600 ◦C to 800 ◦C, a total yield that increases from 68.4% to 88.6% is recorded. This observation is largely attributed to the fact that HT precursors would not be able to completely convert to MMO at a temperature lower than 500 ◦C, which results in higher catalytic activity for subsequent aldol condensation [45,46]. On the other hand, Ni nanoparticle is expected to catalyze the dehydrogenation of alcohols, while Ni2+ shows low dehydrogenation activity.

To verify the abovementioned hypothesis, XPS is used to further characterize the catalysts reduced at various temperatures. Binding energy values of metallic Ni are 852.7 eV (Ni 2p3/2) and 870.5 eV (Ni 2p1/2), while those of NiO are 854.0 eV (Ni 2p3/2) and 872.5 eV (Ni 2p1/2) [46,47]. As shown in Figure 8, as the temperature used in the reduction of the catalyst decreases to the range of 400 to 500 ◦C, a peak near 854.6 eV is observed, which indicates that Ni species exists primarily in the form of NiO. On the other hand, as the temperature increases from 600 to 800 ◦C, a peak around 852.6 eV can be clearly observed. The shift in the binding energy toward lower values may be attributed to the change in the configuration of Ni in the MgO-Al2O3 matrix. As indicated in Table 2, the amount of Ni nanoparticles in the catalyst increases from 7% to 87% as the temperature used in the reduction of catalyst increases from 400 to 800 ◦C.

**Figure 8.** Ni 2p XPS spectrums of Ni-MgO–Al2O3 catalysts reduced at various temperatures.

**Table 2.** Quantity of oxidized and metallic Ni element in the catalysts reduced at various temperatures.

It is well accepted that the acid–base properties of the catalyst can play a key role in influencing the aldol condensation activity. The weak Brønsted basic sites of MgAl-MMO are related to the residual surface hydroxyl groups after activation, the moderate strength Lewis sites are related to Mg2−O2− and Al3+O2− acid–base pairs, and the strong Lewis base sites are due to the existence of low coordinated O2 species. The lower Al dopant content and the higher Mg content led to the increase in the basic center density, which is due to the formation of coordinated unsaturated oxygen sites. Materials with high Al contents are beneficial to the dehydration of alcohols rather than dehydrogenation and condensation. Ref. [48] by varying the Mg/Al-ratios, which changes the number and strength of the acid-base sites, the selectivity can be optimized towards dehydrogenation, aldolization, and hydride-shifts. Thus, the effect of the Mg/Al ratio on the catalytic performance of the catalyst is investigated. As shown in Figure 9, as the Mg/Al ratio increases from 1 to 9, ABE conversion yield remains constant at 88.4%. However, significant changes in the product distribution are observed across the varying Mg/Al ratio. Note that as the Mg/Al ratio increases from 1 to 9, the selectivity for C5 and 2-C7 decreases and more C8–C15 are obtained. This result indicates that double alkylation is preferable at higher Mg/Al ratios. Figure S6 shows the results of the product distribution for the catalytic coupling of the ABE mixture.

**Figure 9.** Effect of Mg/Al ratio on catalytic performance. Reaction conditions: 15 g of ABE mixture and 1.5 g of Ni-MgO-Al2O3 catalyst, Ni loading amount: 6 wt.%, temperature used in the reduction of catalyst: 700 ◦C, 240 ◦C, 20 h.

The acid and base properties of catalysts with various Mg/Al ratios are investigated using NH3-TPD and CO2-TPD, respectively. As shown in Figure 10a, catalysts with various Mg/Al ratios exhibit a similar profile with observable broad peaks at around 120 ◦C, which indicates that Ni-MgO-Al2O3 catalysts only contain weak acidic sites. As shown in Figure 10b, CO2-TPD profiles are composed of two overlapping desorption peaks centered around 150 ◦C (peak I) and 260 ◦C (peak II), which correspond to weak and moderate basic sites, respectively.

**Figure 10.** (**a**) NH3-TPD and (**b**) CO2-TPD profiles of Ni-MgO-Al2O3 catalysts with various Mg/Al ratios.

The concentrations of the acidic sites of the catalysts with various Mg/Al ratios are listed in Table 3. The concentration of acidic sites gradually decreases to a minimum value of 0.27 μmol/g with an increase in Mg/Al ratio. On the other hand, with the increase in Mg/Al ratio, the density of weak basic site decreases from 0.69 to 0.61 μmol/g, while densities of moderate basic site and the total basic site gradually increase. The weak acidic sites in the catalyst are beneficial toward the dehydration of unstable aldol products. However, the presence of strong acid sites could potentially result in side reactions such as dehydration of alcohols to olefins. The basicity and number of basic sites play an important role in determining the product distribution. With the consideration of the results presented in Figure 9, it is suggested that there is a preferential double alkylation of acetone when using catalysts with higher basicity and more basic sites. This may explain the need for large amounts of alkali such as K3PO4, KOH, and K2CO3 in other works [27,28].

**Table 3.** Acid and base properties of Ni-MgO-Al2O3 catalysts with various Mg/Al ratios.

#### *2.3. Regeneration Performance*

The reusability of the as-prepared catalyst is studied, and the result is shown in Figure 11. It can be observed that ABE conversion yield decreases from 89.2% to 72.1% after four runs. To provide some insights into the decrease in the ABE conversion yield with runs, the spent catalysts are investigated with XRD, BET, and TEM. As shown in Figure S2, there is no distinct difference between the XRD spectrums of the fresh and spent catalysts, which indicates that the crystal structure and phase of the catalyst remain unchanged after the operation. The pore size distribution and N2 adsorption–desorption isotherms of the spent catalysts are depicted in Figure S3, with the rest of the BET results listed in Table S1. With the increase in the number of recycling runs, the specific surface area and pore volume of the spent catalyst decrease gradually. Such a result may be the key factor towards the observed catalytic activity loss. TEM image of the spent catalyst is shown in Figure S4a, and the histogram of the size distribution of Ni nanoparticles is shown in Figure S4b. The mean diameter of Ni nanoparticles increases to 8.1 nm after several cycles, which is another factor that poses a detrimental effect on the catalytic performance of the catalyst. The base density of the used catalyst decreased significantly from 1.24 to 0 μmol/g, which may be caused by the formation of MgCO3. The main peaks located at 400 ◦C and 540 ◦C can be attributed to the release of CO2 from the decomposition of MgCO3. The decrease in the base density and surface area of the activated catalyst may lead to a decrease in its catalytic activity.

**Figure 11.** Regeneration performance of Ni-MgO-Al2O3 catalyst (reaction conditions: 15 g of ABE mixture and 1.5 g of Ni-MgO-Al2O3 catalyst, molar ratio of Mg/Al: 3, Ni loading: 6 wt.%, temperature used in the reduction of catalyst: 700 ◦C, 240 ◦C, 20 h).

#### **3. Experimental Section**

*3.1. Materials*

Al(NO3)3·9H2O, Mg(NO3)2·6H2O, and Ni(NO3)2·6H2O were purchased from Sigma-Aldrich Co. (Sigma-Aldrich, St. Louis, MO, USA) Ethanol (99.9%), acetone (99.9%), and 1-butanol (99.9%) were purchased from Fuchen Chemical Plant (Tianjin, China). Deionized water was used in all reactions. All chemicals were used as received, without further purification.

#### *3.2. Preparation of the Catalyst*

Ni-MgO-Al2O3 mixed metal oxide (MMO) was prepared via co-precipitation. In a typical preparation process, an aqueous solution (0.1 L) of Na2CO3(0.2 mol, 2.12 g) solution was added into an aqueous solution (0.1 L) containing Mg(NO3)2·6H2O (1.05 mol, 26.92 g), Al(NO3)3·9H2O(0.35 mol, 13.13 g), and Ni(NO3)2·6H2O(0.09 mol, 2.62 g), and the mixture was mixed via vigorous stirring. The pH of the mixture was carefully maintained in the range of 9 to 10 using an aqueous 0.3 M NaOH solution. After continuously stirring and aging overnight, the precipitate (Mg-Al MMO) was filtered and washed with deionized water until the pH of the precipitate reached 7, and the washed precipitate was dried at 100 ◦C overnight. The as-prepared catalyst was subsequently dried at 80 ◦C overnight. Finally, the catalyst was reduced at 700 ◦C in the presence of H2.

#### *3.3. Characterization Techniques*

XRD spectrum of the sample was recorded using a Shimadzu XRD-6000 diffractometer (Shimadzu, Tokyo, Japan) with Cu K*α* radiation (*λ* = 1.5418 Å). A 2-theta value range of 5◦ ≤ 2θ ≤ 90◦ was used in the XRD measurement. XPS spectrum of the sample was measured using a Thermo VGESCALAB 250 spectrometer (Thermo, Waltham, MA, USA), with Mg K*α* (1253.6 eV) radiation as the X-ray source. All binding energies were calibrated with reference to the position of C1s peak at 284.6 eV. The morphology of the sample was observed under a ZEISS SUPRA55 SEM (ZEISS, Jena, Germany), with an accelerating voltage of 2.0 kV. The structure, size, and lattice fringes of the sample were examined under a JEOL JEM-2011 TEM (JEOL, Tokyo, Japan), equipped with an energy-dispersive X-ray spectrometer. The specific surface area of the sample was measured (Bruker, Karlsruhe, Germany) according to the Brunauer–Emmett–Teller (BET) method based on N2 adsorption isotherm. All samples were degassed at 180 ◦C for 4 h prior to the BET measurement. The acid–base properties of the catalyst were determined using NH3-TPD and CO2-TPD, respectively, which are both equipped with a thermal conductivity detector (TCD, Bruker, Karlsruhe, Germany). 0.2 g sample was pretreated in a U-tube glass under a 25 mL/min He flow at 350 ◦C for 1 h, and it was then cooled to 100 ◦C. After completing the degassing of the sample, the adsorption gas was switched to CO2 or NH3, which was used to flush the U-tube glass for 30 min. After which, the temperature was increased from 100 to 850 ◦C at a heating rate of 10 ◦C/min, under a pure He atmosphere, to record the TCD signals.

#### *3.4. Catalytic Conversion of Feedstock to Biofuel*

A mixture of acetone, *n*-butanol, and ethanol with a molar ratio of 2.3:3.7:1 was used as a model of ABE fermentation. The catalytic conversion was conducted in a high-pressure reaction vessel, which was equipped with a magnetic stirring bar (IKA, Köln, Germany). In a typical reaction process, 1.5 g of the as-prepared catalyst and 15 g ABE mixture were added into the reactor, and the reactor was then heated to 240 ◦C for 20 h under a stirring speed of 800 rpm. After which, the reactor was cooled to room temperature by immersing it into an ice-water bath. The reaction product is analyzed using Shimadzu GC-2014 Chromatograph (Shimadzu, Tokyo, Japan) and Agilent GC-MS (Agilent, Palo Alto, Santa Clara, CA, USA) with DB-5 column, according to our previous work [37].

#### **4. Conclusions**

In summary, the potential of Ni-MgO-Al2O3 as a heterogeneous catalyst in biofuel production is investigated in this work. The as-prepared catalyst shows high efficiency in upgrading ABE mixture into long-chain (C5–C15) ketones and alcohols, which are important biofuel precursors. With the increase in Ni loading from 2 to 10 wt.%, the specific surface area of the catalyst decreases from 256.1 to 227.2 m2/g, while the mean diameter of Ni nanoparticles increases from 6.7 to 8.5 nm. The acid–base properties of the as-prepared catalyst can be controlled by adjusting the Mg/Al molar ratio. Based on the result, catalysts with Mg/Al molar ratio in the range of 1 to 9 all show weak acidic sites, with a decreasing concentration of these acidic sites from 0.41 to 0.27 μmol/g. In contrast, as the Mg/Al molar ratio increases from 1 to 9 and the concentration of basic site increases from 1.09 to 1.45 μmol/g, with more moderate basic sites being generated. Furthermore, it is shown that higher conversion and greater preferential for double alkylation can be realized with higher Ni loading and higher temperature used in the reduction of catalyst. The Mg/Al molar ratio has little effect on the conversion yield, but it plays a significant role in influencing the product distribution. When employing a catalyst with a high amount of Mg, significant enhancement in the selectivity for 4-C7 to C15 hydrocarbons is observed. The catalyst, prepared with Mg/Al ratio of 3 and 6 wt.% Ni loading, and reduced at 700 ◦C, can achieve a conversion yield of 89.2% with the total C5–C15 compounds yield of 79.9%. The cyclic performance of the catalyst is also investigated, whereby there is a 17.1% decrease in the conversion yield after 4 runs. This loss in the activity may be a result of the decrease in the surface area and increase in the mean Ni particle size.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2073-434 4/11/4/414/s1.

**Author Contributions:** Conceptualization, Z.W. and T.T.; writing—original draft preparation, Z.W., P.W. and J.W.; writing—review and editing, Z.W., P.W. and J.W.; funding acquisition, T.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** The work described above was supported by the National Nature Science Foundation of China (21606008, U1663227), the Fundamental Research Funds for the Central Universities (ZY1630, JD1617, buctrc201616), and the State Key Laboratory of Chemical Engineering (SKL-ChE-17A02).

**Data Availability Statement:** The data presented in this paper are from published sources.

**Acknowledgments:** We gratefully acknowledge the support of the National Nature Science Foundation of China (21606008, U1663227), the Fundamental Research Funds for the Central Universities (ZY1630, JD1617, buctrc201616), and the State Key Laboratory of Chemical Engineering (SKL-ChE-17A02).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

### *Article* **A Novel and Efficient Method for the Synthesis of Methyl (***R***)-10-Hydroxystearate and FAMEs from Sewage Scum**

**Luigi di Bitonto, Valeria D'Ambrosio and Carlo Pastore \***

Water Research Institute (IRSA), National Research Council (CNR), via F. de Blasio 5, 70132 Bari, Italy<EMAIL_ADDRESS>(L.d.B<EMAIL_ADDRESS>(V.D.)

**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** In this work, the transesterification of methyl estolides (ME) extracted from the lipid component present in the sewage scum was investigated. Methyl 10-(*R*)-hydroxystearate (Me-10-HSA) and Fatty Acid Methyl Esters (FAMEs) were obtained in a single step. A three-level and four factorial Box–Behnken experimental design were used to study the effects of methanol amounts, catalyst, temperature, and reaction time on the transesterification reaction using aluminum chloride hexahydrate (AlCl3·6H2O) or hydrochloric acid (HCl) as catalysts. AlCl3·6H2O was found quite active as well as conventional homogeneous acid catalysts as HCl. In both cases, a complete conversion of ME into Me-10-HSA and FAMEs was observed. The products were isolated, quantified, and fully characterized. At the end of the process, Me-10-HSA (32.3%wt) was purified through a chromatographic separation and analyzed by NMR. The high enantiomeric excess (ee > 92%) of the *R*-enantiomer isomer opens a new scenario for the valorization of sewage scum.

**Keywords:** sewage scum; methyl (*R*)-10-hydroxystearate; FAMEs; biodiesel; estolides

**Citation:** di Bitonto, L.; D'Ambrosio, V.; Pastore, C. A Novel and Efficient Method for the Synthesis of Methyl (*R*)-10-Hydroxystearate and FAMEs from Sewage Scum. *Catalysts* **2021**, *11*, 663. https://doi.org/10.3390/ catal11060663

Academic Editor: Domenico Licursi

Received: 24 April 2021 Accepted: 21 May 2021 Published: 23 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Hydroxy Fatty Acids (HFAs) are valuable raw materials widely used for several industrial applications, including resins, polymers, cosmetics, biofuels, biolubricants, and additives in coatings and paintings [1,2]. They are valuable intermediates for synthesizing chemicals and pharmaceuticals for their antibiotic, anti-inflammatory, and anticancer properties [3,4].

HFAs are ubiquitous as constituents of plants, seeds, insects, animals and other microorganisms [5,6]. Many of these natural sources are found as part of estolides, oligomeric fatty acid esters formed by hydroxy acyl groups bonded together with ester bonds [7]. Estolides are being marketed as biolubricants for automotive and industrial applications for their excellent physicochemical properties as high viscosity and flash point, good resistance and biodegradability [8–10].

Since its first discovery, 10-(*R*)-Hydroxystearic acid (10-HSA) has attracted great industrial interest. It is the natural precursor of γ-(*R*)-dodecalactone, a taste and aroma component used in the flavor and fragrance industry [11–13]. Moreover, it is used in the manufacturing of lubricants and cosmetics for its chemical properties similar to those of Ricinoleic acid (or 12-Hydroxystearic acid) [14,15].

In recent years, different studies have been carried out for the production of 10-HSA, based on the enzymatic hydrolysis of vegetable oils from bacteria and microorganisms, such as *Elizabethkingia meningoseptica* [16], *Enterococcus faecalis* [17], *Lactobacillus plantarum* [18], *Lysinibacillus fusiformis* [19], *Nocardia cholesterolicum* [20], *Selenomonas ruminantium* [21], *Stenotrophomonas nitritireducens* [22], *Stenotrophomonas maltophilia* [23], *Sphingobacterium thalpophilum* [24]. Fatty acid hydratases have shown to be efficient catalysts with a good regio- and stereoselectivity, particularly useful to obtaining pure enantiomeric forms [25,26]. However, their applicability is not competitive with the currently existing conventional diesel-producing technology for a series of drawbacks, including (i) the specificity of the

substrate, which can be a problem for the conversion of some feedstocks, (ii) the instability of the enzymes in the organic solvents, often required for the solubilization of reacting substrate or recovery of the final product, and (iii) their excessive cost [27].

The use of edible oils for human consumption represents a non-sustainable choice from an economic and environmental perspective. The development of new solutions to produce 10-HSA or its derivatives, mainly if based on non-edible feedstocks, could be a challenging goal for economic reasons and environmental and ethical concerns. Although considered a waste, sewage scum can be used as a source of energy and resources, thus replacing non-renewable resources with a considerable environmental impact [28–31].

Identified as CER190809, it is a floatable by-product of the wastewater treatment plants (WWTPs) obtained from the primary and secondary settler tanks. It mainly consists of vegetable oils and grease, animal fats, and food waste deriving from households, restaurants and animal product industries [32]. Due to their low density, these oily materials float on the wastewater surface. They can be easily skimmed off at the beginning of the treatment processes and used for energy purposes. Usually, sewage scum is processed in the anaerobic digester to produce biogas for electricity generation in the same plant [33]. However, the separation of sewage scum is often avoided compared with that of primary and secondary sludge, which instead accounts for about 50% of the total operating costs of a WWTP [34,35]. More frequently, they are directly disposed of in landfills, increasing the cost of treatment facilities and negatively impacting the environment. For these reasons, researchers have focused on developing novel technologies for their full exploitation. The use of sewage sludge as a lipid feedstock for biodiesel production is an alternative and sustainable approach to sludge management and disposal challenges [36].

Biodiesel is a biodegradable and renewable fuel with chemical and physical properties similar to petroleum-based fuels [37–39]. It is a mixture of Fatty Acid Methyl Esters (FAMEs), which can be synthesized by the reaction of different lipid fractions with methanol in the presence of an acid, a base or an enzyme catalyst [40,41]. The main obstacle to its marketing is the raw materials (mostly vegetable oils and animal fats), which constitute about 70–85% of production costs [42,43]. As a result, the use of non-edible alternative oils is constantly growing. Lipids extracted from sewage scum are mainly constituted by Free Fatty Acids (FFAs, 45–55%wt) and calcium soaps of fatty acids (25–30%wt) [28,29]. After the chemical activation with formic acid [44], they can be easily converted into the corresponding methyl esters by direct esterification with methanol using aluminum chloride hexahydrate (AlCl3·6H2O) as a catalyst [45]. However, besides the biodiesel production (75–80%wt), methyl estolides (ME, 15–20%) were also isolated and characterized [28]. These last, obtained as a result of bacteria activity in sewage sludge [46], can be further converted into methyl-10-hydroxystearate (Me-10-HSA) and FAMEs, representing a valuable source for 10-HSA production (Figure 1) through a transesterification reaction.

**Figure 1.** Schematic process of the transesterification reaction of methyl estolides with methanol for the synthesis of Me-10-HSA and FAMEs.

Such a process would contribute to the production of Me-10-HSA from non-edible feedstocks and a complete valorization of the lipid fraction present in the sewage scum. In this study, an acidic transesterification was proposed for the synthesis of Me-10-HSA and FAMEs by direct conversion of ME isolated from sewage scum with methanol. Aluminum chloride hexahydrate (AlCl3·6H2O) and hydrochloric acid (HCl) were used as catalysts. The best operative conditions were determined through a response surface methodology, widely adopted in studies concerning the production of biodiesel for optimizing the transesterification reaction [47–50]. Notably, a Box–Behnken factorial design of experiments was used. The amount of methanol and catalyst, temperature and reaction time were optimized with the aim of maximizing the conversion of ME into Me-10-HSA and FAMEs.

#### **2. Analysis of Results**

#### *2.1. Characterization of the Lipid Component of Sewage Scum and Biodiesel Production*

Biofuels are considered the leading renewable energy sources, presenting several advantages with respect to conventional fossil fuels [51,52]. Nevertheless, the high production costs associated with the raw materials (vegetable oils and animal fats) result in a significant increase in their price [42,43].

Sewage scum can be considered a cheap and available feedstock to synthesize biofuels due to its high lipid content (up to 36–50% of dry weight) [29]. The lipid fraction, very rich in FFAs, can be easily converted into FAMEs by acid-catalyzed direct esterification [53–55]. However, the high water content in the sewage scum (TS = 10–25%wt) represents a significant obstacle to biodiesel production at a commercial scale. The initial stages, from the collection of the raw sludge to the dehydration and drying, are expensive processes, which make the biodiesel production from sewage scum not economically feasible [56]. Furthermore, the subsequent extraction of the lipid fraction requires a significant amount of organic solvent, thus increasing the manufacturing costs [57].

Lastly, the method typically known for biodiesel production from sewage scum is based on homogeneous acid catalysts as H2SO4 [53–55]. Still, it is not competitive with the conventional technologies from triglycerides under alkaline catalysis for a series of drawbacks: (i) the recovery of the catalyst takes place only partially, and (ii) additional steps are required for the purification of the final products. A new methodology was then developed to successfully convert wastewater sewage scum into biodiesel, consisting of four different steps [28]. The overall process is outlined in Figure 2. First, sewage scum was heated at a temperature of 80 ◦C, with the lipid component was recovered by centrifugation at 4000 rpm for 3 min (recoverability > 90%), without the addition of solvents or acids [29]. Subsequently, the lipid extract was activated by adding the stoichiometric amount of formic acid (HCOOH) to calcium soaps (25–30%), thus obtaining their complete conversion into FFAs [44]. Activated lipids (FFAs = 75–80%) were then efficiently converted into the corresponding methyl esters by direct esterification using AlCl3·6H2O as a catalyst [45].

As a result, about 95% FFAs were converted into FAMEs with minimal reactants under mild conditions (molar ratio FFAs:MeOH:catalyst = 1:10:0.02, 72 ◦C, 2 h). Moreover, the use of AlCl3·6H2O favored a convenient separation of products between the two phases: the catalyst was recovered entirely, with the upper methanol phase along with the water produced during the reaction, whereas the methyl esters were present in the lower oily phase [45]. This resultant oily phase was recovered and pure FAMEs (75–80%wt) were collected by vacuum distillation (Figure 2).

The proposed scheme for exploiting sewage scum for biodiesel production has proven to be economically viable and applicable on an industrial scale. Nevertheless, the potential of the lipid component has not yet been fully exploited. After the distillation process, a residue was recovered (20–25%wt), which was analyzed by preparative chromatography. The residue was mainly composed of: ME (50.3%wt), polar compounds (33.8%), FAMEs (6.4%wt) and small quantities of Me-10-HSA (3.6%wt), Mineral oils (2.7%wt), Waxes (1.8%wt), FFAs (1.0%wt) and Methyl-10-ketostearate (0.4%wt). ME already have a potential market value as biolubricants [8–10]; however, to obtain a complete valorization of the lipid component, a further improvement of the reaction by-products could help to improve the economy of the overall process. For these reasons, the transesterification reaction of ME with methanol for the synthesis of Me-10-HSA and FAMEs was investigated, by optimizing the process parameters.

**Figure 2.** Scheme of biodiesel synthesis and purification from sewage scum.

*2.2. Optimization of Transesterification Conditions for the Conversion of ME into Me-10-HSA and FAMEs*

The conversion of ME into Me-10-HSA and FAMEs (according to the scheme reported in Figure 1) was optimized using AlCl3·6H2O and HCl as catalysts. According to the Box–Behnken experimental design described in Section 3.6 experiments were conducted on the distillation residue to find the optimal reaction conditions and study the process parameters' effect in the transesterification reaction. Experimental and predicted values for ME conversion at the design points are reported in Table 1.

A quadratic regression model was used to fit the experimental data, by obtaining the following relationships between factors and response for the two catalysts (Equations (1) and (2)):

*ME conversion AlCl*3·6*H*2*O* (%) = – 45.6167 + 11.6267*C* + 38.4455*cat* + 1.75413*T*+ +0.410478*t* – 1.01146*C*2– 34.8958*cat*2– 0.00761458*T*2– 0.00848126*t* 2– 2.875*Ccat*+ – 0.030625*CT* + 0.0644231*Ct* + 0.203125*catT* + 0.0865385*catt* – 0.00134615*Tt* (1)

*ME conversion HCl* (%) = 57.6733 + 10.5721*C* + 33.0761*cat*– 0.0389744*T*+ +0.684689*t* – 0.404167*C*2–10.4948*cat*2+0.00192708*T*2– 0.000690335*t* 2– 2.4375*Ccat*+ – 0.054375*CT* + 0.00384615*Ct*– 0.034375*catT*–0.177885*catt*– 0.00442308*Tt* (2)

The graphs between the predicted and the experimental ME conversion (%) reported in Figure 3 show that expected values are similar to the observed values, therefore validating the model's reliability in establishing the correlation between the process variables and the ME conversion.

**Table 1.** Box–Behnken design matrix for the four independent variables and the experimental ME conversion (%) using AlCl3·6H2O and HCl as catalysts.

a Denoted as central points. Pred. = predicted values, Exp. = experimental values.

**Figure 3.** *Cont.*

**Figure 3.** Predicted vs. experiment value for ME conversion (%) using (**a**) AlCl3·6H2O and (**b**) HCl as catalysts.

Subsequently, the significance of each parameter was evaluated by the analysis of variance (ANOVA) followed by Fisher's statistical test (F-test) for linear, interaction, and quadratic parameters in the second-order polynomial equations. In this work, the significance of the mathematical model adopted was associated with the *p*-value. A value of 0.05 was considered a suitable threshold with the corresponding significant parameters highlighted with an asterisk. The model's main statistics and the components of the fitting equations are given in Tables 2 and 3.

**Table 2.** The ANOVA summary table for the conversion of ME into Me-10-HSA and FAMEs using AlCl3·6H2O as catalyst.

R2 = 96.95% \* *p* < 0.05 indicates model is significant. R2(adjusted for d.f.) = 93.39% \*\* *p* < 0.05 indicates model terms are significant

**Table 3.** The ANOVA summary table for the conversion of ME into Me-10-HSA and FAMEs using HCl as catalyst.

R2 = 93.32% \* *p* < 0.05 indicates model is significant. R2(adjusted for d.f.) = 90.46% \*\* *p* < 0.05 indicates model terms are significant.

The *p*-associated values for the models adopted were less than 0.05, indicating that the model used to describe the transesterification reaction of ME with methanol was statistically significant. All linear parameters were substantial in the transesterification process. In particular, it was noted that there was a considerable difference in the relationship between the independent variables and their effects on the response variable (ME conversion) for the two catalysts. Using AlCl3·6H2O as a catalyst, temperature showed the most significant impact in the transesterification reaction followed by the amount of the catalyst, methanol and reaction time (Table 2). Instead, in the case of HCl, the amount of catalyst and methanol were the most significant variables with respect to temperature and reaction time (Table 3). As for the other terms (interaction and quadratic parameters), only *Ccat* (the interaction between the amount of methanol and catalyst), *C*2 (the quadratic term associated with the methanol amount) and *cat*2 (the quadratic term related to the amount of catalyst) were significant for both catalysts. Then, the goodness of fit of the models was checked by the coefficient of determination *R*2. The value obtained of 0.9339 and 0.9046, respectively, for AlCl3·6H2O and HCl in its adjusted form, confirmed the efficacy of the model adopted.

Finally, response surface plots were generated to investigate the influence of the process parameters in the conversion of ME and identify the optimal experimental conditions required for both catalysts. Figure 4a shows the combined effect of methanol and catalyst, at a fixed temperature of 100 ◦C and a reaction time of 17 h. By increasing the amount of methanol and catalyst, an increase of conversion of ME was observed: 95 and 100%, were respectively obtained for AlCl3·6H2O and HCl with 5 mL of methanol and 1 mmol of catalyst. In Figure 4b, the effect of the temperature with the reaction time was investigated (methanol = 3 mL, catalyst = 0.6 mmol). In this case, the key role played by temperature in the transesterification reaction for the two systems studied is clear. At 120 ◦C, for AlCl3·6H2O, the transesterification process's kinetic was particularly slow and long reaction times (30 h) were required for the full conversion of ME. In contrast, the reaction was complete after a few hours (4–6 h) using HCl. Figure 4c,d show the combined effect of temperature and catalyst (methanol = 3 mL, time = 17 h) and reaction time and methanol (catalyst = 0.6 mmol, temperature = 100 ◦C), respectively. These combinations of factors positively influenced the conversion of ME, obtaining a value close to 100%.

**Figure 4.** Response surface plot of the combined effects of: (**a**) methanol and catalyst amount (temperature = 100 ◦C, time = 17 h), (**b**) temperature and reaction time (methanol = 3 mL, catalyst = 0.6 mmol), (**c**) temperature and catalyst (methanol = 3 mL, time = 17 h), (**d**) time and methanol (catalyst = 0.6 mmol, temperature = 100 ◦C) for AlCl3·6H2O and HCl.

Based on these results, the optimal conditions were determined and directly applied in the transesterification of ME with methanol. The results obtained are reported in Table 4. Predicted responses are found to be in good agreement with the experimental results. In detail, using AlCl3·6H2O as a catalyst (0.76 mmol), a ME conversion of 99.6% was obtained at 115 ◦C after 30 h of reaction and 3.9 mL of methanol. Instead, the reaction catalyzed by HCl (1 mmol) was much faster: a ME conversion of 99.8% was achieved at 120 ◦C with a reduced amount of methanol (2.1 mL) after 4 h. In the absence of the catalyst, using the highest amount of methanol (5 mL) at 120 ◦C, a ME conversion of only 3% was obtained, confirming the efficiency of both catalysts.

**Table 4.** Results of the model validation under optimum conditions using AlCl3·6H2O and HCl as catalysts.

Pred. = predicted values, Exp. = experimental values.

The transesterification in methanol of stearyl stearate was also evaluated to compare the different reactivity between HCl and AlCl3·6H2O vs. a fatty ester less congested sterically.

As can be seen from Table 5, for both the catalysts, a total conversion of stearyl stearate in methyl stearate was obtained at 100 ◦C after 24 h. In the case of HCl catalysis, the conversion is high even after 2 h at 70 ◦C (80%), showing a higher reaction rate than that of AlCl3·6H2O, for which the conversion was only about 20%. Furthermore, regarding AlCl3·6H2O, the results obtained clearly show that the amounts of methanol and catalyst greatly influence the transformation of stearyl stearate. This different behavior could be ascribed to the different strength of acidity among these two catalysts and the higher steric hindrance related to the hexa-aquo complex of aluminum chloride [45]. In fact, it was already demonstrated that the partial substitution of water coordinated to the aluminum center in AlCl3·6H2O produces a mixed-aquo-alcohol complex, which acts as a Brønsted acid.

**Table 5.** Stearyl stearate conversions under HCl and AlCl3·6H2O catalysis.

#### *2.3. Analysis of the Reaction Products*

Once identified the optimal experimental conditions required for the complete conversion of ME, the organic phase was processed by column chromatography [28] and the products obtained were isolated, analyzed and quantified (Figures S1–S8). Based on 100 g of distillation residue, 28.2 g FAMEs and 32.3 g Me-10-HSA were respectively achieved. A detailed analysis of the fatty acids (FAs) profile was carried out by comparing the chemical composition of the methyl esters obtained with that of biodiesel previously recovered from FAME distillation. The results are reported in Figure 5.

**Figure 5.** Comparison of FAs profile (%wt.) of methyl estolides and lipids extracted from sewage scum.

ME contained predominately oleic acid (C18:1) and linoleic acid (C18:2), totaling 73.9% of fatty acids present (AMW FAs = 276.8 g/mole).  This difference in FAs profile can be attributed to a possible origin of estolides as reaction products between 10-HSA (obtained from the enantioselective microbial hydration of oleic acid [25]) the subsequent esterification and/or transesterification with the oils and fats present in the stream. Considering only the market value of biodiesel produced (EUR 0.8 kg−1 [58]), a potential gain of EUR 225 could be obtained for each ton of the sample treated, leading to a further enhancement of sewage sludge to produce biofuels and biochemicals. However, the greatest profits would be obtained from Me-10-HSA produced during the process.

A high enantiomeric excess (ee > 92%) of R-enantiomeric form was observed (Figure S10), compound employed as the precursor to produce biochemicals, namely γ-(R)-dodecalactone. Since its current market value ranges from EUR 800 to 3000 kg−1 [59], the economy of the whole process would be greatly improved. As described above, the synthesis of 10-HSA generally requires the use of enzymes. The use of HCl or AlCl3·6H2O as a catalyst not only is significantly cheaper (EUR 0.8 kg−1 [60]), but in the case of AlCl3·6H2O at the end of its use, it could potentially be used in WWTPs as a coagulant, further contributing to the overall economy of the process.

#### **3. Materials and Methods**

#### *3.1. Reagents and Instruments*

All chemical reagents used in this work were of analytical grade and were used directly without further purification or treatment. Hexane (C6H14, 99%), toluene (C6H6, 99%), methanol (CH3OH, 99.8%) and methyl heptadecanoate (C18H36O2, ≥99%) were purchased from Sigma-Aldrich. Aluminum chloride hexahydrate (AlCl3·6H2O, 99%) was obtained as pure-grade reagent from Baker. Ethanol (C2H5OH, ≥99.8%), diethyl ether ((C2H5)2O, 99%), formic acid (HCOOH, 99%), sulfuric acid (H2SO4, 98%), hydrochloric acid (HCl, 37%) and potassium hydroxide (KOH, 85%) were purchased from Carlo Erba.

A Rotofix 32 Hettich Centrifuge was used for the centrifugation experiments.

Identification of ME, Me-10-HSA and FAMEs was carried out by gas chromatographymass spectroscopy (GC-MS) using a Perking Elmer Clarus 500 equipped with a Clarus spectrometer. Quantitative determinations were performed using a Varian 3800 GC-FID. Helium was used as a carrier gas with a flow of 1.3 mL min−1. Both instruments were configured for cold on-column injections with a HP-5MS capillary column (30 m; Ø 0.32 mm; 0.25 μm film). The same temperature program was employed for the injector and the oven. The initial temperature was set to 60 ◦C and kept constant for 2 min. Then, it increased to 300 ◦C with a 15 ◦C min−1 ramp and kept constant for other 20 min. The temperature of detector (FID) was set to 300 ◦C. For GC-MS, the ion source was set to 70 eV and maintained at 250 ◦C.

FTIR spectra were recorded by a Perkin Elmer FTIR Spectrum BX instrument using KBr cells (neat compounds).

1H NMR spectra were recorded on a Bruker AV-400 spectrometer using the residual solvent peak as a reference [61].

#### *3.2. Sewage Scum*

Sewage scum was collected from WWTPs of Bari West (240,000 Population Equivalent, PE), located in South of Italy. Samples were immediately processed to avoid long storage time (within two days, 4 ◦C) and characterized in terms of total solids, lipids, proteins, cellulose, lignin and ashes [29].

#### *3.3. Experimental Procedure for Lipids Characterization*

#### 3.3.1. Determination of FFAs and Soaps

FFAs were determined by titration of the acidity present with a 0.1 N KOH solution and phenolphthalein (≥99%, Sigma-Aldrich) as an indicator. A total of 1 g of the sample collected was previously dissolved into 150 mL of a 1:1 *v*/*v* diethyl ether:ethanol mixture. Using the same experimental conditions, soaps were determined by titration with a 0.1 N HCl solution and methyl red (99%, Sigma-Aldrich) as an indicator.

#### 3.3.2. Determination of Fatty Acids Profile and Average Molecular Weight

In a glass Pyrex reactor of 5 mL, 0.02 g of sample were dissolved with 2 mL of a 2:2:0.01 *v*/*v*/*v* toluene:methanol:concentrated H2SO4 solution. The system was closed and placed into an ultrasonic bath at 70 ◦C for 5 h. Then, 1 mL of methyl heptadecanoate toluene solution (1000 ppm) was added as internal standard and the resulting solution gaschromatographically analyzed (1 μL). Average molecular weight (AMW) was determined according to the following equation (Equation (3)):

$$\text{AMW} = \frac{\sum \text{A}\_{\text{i}} \text{MW}\_{\text{i}}}{\sum \text{A}\_{\text{i}}} \tag{3}$$

where Ai and MWi are the area and molecular weight of FFAs identified, respectively. Then, FAMEs content (%wt.) was calculated respect to methyl heptadecanoate as follows (Equation (4)):

$$\text{FAMEs content} = \frac{\sum \text{A}\_{\text{i}}}{\text{A}\_{\text{sdt}}} \times \frac{\text{m}\_{\text{sdt}}}{\text{m}\_{\text{sample}}} \times 100\tag{4}$$

where Asdt and msdt are the area and the mass of standard (methyl heptadecanoate), respectively, and msample is the amount of sample analyzed.

#### *3.4. Extraction of Lipid Fraction from Sewage Scum and Chemical Activation*

In a glass Pyrex reactor of 250 mL, 100 g of sewage scum were placed and closed. The system was heated in an oven at 80 ◦C. After this thermal treatment, the sample was rapidly centrifuged at 4000 rpm for 3 min by obtaining a three-phasic system consisting of: (i) an upper organic brown oily phase, (ii) a lower phase of wet residual solid, and (iii) an aqueous intermediate phase. The oily phase was recovered and stored at 4 ◦C for the subsequent operations. The isolated product was mainly constituted by FFAs (51.7%wt) and calcium soaps of fatty acids (30.4%wt). Then, the stoichiometric amount of HCOOH respect to the calcium soaps was added (4.8 g for 100 g of raw lipids) and the activated lipids recovered as clear oil, after centrifugation (4000 rpm, 1 min) at 80 ◦C [44].

#### *3.5. Conversion of Activated Lipids into Methyl Esters of Recovery of Biodiesel Produced by Distillation Process*

Activated lipids extracted from sewage scum were converted into the corresponding methyl esters, by direct esterification with methanol using AlCl3·6H2O as a catalyst [45]. The reaction was carried out at 72 ◦C for 2 h with a molar ratio FFAs:MeOH:catalyst = 1:10:0.02. At the end of the process, the reagent mixture was cooled to room temperature with the formation of bi-phasic system consisting of: (i) a light methanol layer (in which the catalyst was present) and (ii) a lower oily layer composed of methyl esters. The oily phase was recovered, and the residual methanol removed under vacuum (60 ◦C, 700 mmHg). Finally, biodiesel (75–80%wt, purity > 99%) was collected by subsequently vacuum distillation (160–180 ◦C, 50 mm Hg). The distillation residue (20–25%wt) was instead recovered, dried under nitrogen flow and analyzed by column chromatography [29], obtaining the chemical composition reported in Table 6.

**Table 6.** Chemical composition of the distillation residue obtained after direct esterification of the lipid fraction with methanol and recovery of biodiesel produced by distillation process.

#### *3.6. Transesterification Reaction of Methyl Estolides (or Stearyl Stearate) with Methanol*

In a typical reaction, 0.1 g of sample (the distillation residue recovered in the previous step, ME content = 50.3% or stearyl stearate) were placed with methanol (1 mL) and 0.2 mmol of catalyst (HCl or AlCl3·6H2O) in a glass Pyrex reactor of 15 mL. The system was closed and placed into a thermostatic bath at 80 ◦C for 4 h under agitation (250 rpm), using a magnetic stirring. Then, it was cooled to room temperature and the residual methanol was removed under nitrogen flow. Where it was possible, the catalyst was recovered by centrifugation and the organic phase was analyzed by gas chromatography for the determination of FAMEs content. Following the same procedure, Me-10-HSA and ME were also determined with the calibration curves obtained from the pure product, previously isolated by column chromatography [28].

#### Optimization of Transesterification Conditions

A three-step approach was used to investigate the effects of the process variables in the conversion of ME into Me-10-HSA and FAMEs and maximize their yield [44]. Methanol (C) and catalyst (cat) amount, temperature (T) and reaction time (t) were selected as independent variables (factors), while methyl estolides (ME) conversion was set as dependent variable (response). The experimental range of the levels and the independent variables considered in this study are presented in Table 7.

a Step change values.

A total of 27 experiments (including three replicates for the center point), were used for fitting a second-order response surface. The effects of factors on the response were analyzed according to the following quadratic function (Equation (5)):

$$\mathcal{Y} = \beta\_0 + \sum\_{i=1}^{n} \beta\_i X\_i + \sum\_{i=1}^{n} \beta\_{i\bar{i}} X\_i^2 + \sum\_{i=1}^{n} \sum\_{i$$

where Y represents the ME conversion (%), *Xi* and *XJ* are the independent variables, *β*0, *βi*, *βij* and *βii* are the offset term, linear, interaction, and quadratic parameters, respectively. Statgraphycs® Centurion XVI was used for the regression analysis and the plot response surface. Then, to verify the validation of the overall fit of the developed regression model, the data obtained were processed by the analysis of variance (ANOVA). The adequacy of the polynomial model to fit experimental data were expressed as R2 (coefficient of determination) and in its adjusted form. The statistical significance of R2 was checked by the F-test at a confidence level of 95%.

Finally, the optimization of reaction conditions was carried out using response surface methodology (RSM) combined with the desirability function approach to form the desirability optimization methodology (DOM) [45].

#### **4. Conclusions**

In this work, for the first time, an efficient method was proposed for the synthesis of Me-10-HSA and FAMEs by direct conversion of methyl estolides isolated from sewage scum. A response surface methodology was applied to investigate the effect of the process variables on the methyl estolides conversion and maximize the final yield. AlCl3·6H2O and HCl were used as effective catalysts in promoting transesterification with methanol. HCl is more active in promoting the transesterification of methyl estolides to produce FAMEs and Me-(*R*)-10-HSA: a total conversion was in fact obtained already after 4 h. In the case of AlCl3·6H2O, under similar reactive conditions, 20 h were necessary to achieve a conversion of 99.4%. On the other side, AlCl3·6H2O is a solid catalyst, easy to manage and less corrosive than mineral conventional acids [62]. AlCl3·6H2O-catalyzed reaction resulted principally affected by temperature, whereas in the case of HCl, the amount of catalyst and methanol were the most significant variables. The obtainment of Me-(*R*)-10-HSA, in its (almost) pure enantiomeric form, increases the potential of sewage scum. For the specific case of AlCl3·6H2O, the possible final use of the relevant residues in WWTPs as a coagulant results in a new scheme of valorization of a special waste, namely sewage scum, in which no secondary waste was generated. The transesterification of methyl estolides could actually implement the scenario of the full valorization of sewage scum towards a multi-products biorefinery. With the use of a limited number of reagents namely MeOH and AlCl3·6H2O, and an integrated network of processes, biodiesel, methyl estolides, and Me-(*R*)-10-HSA would be effectively obtained from sewage scum in a sustainable way.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/catal11060663/s1, Figure S1: GC-chromatogram of methyl estolides isolated by distillation process, Figure S2: FTIR spectra of methyl estolides, Figure S3: GC-MS chromatogram of 10-(palmitoyloxy)-stearic methyl ester, Figure S4: GC-MS chromatogram of 10-(stearoyloxy)-stearic methyl ester, Figure S5: GC-chromatogram of methyl 10-(*R*)-hydroxystearate isolated, Figure S6: FTIR spectra of methyl 10-(*R*)-hydroxystearate, Figure S7: GC-MS chromatogram of methyl 10- (*R*)-hydroxystearate, Figure S8: 1H NMR of isolated methyl 10-(*R*)-hydroxystearate, Figure S9: Comparison of chromatographic profiles of FAs obtained from methyl estolides and lipids extracted from sewage scum, Figure S10: Chemical structures of derivatizing agents (D1, D2 and D3) used for the determination of the absolute configuration of the Methyl 10-Hydroxy stearic acid (M10-HSA) isolated from sewage scum.

**Author Contributions:** L.d.B.: Methodology, Writing—original draft, Investigation, Validation. V.D.: Methodology, Writing—original draft, Investigation. C.P.: Conceptualization, Methodology, Investigation, Resources, Writing—review & editing, Supervision, Project administration, Funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was financially supported by MIUR (ERANETMEDNEXUS-14-035 Project WE-MET). This research was partially supported by European Union—FESR "PON Ricerca e Innovazione 2014–2020. Progetto: Energie per l'Ambiente TARANTO—Cod. ARS01\_00637".

**Data Availability Statement:** The data presented in this study are contained within the article and the supplementary material.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

### *Article* **MgO Catalysts for FAME Synthesis Prepared Using PEG Surfactant during Precipitation and Calcination**

**Valdis Kampars \*, Ruta Kampare and Aija Krumina**

Institute of Applied Chemistry, Faculty of Material Sciences and Applied Chemistry, Riga Technical University, LV-1048 Riga, Latvia<EMAIL_ADDRESS>(R.K<EMAIL_ADDRESS>(A.K.)

**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** To develop a method for the preparation of MgO nanoparticles, precatalyst synthesis from magnesium nitrate with ammonia and calcination was performed in presence of PEG in air. Without PEG, the catalysts are inactive. The conversion to hydroxide was performed using a PEG/MgO molar ratio of 1, but, before the calcination, excess of PEG was either saved (PEG1) or increased to 2, 3, or 4 (PEG 2–4). Catalysts were calcined at 400–660 ◦C and characterized using XRD, N2 adsorption-desorption, TGA, FTIR, and SEM. The FAME yield in the reactions with methanol depend on the PEG ratio used and the calcination temperature. The optimal calcination temperature and highest FAME yield in the6hreactions for catalysts PEG1, PEG2, PEG3 and PEG4 were 400 ◦C, 74%; 500 ◦C, 80%; 500 ◦C, 51% and 550 ◦C, 31%, respectively. The yield dependence on calcination temperature for catalysts with a constant PEG ratio is similar to that of a bell curve, which becomes wider and flatters with an increase in PEG ratio. For most catalysts, the FAME yield increases as the size of the crystallites decreases. The dependence of FAME and the intermediate yield on oil conversion confirms that all catalysts have strong base sites.

**Keywords:** heterogeneous catalysts; combustion; PEG; transesterification; biodiesel

#### **1. Introduction**

Biodiesel is one of the three main biofuels and is the main biofuel in the EU. The European biodiesel market has been stabilized at around 10 million tons/year [1]. Biodiesel is currently produced from high-quality vegetable oil (EU rapeseed oil) by transesterification with methanol at 65 ◦C in the presence of a homogeneous basic catalyst using a methanol/triglyceride molar ratio of 6/9. Reaction time does not exceed 1h. In parallel with the production of biofuels from high-quality edible oils, there is a growing trend towards the use of cheap and non-edible raw materials for the production of advanced biodiesel [2]. The RED II Directive provides for a gradual transition for Member States from conventional to advanced biofuels, with the following shares of energy consumption in the transport sector: 0.2% in 2022, 1% in 2025, and 3.5% in 2030 [3]. The production of advanced biofuels makes it possible to use local raw materials, including waste, and to reduce the extremely harmful effects of the transportation sector on the environment and climate.

Regardless of the raw material, the currently dominant acquisition process involves esterification in the presence of a homogeneous catalyst, and has various significant drawbacks:

**Citation:** Kampars, V.; Kampare, R.; Krumina, A. MgO Catalysts for FAME Synthesis Prepared Using PEG Surfactant during Precipitation and Calcination. *Catalysts* **2022**, *12*, 226. https://doi.org/10.3390/ catal12020226

Academic Editor: Domenico Licursi

Received: 1 December 2021 Accepted: 11 February 2022 Published: 16 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

135

• The production of biodiesel from low-quality feedstock requires pre-treatment and the resulting raw material has increased the content of free fatty acids (FFA), so the use of homogeneous catalyst causes soap formation.

All the above-mentioned problems could be solved by using heterogeneous basic catalysts [4,5]. The development of these catalysts was intensified by the RED II Directive requirements, but the achieved results so far are unsatisfactory. Transesterification reactions in the presence of active basic heterogeneous catalysts usually take place at 90–150 ◦C for 4–8 h [6,7]. The only heterogeneous catalyst developed by the AXENS corporation, which has proven to be good and durable in industrial operations, works at a temperature of 190–220 ◦C and pressure of 40–70 bar [8]. As the currently dominant biodiesel production technology is implemented at atmospheric pressure, it is not expected that developments will take place in terms of the complexity of the production infrastructure and there is a significant increase in production cost by using high temperature and pressure. Undoubtedly, the most promising direction of development in the industry is processing at low temperature in the presence of more active and robust heterogeneous catalysts.

Biodiesel synthesis from triglyceride (TG) proceeds accordingly to the balanced summary equation (Equation (1)):

$$\text{TG} + \text{3 MeOH} \rightleftharpoons \text{3 FAME} + \text{G} \tag{1}$$

where MeOH—methanol; FAME—mixture of fatty acid methyl esters (biodiesel); and G—glycerol.

Reaction (1) consists of three reversible stages and proceeds with the production of intermediates, such as diglycerides (DG) and monoglycerides (MG). In the presence of homogeneous basic catalysts, reaction is initiated by generation of methoxide anion (2), which attacks the carbonyl group of TG and causes fast FAME production without losing the active anion intermediates:

$$\text{MeOH} + \text{B}^- \rightleftharpoons \text{MeO}^- + \text{BH} \tag{2}$$

where B− are HO− or RO−.

All reactions are reversible, therefore, the partitioning of glycerol in the form of separate layers is very important for shifting the equilibrium (1) to the right. In order for the reactions to proceed rapidly and selectively, a necessary minimum B− concentration must be maintained [9]. In the presence of basic heterogeneous catalysts, the mechanism is considered to be similar, but the role of the base is to create strong base sites on the surface of the catalysts. Similar to homogeneous catalysis, the reaction is initiated by methoxide anion generation and the activity of the heterogeneous catalyst should be determined by the density of the strong base sites on its surface [10–13]. Most of the successfully investigated metal oxide basic catalysts for transesterification reactions belong to the M(II)O group and are ionic. The surface of these catalysts is terminated by metal cations and oxide anions (O2−), and contains different types of defects and environments (kinks, steps, terraces), which play a determining role in the catalytic phenomenon [14]. Successfully tested catalysts for biodiesel production are BeO, MgO, CaO, SrO, and BaO [15]. From these, calcium oxide is favored as it is highly active, cheap, and eco-friendly [16]. Unfortunately, this catalyst is leached during reaction and loses its activity in contact with air, it therefore cannot be considered as a prospective choice [15]. More stable and promising is MgO, which is used in medicine as a catalyst and an adsorbent [17,18]. The polarizing power of magnesium ion (3.9) is remarkably stronger than that of calcium ion (2.0), which would make the building of strong base sites more difficult than in case of CaO [19]. This is confirmed by the high dependence of MgO catalyst activity on manufacturing conditions [20]. Solution combustion synthesis of catalysts increases activity by employing metal salts as oxidants and different organic compounds as fuels in order to use the heat of the redox reaction [21–23]. The characteristic stages of catalyst synthesis according to this method are: sol solution, viscous gel, dried gel, and calcined product preparation. The most active oxidants are nitrates, but their mixture with organic materials is explosive and there are less prospective industrial uses. To prevent the explosiveness proceeding the calcination stage, conversion from the nitrates to the hydroxides is widely used [17]. The present work is undertaken to investigate the synthesis of active MgO catalysts, and avoiding explosion during calcination by replacing strong oxidants with a weak oxidant, such as air.

#### **2. Results and Discussion**

#### *2.1. Preparation of Catalysts and Their Characteristics*

Magnesium salts, such as nitrate, sulfate, chloride, carbonate, acetate, and citrate, are most often used as feedstocks for the synthesis of MgO. Often, these salts are not used directly as a source of magnesium, but are converted into a magnesium hydroxide precatalyst. This is based on the considerations that low-temperature decomposition of magnesium hydroxide yields pure magnesium oxide. Thus, it is possible to perform calcination without harmful gases. The addition of different organic compounds as fuels before calcination prevents the formation of explosive mixtures. Thermal decomposition of magnesium hydroxide is well investigated. Its low decomposition temperature (300–400 ◦C) allows easy activation of the precatalyst [20,24]. As presented in Figure 1, the calcination of Mg(OH)2 at 375 ◦C is sufficient for obtaining the polycrystalline cubic structure of MgO nanoparticles.

**Figure 1.** XRD patterns of PEG1, calcined at 375 ◦C for 3 h, obtained using Cu K-alpha radiation (0.15406 nm).

The peaks at 37◦, 43◦, 62.4◦, and 74.7◦ correspond to (111), (200), (220), and (311) reflections, which reveal the formation of the polycrystalline structure of MgO nanoparticles [25]. No peaks from impurities were detected in the registered patterns. The sharp diffraction peaks indicate the good crystallinity of the MgO products.

It is known that higher activity of catalysts is obtained when fuel and surfactants are present in both precatalyst synthesis and in the activation stages [26–28]. During the preparation of a precatalyst, PEG 6000 works as a surfactant, but during the calcination it is as a surfactant and fuel. Our research shows that the presence of PEG during the synthesis of the precatalyst in a molar ratio of MgO of 1 creates a small effect, but a further increase in PEG shows no improvement up to 4. Therefore, in all cases, we performed the first step using a molar ratio of PEG 6000 to MgO of 1. The presence of surfactant and fuel at the calcination stage changes the structure and activity of the resulting catalyst and the effect depends on the ratio to catalyst [28]. Thermal analysis up to 700 ◦C (Figure 2) of precatalysts was performed to examine PEG 6000 effects in an inert atmosphere.

From the TG curves, it can be seen that samples PEG1 and PEG4 display a very notable mass loss near 400 ◦C, which is related to the decomposition of PEG 6000. In the row PEG1–PEG2–PEG3–PEG4, gradual changes in the TG curves occur, suggesting that the calcination temperature at 400 ◦C would be sufficient to obtain active MgO nanoparticles.

**Figure 2.** Thermogravimetric analysis of precursors of PEG1 and PEG4.

As shown in Figure 2, a slight weight loss is still observed in the temperature range of 430 °C to 730 °C, which can be explained by the elimination of adsorbed carbonate species [20].

The nitrogen adsorption–desorption isotherms and specific surface area, total pore volume, the pore sizes of the catalysts are shown in Figure 3 and Table 1, respectively. All catalysts have large pores with an average pore diameter of around 5 nm. As seen from Figure 3, the isotherms of MgO are type IV with an H3 hysteresis loop.

**Figure 3.** Nitrogen adsorption–desorption isotherms of PEG2 calcined at 400 ◦C and 450 ◦C.

As seen in Table 1, the change in the textural characteristics is small and the number of performed experiments does not allow to draw conclusions about the effect of texture on the activity of the catalysts.

Characteristic SEM images of the obtained MgO are presented in Figure 4. Two different forms of aggregates dominate. These images indicate that the powder sample is an agglomeration of nanoparticles. Irregular aggregated flake-like particles with a rough surface represented the active catalysts, while MgO composed of many irregular nanoparticles did not catalyze the interesterification reactions. A similar phenomenon, with a significant morphological effect on the activity of catalysts or adsorbents, has also been observed in other works [29,30]. The aggregation process with the formation of a unique morphology may play a crucial role in ensuring catalytic activity.

**Figure 4.** SEM image of active (**left**) and inactive (**right**) MgO catalysts.

#### *2.2. Catalytic Performance*

Initialization of the transesterification reaction proceeded according to Equation (2). The catalytic activity of heterogeneous catalysts depends on the density of strong base sites on the catalyst surface. This depends on the morphology, number, and types of defects [10–13]. The presence of PEG improved MgO activity. Catalysts synthesized without PEG under experimental conditions did not provide a FAME yield above 3%.

#### 2.2.1. Effects of PEG Content and Calcination Temperature

To determine the effect of PEG content and calcination temperature on catalytic performance, each PEG1–PEG4 precatalyst was calcined at a different temperature. As shown in Figure 5, sharp maxima of the FAME yield were registered for the PEG1 catalyst at 400 ◦C, for the PEG2 catalyst at 500 ◦C, for the PEG3 catalyst at 500 ◦C, and there was a flatter maximum for PEG4 at 550 ◦C.

**Figure 5.** Effect of PEG content and calcination temperature on FAME yield in transesterification reactions at 65 ◦C after 6h.

The repetition of individual points showed that the standard deviations of the FAME yield of the PEG1 catalyst at 400 ◦C reached 15.5%. It also showed that the density of strong base catalytic sites depends on more than just the calcination temperature. It was also affected by minor changes in the calcination process, including air circulation, crucible form, the volume of precatalyst in the crucible, and location in the muffle furnace. As the combustion process proceeds by the reaction of fuel with oxygen in the air, this seems understandable and requires a more precise process for providing airflow control. The standard deviation of the results was lowered by increasing the PEG content, but a PEG content above 3 lowered the activity. The shape of the distribution of the experimental points from the calcination temperature for the catalyst with a constant PEG ratio is similar to a bell curve and becomes wider and lower with an increase in PEG content. PEG2 provides the best catalysts.

The dependence of the activity of MgO catalysts on small changes in the manufacturing process is well known and the safe production of an active MgO catalyst for practical use is indeed a serious problem. Babak et al. concluded that unsupported MgO was not a suitable catalyst, but Li et al. showed that mesoporous-supported MgO catalyst only becomes active at 220 ◦C [31,32]. At the same time, works on very high activity MgO catalysts have been published [6,26,27]. The problem of MgO catalyst activity has been analyzed using specific surface properties in the form of research and quantum mechanics calculation methods. Di Cosimo et al. confirmed that the surface of the magnesium oxide catalyst obtained by the calcination of magnesium hydroxide contains three types of sites with different basicity: surface sites of strong (low coordination O2− anions), medium (oxygen in Mg2+-O2− pairs), and weak (OH- groups) basicity [20]. Decomposition of Mg(OH)2 at 400 ◦C generates hydroxylated MgO, mainly containing strong O2− basic sites located in surface defects, such as the corners and edges of the crystalline solid surface. An increase in the calcination temperature removes the OH groups and also surface solid defects, creating more stable structures that contains a higher concentration of medium-strength Mg2+-O2− basic pair sites. Thus, an increase in the calcination temperature drastically decreased the density of strong base sites and, to a lesser extent, that of weak OH groups, while it slightly increased that of medium-strength base sites; therefore, a calcination temperature of 400 ◦C seems to be optimal [20]. According to Montero et al., water and methanol chemisorb preferentially over defects and edge sites over NanoMgO-700 through the conversion of surface O2− sites to OH− and the coincident creation of Mg-OH or Mg-OCH3 moieties, respectively [33]. According to this work, the best calcination temperature for magnesium hydroxide would be 700 ◦C [33]. It could be considered that both publications indicate the area fir the best calcination temperatures and the results for PEG catalysts, which narrow this area to 400–600 ◦C (Figure 5).

According to Vedrine, the main role in creating of strong base sites is different for different types of defects [14]. If this is so, then reducing the size of the crystallites should increase the density of the defects and also catalyst activity. As follows from Figure 6, in general, the FAME yield and, thus, the density of strong base sites, increase as the size of the crystallites decreases. This could indicate an association between the defects of the boundary surfaces of these crystallites with the number of strong basic centers.

**Figure 6.** Relationship between FAME yield and crystallite size. The main crystallite size is calculated using (200) and (220) reflections using the Scherrer equation (Table S1).

However, two catalysts fall out of this relationship (PEG1, FAME content 22.5 wt.% and PEG2, FAME content 73.9 wt.%), which confirms that the size of the crystallites does not always determine the density of strong base sites, and other phenomena can rule out these relationships. An unexpected decrease in catalyst activity could be caused by partial blocking of the MgO surface with combustion products, or products resulting from incomplete calcination. To determine if this was the case, FTIR spectra were recorded for individual catalysts. The obtained FTIR spectra confirmed that not all catalysts have a clean MgO surface (Figure 7), although XRD and SEM-EDS analyses did not indicate this. Absorption bands near 1420 are assignable to magnesium carbonate or hydromagnesite [34], which is not uncommon for oxide catalysts. Therefore, catalyst surfaces control, determined with FTIR, should be included in determining the characteristics [35].

**Figure 7.** FTIR spectra of active (PEG2 crystallite size 13 nm, FAME 73.5%) and inactive (PEG1 crystallite size 8 nm, FAME 22.5%) catalysts.

#### 2.2.2. Strong Base Sites and Reaction Proceeding

In reality, reaction (1) proceeds in three reversible stages:

$$\text{TG} + \text{MeOH} \rightleftharpoons \text{FAME} + \text{DG} \tag{3}$$

where DG are diglycerides,

$$\text{LDG} + \text{MeOH} \rightleftharpoons \text{FAME} + \text{MG} \tag{4}$$

where MG are monoglycerides, and

$$\text{MG} + \text{MeOH} \rightleftharpoons \text{FAME} + \text{G} \tag{5}$$

where G is glycerol.

Chromatographic analysis shows that the main constituents of the obtained reaction mixtures were FAME, MG, DG, and TG, if the amount of G did not exceed 1 wt.%. The sum of these components was usually 97–100%, which allowed us to consider that the amount of unknown by-product was insignificant. Assuming that the reaction mechanism was provided by the same active sites in all cases, the change in the composition of the reaction products depending on oil conversion should reflect the gradual realization of the transesterification. As follows from Figure 8, the experimental points form logical curves that confirm the presence of the same active sites in all cases.

 **Figure 8.** Composition of reaction mixture as a function on the conversion of oil.

The contents of MG and DG go through a maximum and approach zero above 95% conversion. DG content reaches a maximum with oil conversion of about 55%. However, the maximum content of MG was observed at an oil conversion of 70%. Up to 40% oil conversion, FAME content increases relatively slowly. As oil conversion increases, FAME content grows faster. In the conversion region, 40–80% FAME content increases at least twice as efficient than below 40%. Most likely, this is due to changes in the reaction rate of the reverse reactions caused by the gradual release of the glycerol in a separate layer.

#### **3. Materials and Methods**

#### *3.1. Materials*

Refined rapeseed oil was purchased from a local producer Iecavnieks & Co Ltd (Iecava, Latvia). The average molecular weight of the oil was 896 g/mol, density–0.92 g/mL at 20 ◦C, saponification value–186.7 mg KOH/g, and acid value–0.12 mg KOH/g. The percentages of fatty acids in the oil were: palmitic–4.1%, stearic–1.4%, oleic–62.5%, linoleic– 21.7%, linolenic–8.7%, arachidic–0.4%, and other fatty acids–1.2 wt.%. The content of MG (monoglycerides) was 0.0 wt.%, DG (diglycerides)–0.4 wt.%, and TG–99.6 wt.%.

Magnesium nitrate hexahydrate, ammonia solution 25%, PEG 6000 and methanol were supplied by Sigma-Aldrich (St. Louis, MO, USA). Materials for GC analysis—1,2,4 butanetriol (96%) and MSTFA (*N*-Methyl-*N*-(trimethylsilyl)trifluoroacetamide (97%)–were purchased from Alfa Aesar (Haverhill, MA, USA), tricaprin (98%)—from TCI Europe (2070 Zwijndrecht, Belgium), pyridine (99.5%)—from Lach-Ner (Neratovice, Czech Republic), and dichloromethane (99.5%) was supplied by Chempur (Karlsruhe, Germany). Grade 1Whatman filter paper was supplied by Sigma-Aldrich.

#### *3.2. Catalyst and Biodiesel Synthesis*

Catalyst synthesis: 100 mL of 1M magnesium nitrate solution (25.6 g/100 mL) was added to a 250 mL Erlenmeyer flask and 4.4 g of PEG 6000 was then dissolved. The obtained mixture was stirred at 500 rpm for 1 h (homogenization stage N1). Then, it was filtrated with a NH4OH (25% NH3 basis) solution in a burette to adjust the pH up to 10. The NH4OH solution was dripped from the burette slowly in a drop-wise manner. After reaching pH 10, the second homogenization process continued for 2h by stirring (homogenization stage N2) and then hydrothermal treatment was performed by leaving the solution overnight. The material obtained after centrifugation was suspended in 100 mL of PEG 6000 solution (PEG1 4.4 g/100 mL; PEG2 8.8 g/100 mL; PEG3 13.2 g/100 mL and PEG4 17.6 g/100 mL) using an ultrasound environment. Water was slowly removed using a rotary evaporator. The obtained material was placed in oven at 80 ◦C for 5 h to remove excess water moisture. The dried sample was placed in a crucible, which was placed inside a muffle furnace, for calcination at the selected temperature for 3 h. The temperature of the furnace was increased at the very slow rate of 1 ◦C·min−1 until the desired temperature of calcination was attained. Fine amorphous MgO nanoparticles were prepared and collected.

Biodiesel synthesis: 0.64 g of MgO catalyst (7% by weight of oil) was added to a 100 mL round bottom flask containing 11 mL of methanol (methanol to oil molar ratio of 27). The mixture was allowed to reflux at 65 ◦C for 0.5 h at 600 rpm, using a hotplate with a magnetic stirrer. Then, 10 mL of oil (10.3 mmol) diluted with 10 mL of co-solvent THF was added and refluxed at 65 ◦C at 600 rpm for 6 h. The obtained product was allowed to settle for 3 h. The crude biodiesel was separated from glycerol using a separating funnel. FAME was filtered using Whatman filter paper and, after removing the THF and excess methanol, stored in a glass container for analysis.

#### *3.3. Catalyst Characterization*

The samples were out-gassed at 150 ◦C for 24 h before measurements. The total surface area was estimated using the Brunauer–Emmett–Teller (BET) method. Pore diameters were derived from desorption isotherms using the Barrett–Joyner–Halenda (BJH) method.

XRD analysis was performed using an X-ray diffractometer (Bruker AXS D8 AD-VANCE, Billerica, MA, USA) in the range of 10–75◦ (2θ) with Cu K-alpha radiation (0.15406 nm) and a step size of 0.02◦.

The surfaces of the catalysts were studied with a Tescan MIRA3 LMU (Tescan, Brno, Czech Republic) scanning electron microscope. Prior to analysis, samples were coated with a gold layer using an Emitech K550X sputter coater.

Universal attenuated total reflectance-Fourier transform infrared spectroscopy (UATR-FTIR) was used for surface control of the catalysts. Measurements were carried out on a *PerkinElmer Spectrum 100* spectrometer connected to a *Universal ATR Sampling Accessory*. A spectral range 650–4000 cm−1 was selected.

TGA analysis was performed using a thermogravimetric analyzer (PerkinElmer STA 6000). The heating rate was 10 ◦C/min and experiments were performed under a pure nitrogen flow of 20 mL/min, supervised using a mass-flow controller.

#### *3.4. Biodiesel Characterization*

Analysis of all components from each sample of raw biodiesel was carried out by using an Analytical Controls (Rotterdam, The Netherlands) biodiesel analyser, based on the Agilent Technologies (Santa Clara, CA, USA) gas chromatograph 7890A. All compounds were analyzed using DB5-HT column (15 m, 0.32 mm, 0.10 μm) under conditions similar to those prescribed by standard EN 14105. Glycerol (G), monoglicerides (MG), diglicerides (DG), triglycerides (TG), and FAME were quantified as in our previous report [36]. The oven temperature was initially set to 50 ◦C for 5 min. Then, the temperature was first increased to 180 ◦C at a rate of 15 ◦C/min, then, to 230 ◦C at a rate of 7 ◦C/min, and, finally, to 370 ◦C at a rate of 10 ◦C/min. Helium was used as a carrier gas and the detector temperature was set to 390 ◦C. The reaction mixture was characterized by the mass percent content of the mentioned groups of compounds. The standard deviation for all analyzed compounds was not higher than 0.5 wt.%, but FAME and TG were not higher than 0.9 wt.%.

All experiments were repeated triplicate in order to determine the variability of the results and to assess experimental errors in GC analysis. Arithmetic averages and standard deviations were calculated for all results. Statistical analyses were performed using Microsoft Excel (2013). The yield of FAME was assumed to be equal to the wt.% of FAME content in the reaction mixture after removing glycerol, methanol, and the co-solvent.

#### **4. Conclusions**

From the alkaline earth metal oxides, MgO can be considered as the most promising catalyst for biodiesel synthesis. The chemical bond in MgO is less ionic than that in CaO, which makes it more stable but poses problems in the formation of a high density of strong base sites on the surface of nanoparticles to ensure a high catalytic activity. It is possible to significantly increase the activity of MgO nanoparticles using PEG as a surfactant and fuel, as well as with an optimal calcination temperature. FAME yield dependence on calcination temperature for catalysts obtained in presence of a constant molar PEG/MgO ratio is like a bell curve, the width, height, and position of which depend on that molar ratio. Increasing the PEG/MgO ratio has an optimum catalyst activity at 2. With the presence of catalysts PEG1, PEG2, PEG3, and PEG4, the maximum yield of FAME was achieved as 400 ◦C, 74%; 500 ◦C, 80%; 500 ◦C, 51%, and 550 ◦C, 31%, respectively. The narrowest zone of high activity was for the PEG1 catalyst and the widest and flattest was for PEG4. The PEG2 catalyst remaining the most prospective.

For most of the catalysts, the FAME yield increased as the size of the crystallites of catalyst decreased. The use of FTIR spectra showed that deviations in this relationship may be due to the retention of incomplete calcination products on the surface of MgO. FAME and intermediate yield dependence on oil conversion confirmed that all catalysts had the same types of strong base sites that were necessary for initialization of transesterification reactions.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/catal12020226/s1, Table S1. Reaction products by using different catalysts; Figure S1. XRD analysis of PEG1 catalyst Calcined at 400 ◦C; Figure S2. XRD analysis of PEG2 catalyst Calcined at 400 ◦C.

**Author Contributions:** Conceptualization, funding and final approval V.K.; methodology V.K., R.K. and A.K.; original draft preparation, editing V.K. and R.K., performed experiments and data collection R.K. and A.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was conducted as the project of the Latvian Council of Science Izp-2020/2-0194.

**Data Availability Statement:** Data are contained within the article and the Supplementary Materials.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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